WO2023220694A1 - Compositions de plaquettes chargées d'agent d'irm et leurs procédés de préparation et d'utilisation - Google Patents

Compositions de plaquettes chargées d'agent d'irm et leurs procédés de préparation et d'utilisation Download PDF

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WO2023220694A1
WO2023220694A1 PCT/US2023/066904 US2023066904W WO2023220694A1 WO 2023220694 A1 WO2023220694 A1 WO 2023220694A1 US 2023066904 W US2023066904 W US 2023066904W WO 2023220694 A1 WO2023220694 A1 WO 2023220694A1
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Prior art keywords
platelets
loaded
mri agent
composition
agent
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PCT/US2023/066904
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English (en)
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Daniel Allen SHEIK
Keith Andrew MOSKOWITZ
Benjamin J. KUHN
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Cellphire, Inc.
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Publication of WO2023220694A1 publication Critical patent/WO2023220694A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/06—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations
    • A61K49/18—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes
    • A61K49/1896—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes not provided for elsewhere, e.g. cells, viruses, ghosts, red blood cells, virus capsides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/19—Platelets; Megacaryocytes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes

Definitions

  • compositions and methods for use of platelets, platelet derivatives, or thrombosomes e.g., freeze-dried platelet derivatives
  • biological carriers of cargo such as MRI agents, also referred to herein as MRI agent-loaded platelets, platelet derivatives, or thrombosomes.
  • methods of preparing platelets, platelet derivatives, or thrombosomes loaded with the MRI agent of interest are also provided herein.
  • MRI agent-loaded platelets described herein can be stored under typical ambient conditions, refrigerated, cryopreserved, for example with dimethyl sulfoxide (DMSO), and/or lyophilized after stabilization (e.g., to form thrombosomes)
  • DMSO dimethyl sulfoxide
  • Blood is a complex mixture of numerous components.
  • blood can be described as comprising four main parts: red blood cells, white blood cells, platelets, and plasma.
  • the first three are cellular or cell-like components, whereas the fourth (plasma) is a liquid component comprising a wide and variable mixture of salts, proteins, and other factors necessary for numerous bodily functions.
  • the components of blood can be separated from each other by various methods. In general, differential centrifugation is most commonly used currently to separate the different components of blood based on size and, in some applications, density.
  • Unactivated platelets which are also commonly referred to as thrombocytes, are small, often irregularly-shaped (e.g., discoidal or ovoidal) megakaryocyte-derived components of blood that are involved in the clotting process. They aid in protecting the body from excessive blood loss due not only to trauma or injury, but to normal physiological activity as well. Platelets are considered crucial in normal hemostasis, providing the first line of defense against blood escaping from injured blood vessels. Platelets generally function by adhering to the lining of broken blood vessels, in the process becoming activated, changing to an amorphous shape, and interacting with components of the clotting system that are present in plasma or are released by the platelets themselves or other components of the blood.
  • irregularly-shaped (e.g., discoidal or ovoidal) megakaryocyte-derived components of blood that are involved in the clotting process. They aid in protecting the body from excessive blood loss due not only to trauma or injury, but to normal physiological activity as well. Platelets are considered crucial in normal hemostasis
  • Purified platelets have found use in treating subjects with low platelet count (thrombocytopenia) and abnormal platelet function (thrombasthenia). Concentrated platelets are often used to control bleeding after injury or during acquired platelet function defects or deficiencies, for example those occurring during surgery and those due to the presence of platelet inhibitors.
  • the present disclosure provides, at least in part, a composition comprising MRI agent-loaded cryopreserved platelets, or a composition comprising MRI agent-loaded platelet derivatives. Also provided herein are methods for preparing the compositions as disclosed herein along with the use of the compositions. Methods for delivering an MRI agent to a subject are also provided along with method for detecting a site of inflamed, diseases or compromised blood vessels in the subject.
  • composition comprising MRI agent-loaded cryopreserved platelets, wherein the MRI agent-loaded cryopreserved platelets comprise an MRI agent complex covalently bonded to the surface of the cryopreserved platelets, wherein the MRI agent complex comprises an MRI agent, and a chelator.
  • composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the platelet derivatives, and wherein the MRI agent complex comprises an MRI agent, and a chelator.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder, comprising:
  • a method for preparing a composition comprising:
  • FIG. 1 shows pooled apheresis platelets incubated with FITC labeled TAT peptide in loading buffer.
  • FIGs. 2A-C shows platelets analyzed by flow cytometry for FITC-TAT loading in either HMTA or loading buffer at two concentrations (25 pM or 50 pM) by mean fluorescence intensity (FIG. 2A).
  • FIGs. 2B and 2C show pooled apheresis platelets incubated FITC-labeled TAT peptide in either HMTA or loading buffer at either 50 pM FITC-labeled TAT (FIG. 2B) or 25 pM FITC-labeled TAT (FIG. 2C).
  • FIG. 3 is a flow cytometry histogram of samples incubated with 100 pM FITC-TAT in the presence of platelet anti-aggregation compounds PGE1, GR144053, and eptifibatide.
  • PGE1 appears to be associated with improved platelet loading little to no effect is observed with GR144053 or eptifibatide on the distribution of FITC-CPP.
  • FIG. 4 shows the effect of different buffers on FITC-TAT loading into platelets as measured by fluorescence intensity.
  • FIGs. 5A-C shows brightfield, FITC, and overlaid microscopy images of non-loaded platelets (FIG. 5A), 100 pM fluorescein (FIG. 5B), and 100 pM FITC-labeled TAT (FIG. 5C).
  • FIG. 6 is a flow cytometry histogram of SAMPLS incubated with either loading buffer (left peak) or a solution of FITC-labeled magnetic nanoparticles (right peak).
  • FIGs. 7A-D shows Texas Red (FIG. 7A), FITC (FIG. 7B), brightfield (FIG. 7C), and overlaid (FIG. 7D) images of samples incubated with FITC-labeled magnetic nanoparticles.
  • FIG. 8 is a flow cytometry histogram of samples incubated with either loading buffer (left peak) or a 50 pM FITC-CPP-Gd-DOTA solution (right peak) for 30 minutes.
  • FIGs. 9A-9B show a schematic (FIG. 9A) and magnetic resonance imaging (FIG. 9B).
  • samples 1A, IB. and 2B are negative controls
  • sample 2A includes Gd-DOTA- FITC-CPP with platelets (400K/pL)
  • samples indicated with 100 mM or 100 pM GdCf are positive controls.
  • FIG. 10 is a graph showing post-cry opreservation occlusion time of platelets loaded with Gd- DOTA-FITC-CPP with plasma only (negative control), pooled, unloaded platelets (positive control), and Gd-DOTA-FITC-CPP loaded platelets.
  • FIG. 11 shows exemplary flow cytometry data of thrombosomes unstained (dark data points) or stained (light data points) with an anti-CD-41 antibody.
  • FIG. 12 shows an exemplary histogram of thrombosomes incubated with annexin V with (light data points) and without (dark data points) calcium.
  • FIG. 13 shows an exemplary histogram of thrombosomes incubated with an anti-CD62 antibody (light data points) or with an isotype control (dark data points).
  • FIG. 14 shows a plot of thrombin peak height for thrombosomes in the presence of PRP Reagent containing tissue factor and phospholipids (solid line and long dashes) and control cephalin (dots).
  • FIG. 15A shows the aggregation of 250,000/pL TFF thrombosomes in buffer vs. agonist, demonstrating the aggregation response of thrombosomes in the presence of agonists, but in the absence of fresh platelets.
  • FIG. 15B shows the aggregation of 250,000 platelets/ pL of platelet rich plasma (PRP), demonstrating the aggregation response of platelet-rich plasma (PRP) in the presence of agonists, but in the absence of fresh platelets.
  • PRP platelet rich plasma
  • FIG. 15C shows an ADP comparison, demonstrating the comparison of aggregation of thrombosomes and PRP in the presence of 20 pM ADP.
  • FIG. 15D shows a collagen aggregation comparison, demonstrating a comparison of aggregation of thrombosomes and PRP in the presence of 10 pg/ml collagen.
  • FIG. 15E shows an epinephrine aggregation comparison, demonstrating the comparison of aggregation of thrombosomes and PRP in the presence of 300 pM epinephrine.
  • FIG. 15F shows the comparison of aggregation of thrombosomes and PRP in the presence of 1 mg/ml ristocetin.
  • FIG. 15G shows a Thrombin Receptor Activator Peptide 6 (TRAP-6) aggregation comparison, demonstrating the comparison of aggregation of thrombosomes and PRP in the presence of 10 pM TRAP-6.
  • TRAP-6 Thrombin Receptor Activator Peptide 6
  • FIG. 15H shows an arachidonic acid (AA) aggregation comparison data and graph, showing the comparison of aggregation of thrombosomes and PRP in the presence of 5 mg/ml arachidonic acid.
  • AA arachidonic acid
  • FIG. 16A is a representative graph of experiments where apheresis platelets were used to confirm TRAP activity, showing that TRAP-6 peptide is capable of promoting platelet activation by observing expression of CD62P on the apheresis platelets.
  • FIG. 16B shows that TRAP does not increase AV binding to TFF Thrombosomes, demonstrating that TRAP-6 peptide is not able to increase the expression of CD62P on FDPDs.
  • FIG. 17 shows the measurement of thrombospondin (TSP-1) by flow cytometry in terms of mean fluorescent intensity (MFI) in resting fresh platelets, activated fresh platelets, and different lots of thrombosomes.
  • MFI mean fluorescent intensity
  • FIG. 18 shows the measurement of von Willebrand factor (vWF) by flow cytometry in terms of mean fluorescent intensity (MFI) in resting fresh platelets, activated fresh platelets, and different lots of thrombosomes.
  • MFI mean fluorescent intensity
  • FIG. 19A shows the measurement of vWF by flow cytometry in terms of mean fluorescent intensity (MFI) in fixed lyophilized platelets, and thrombosomes.
  • MFI mean fluorescent intensity
  • FIG. 19B shows the measurement of TSP-1 by flow cytometry in terms of mean fluorescent intensity (MFI) in fixed lyophilized platelets, and thrombosomes
  • FIG. 19C shows the measurement of fibrinogen by flow cytometry in terms of mean fluorescent intensity (MFI) in fixed lyophilized platelets, and thrombosomes.
  • MFI mean fluorescent intensity
  • FIG. 20 shows the forward scatter (FSC) measured by flow cytometry of apheresis platelets, and thrombosomes.
  • FIG. 21 is a comparison of the pre-lyo, CPP, and FDPD platelet count for the six sample group using Beckman Coulter Ac T DifE2 Hematology Analyzer.
  • FIG. 22 shows the flow cytometry measurements of mean fluorescence intensity (MFI) in the FITC channel for all six samples of FDPDs and CPP. Average values are presented with the unloaded product value subtracted out as background. Error bars are the standard deviation of measurements. NovoCyte Quanteon flow cytometer was used to take measurements.
  • MFI mean fluorescence intensity
  • FIG. 23 is a graph of the Thrombin generation results for all six samples of FDPDs and CPP using the CLARIOstar Plus microplate reader.
  • Thrombin generation potency (TGPU) are equivalent to NIH Units of Thrombin per 1 million particles.
  • FIG. 24 shows the flow cytometry measurements of forward scatter for all six samples of FDPDs and CPP. Average values are presented. Error bars are the standard deviation of measurements. NovoCyte Quanteon flow cytometer was used to take measurements.
  • FIGs. 25 A and 25B shows the Total Thrombus System (T-TAS®) results for the six samples of cryopreserved platelet (FIG. 25 A) and six samples of FDPDs (FIG. 25B).
  • T-TAS® Total Thrombus System
  • platelets has its ordinary meaning in the art.
  • cryopreserved platelets are frozen platelets that when thawed are in a liquid state regardless of whether any liquid is added to the frozen platelets after thawing. Accordingly, cryopreserved platelets are not fresh platelets and they are not freeze-dried platelet derivatives. During processing cryopreserved platelets are not dried.
  • the term “cryopreserved platelets” does not imply any minimum length of time such platelets are present in a frozen state. However, cryopreserved platelets are typically stable for at least 1, 2, 3, 4, 5, 6, 9, or 12 months, and in illustrative embodiments are stable for at least 18, 24, 36, or 48 hours. Cryopreserved platelets are typically suspended in a cryoprotectant in a frozen state, until thawing before use.
  • hemostatic properties include the following properties: (a) the ability to generate thrombin in a thrombin generation assay, for example in the presence of tissue factor and phospholipids; (b) the ability to occlude a collagen-coated microchannel in vitro, for example under conditions in which fresh platelets can occlude a collagen-coated microchannel in vitro-, (c) the capability of thrombin-induccd trapping in the presence of thrombin.
  • platelet derivatives such as a freeze-dried platelet derivatives (e.g., thrombosomes) herein
  • platelet derivatives are hemostats, and thus have one, two, or all of the aforementioned hemostatic properties.
  • MRI agent-loaded platelets is inclusive of MRI agent-loaded cryopresereved platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes, unless the context clearly dictates a particular form.
  • platelet derivatives are particles that have some characteristics of fresh platelets but are surrounded by a compromised plasma membrane (i.e. , lack an integrated membrane around them), and as such include pores that are larger than pores found in living platelets. Thus, in illustrative embodiments, platelet derivatives herein exhibit an increased permeability to IgG antibodies. In illustrative embodiments, platelet derivatives, or aggregates thereof found in platelet compositions, are at least 0.5 pm or between 0.5 pm and 25 pm in diameter as determined by dynamic light scattering. Thus, such subsets of platelet-derivative particles are distinguishable from platelet-derivative microparticles, which have a diameter of less than 0.5 pm.
  • platelet derivatives herein have a reduced ability to, or are unable to transduce signals from the external environment into a response inside the particle that are typically transduced in living platelets.
  • platelet derivatives herein e.g., thrombosomes
  • LDH lactate dehydrogenase
  • particle size refers to the diameter of a particle, unless indicated otherwise.
  • the size of the particles is determined after rehydrating the platelet derivative composition with an appropriate solution.
  • the amount of solution for rehydrating a platelet derivative composition is equal to the amount of buffer or preparation agent present at the step of freeze-dry ing.
  • the particle size distribution and microparticle content of a composition can be measured by any appropriate method, for example, by flow cytometry using sizing standards, or in illustrative embodiments by dynamic light scattering (DLS).
  • a content (e g., ratio or percent) of microparticles in illustrative embodiments refers to the microparticle content based on the scattering intensity of all particles from about 1 nm to about 60,000 nm in radius in the composition.
  • the viscosity of a sample used for DLS can be at about 1.060 cP (or adjusted to be so), as this is the approximate viscosity of plasma. It will be understood that the measured size of particles can vary depending on the technology used to perform the measurement. Particle sizes provided herein are typically as determined by DLS unless the context indicates otherwise.
  • the platelet derivative composition as per any aspects, or embodiments comprises a population of platelet derivatives greater than 0.5 pm, and microparticles, wherein the numerical ratio of platelet derivatives to the microparticles is at least 90:1, 91:1, 92:1, 93:1, 94: 1, 95:1, 96:1, 97: 1, 98:1, or 99:1.
  • illustrative or target platelet derivatives have a diameter in the range of 0.5-2.5 pm using flow cytometry, or a diameter of 0.5-25 pm using DLS, and microparticles have a diameter less than 0.5 pm by either method.
  • thrombosomes are platelet derivatives that have been contacted with an incubating agent (e.g., any of the incubating agents described herein) and lyopreserved (e.g., freeze-dried).
  • an incubating agent e.g., any of the incubating agents described herein
  • lyopreserved e.g., freeze-dried
  • thrombosomes are illustrative or target freeze-dried platelet derivatives (FDPDs).
  • Illustrative or target freeze-dried platelet derivative compositions herein typically have at least 1 hemostatic property, and thus can function as hemostatic agents.
  • illustrative or target FDPDs and compositions herein comprising the same can also be referred to as hemostat(s), hemostatic product(s), freeze-dried platelet derived hemostat(s) (FDPDH or FPDH), freeze-dried platelet hemostat(s) (FDPH or FPH), or dry platelet derivative hemostat(s) (PDH).
  • illustrative or target FDPDs such as thrombosomes can be prepared from pooled platelets.
  • Illustrative or target FDPDs such as thrombosomes can have a shelf life of 2-3 years in dry form at ambient temperature and can be rehydrated with sterile water within minutes (e.g.
  • thrombosomes are THROMBOSOMES® freeze-dried platelet derivatives (Cellphire Inc., Rockville, MD), which are in clinical trials for the treatment of acute hemorrhage in thrombocy topenic patients and are a product of Cellphire, Inc.
  • FDPD compositions, or illustrative or target freeze-dried platelet-derivative i.e.
  • FDPD FDPD compositions herein, such as those prepared according to Example 2 herein, are compositions that include a population of platelet derivatives having a reduced propensity to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and in illustrative embodiments, no divalent cations.
  • illustrative or target platelet derivatives typically have the ability to generate thrombin in an in vitro thrombin generation assay and/or have the ability to occlude a collagen-coated microchannel in vitro.
  • illustrative or target platelet derivatives are CD41 positive and/or CD42 positive.
  • Platelets derivatives e.g., thrombosomes
  • dry platelet derivatives or dry platelet derived particles.
  • dry platelet derivatives are typically present in a dried substance that includes other components (e.g.., saccharides such as, for example, trehalose and/or polysucrose) present along with the platelet derivatives when they were dried.
  • less than 5% of the particles are microparticles having a diameter of less than 0.5 pm.
  • at least 90% of the particles therein arc at least 0.5 pm in diameter.
  • between 75% and 95% of the platelet derivatives or particles therein are CD41 positive
  • between 75% and 95% of the platelet derivatives or particles therein are CD42 positive
  • less than 5% of the CD 41-positive platelet derivatives or particles therein are microparticles having a diameter of less than 0.5 pm. It will be understood that in such percent calculations, particles are only intended to cover those that can be detected for example by the instrument (e.g., flow cytometer) used to detect CD41 or CD42 or any surface marker.
  • the platelet derivatives have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • FDPD compositions, or illustrative FDPD compositions herein, such as those prepared according to Example 2 herein are compositions that include illustrative or target platelet derivatives, wherein at least 50% of the platelet derivatives are CD 41-positive platelet derivatives, wherein less than 15%, 10%, or in further, non-limiting illustrative embodiments less than 5% of the CD 41-positive platelet derivatives are microparticles having a diameter of less than 0.5 pm, and typically such compositions have the ability to generate thrombin in an in vitro thrombin generation assay and/or have the ability to occlude a collagen-coated microchannel in vitro.
  • the platelet derivatives in such compositions have a potency of at least 0.5, 1.0 and in further, non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • the illustrative or target platelet derivatives are between 0.5 and 2.5 pm in diameter by flow cytometry or between 0.5 and 25.0 pm in diameter by dynamic light scattering.
  • fresh platelet can include day of use platelets.
  • stored platelet can include platelets stored for approximately 24 horns or longer before use.
  • a disclosed range of 1-10 includes 1-9, 1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, and so forth.
  • each disclosed range includes up to 5% lower for the lower value of the range and up to 5% higher for the higher value of the range.
  • a disclosed range of 4 - 10 includes 3.8 - 10.5. This concept is captured in this document by the term "about”.
  • Imaging agent-loaded, and in illustrative embodiments magnetic resonance imaging (MRI) agent-loaded platelet derivatives and cryoprcscrvcd platelets provided herein allow targeted delivery of the imaging agent (e.g. MRI agent) to sites of interest.
  • imaging agent-loaded, in illustrative embodiments MRI agent-loaded platelets or platelet derivatives can be used to image blood vessels and inflamed or diseased tissue where blood vessels have become compromised (e.g., “leaky”) or otherwise damaged.
  • MRI agent-loaded platelets such as cryopreserved platelets, or platelet derivatives, such as freeze-dried platelet derivatives (FDPDs), which sometimes can be called freeze-dried platelets herein, can be used to enhance diagnosis of a condition in a subject, especially a condition related to inflamed, diseased, or compromised blood vessels, thus facilitating detection and diagnosis, including in some cases, early or earlier detection and/or diagnosis, thus aiding in higher chances of successful treatment.
  • FDPDs freeze-dried platelet derivatives
  • imaging agent- loaded, in illustrative embodiments MRI agent-loaded platelet derivatives or cryopreserved platelets can be used to treat a subject having a condition/indication/disease as disclosed, wherein the imaging agent (e.g. MRI agent) are useful to detect, analyze in vivo, confirm delivery of, and/or localize the platelet derivatives or cryopreserved platelets.
  • the imaging agent e.g. MRI agent
  • composition comprising imaging agent- loaded, in illustrative embodiments MRI agent-loaded platelets, such as cryopreserved platelets, and platelet derivatives, such freeze-dried platelet derivatives, wherein the imaging agent-loaded, in illustrative embodiments MRI agent-loaded platelets or platelet derivatives are coupled to a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • the present disclosure provides a composition
  • MRI-agent loaded platelets such as cryopreserved platelets
  • platelet derivatives such as freeze-dried platelet derivatives
  • the imaging agent, in illustrative embodiments MRI agent are part of a complex that is covalently bonded to the surface of the platelets, such as the cryopreserved platelets, or the platelet derivatives, such as the freeze-dried platelet derivatives
  • the imaging agent complex or MRI agent complex comprises the imaging agent and/or MRI agent, and a chelator, and a linker in certain embodiments when the MRI agent complex is not covalently attached to a platelet derivative or cryopreserved platelet.
  • the loading of an imaging agent, in illustrative embodiments MRI agent in the platelets can mitigate systemic side effects associated with the imaging agent (e.g. MRI agent) and can shield the imaging agent (e.g. MRI agent) from natural clearance mechanisms during migration to the site of interest, such as a site of injury.
  • the accumulation of imaging agent-loaded platelets or platelet derivatives (e.g. MRI agent-loaded platelets or platelet derivatives) at the site of injury can enhance the resolution of images (e.g. magnetic resonance images) and allow for earlier detection and/or improved disease diagnoses.
  • imaging agent-loaded which in non-limiting illustrative embodiments are MRI agent-loaded platelets, such as cryopreserved platelets, and/or platelet derivatives, such as FDPDs, to image, and/or aid in imaging a site(s) of interest in a subject.
  • a method of detecting, diagnosing, enhancing detection and/or diagnosis of a disease includes administering an effective amount or a therapeutically elfective amount of a composition comprising imaging agent-loaded, in illustrative embodiments, MRI agent-loaded platelets, such as cryopreserved platelets, or platelet derivatives, such as FDPDs of any of the aspects or embodiments disclosed herein, or a composition prepared by a process of any of the aspects or embodiments disclosed herein, and imaging and/or detecting the imaging agent (e.g. MRI agent), thereby detecting or diagnosing, or enhancing detection or and/or diagnosis of, the disease.
  • imaging agent e.g. MRI agent
  • a method for detecting a site(s) of bleeding in a subject comprising: (a) administering an effective amount or a therapeutically effective amount of a composition comprising imaging agent-loaded (e.g. MRI agent-loaded) platelets, such as cryopreserved platelets, or platelet derivatives such as FDPDs, according to any of the aspects or embodiments herein, or prepared by any process disclosed herein, to the subject; and (b) detecting/locating the site of the imaging agent- loaded (e.g. MRI agent-loaded) platelet or platelet derivatives, thereby detecting the site of bleeding in the subject.
  • imaging agent-loaded e.g. MRI agent-loaded
  • imaging agent-loaded platelets comprising: (a) providing platelets; and (b) treating the platelets with an imaging agent (e.g. an MRI agent), to form the imaging agent-loaded (e.g. the MRI agent-loaded) platelets.
  • an imaging agent e.g. an MRI agent
  • a process for preparing MRI agent/imaging agent-loaded platelet derivatives comprising: a) providing platelets; (b) treating the platelets with an MRI agent/imaging agent, to form MRI agent/imaging agent-loaded platelets; and (c) lyophilizing the MRI agent/imaging agent-loaded platelets, to form MRI agent/imaging agent -loaded platelet derivatives.
  • a process for preparing MRI agent/imaging agent-loaded cryopreserved platelets comprising: a) providing platelets; (b) treating the platelets with an MRI agent/imaging agent, to form MRI agent/imaging agent-loaded platelets; and (c) cryopreserving the MRI agent/imaging agent-loaded platelets, to form MRI agent/imaging agent-loaded cryopreserved platelets.
  • a process as provided herein can further comprise a step of rehydrating the MRI agent-loaded platelet derivatives.
  • rehydrating the MRI agent-loaded platelet derivatives includes adding to dried platelet derivatives, an aqueous liquid.
  • the aqueous liquid is water.
  • the aqueous liquid is an aqueous solution.
  • the aqueous liquid is a saline solution.
  • the aqueous liquid is a suspension.
  • the rehydrated platelets have coagulation factor levels showing all individual factors (e.g., Factors VII, VIII and IX) associated with blood clotting at 40 international units (IU) or greater.
  • factors e.g., Factors VII, VIII and IX
  • the dried platelets such as freeze-dried platelets, have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes.
  • the rehydrated platelets have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% crosslinking of platelet membranes via proteins and/or lipids present on the membranes.
  • the MRI agent-loaded platelets and the dried platelets having a particle size (e.g., diameter, max dimension) of at least about 0.2 pm (e.g., at least about 0.3 pm, at least about 0.4 pm, at least about 0.5 pm, at least about 0.6 pm, at least about 0.7 pm, at least about 0.8 pm, at least about 0.9 pm, at least about 1.0 pm, at least about 1.0 pm, at least about 1.5 pm, at least about 2.0 pm, at least about 2.5 pm, or at least about 5.0 pm).
  • a particle size e.g., diameter, max dimension
  • the particle size is less than about 5.0 pm (e.g., less than about 2.5 pm, less than about 2.0 pm, less than about 1.5 pm, less than about 1.0 pm, less than about 0.9 pm, less than about 0.8 pm, less than about 0.7 pm, less than about 0.6 pm, less than about 0.5 pm, less than about 0.4 pm, or less than about 0.3 pm). In some embodiments, the particle size is from about 0.3 pm to about 5.0 pm (e.g., from about 0.4 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • 5.0 pm e.g., from about 0.4 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm.
  • At most 99% e g., at most about 95%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, or at most about 50%
  • the dried platelets such as freeze-dried platelets
  • about 0.3 pm to about 5.0 pm e.g., from about 0.4 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm.
  • about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85, about 70% to about 80%) of platelets and/or the dried platelets, such as freeze-dried platelets, are in the range of about 0.3 pm to about 5.0 pm (e.g., from about 0.4 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • a composition comprising MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a dried powder, wherein the MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets or the MRI agent- loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the platelets, cryopreserved platelets or the platelet derivatives, wherein the MRI agent complex comprises an MRI agent, and a chelator.
  • the MRI agent complex is covalently bonded to proteins on the surface of the platelets.
  • the MRI agent complex comprising an MRI agent, a chelator, and a linker, ty pically, interact with a protein molecule on the surface of platelets, cryopreserved platelets, or platelet derivatives via the linker moiety, and typically, during the interaction the linker moiety, such as NHS gets released and the chelator moiety, such as DOTA gets covalently bonded to the protein via a stable amide bond.
  • the chelator is understood to chelate the MRI agent such that the toxicity of the MRI agent is reduced.
  • an MRI agent complex is covalently bonded to the external surface of platelets, cryopreserved platelets, or platelet derivatives.
  • an MRI agent complex is covalently bonded to the plasma membrane of platelets, cryopreserved platelets, or platelet derivatives. In some embodiments, an MRI agent complex is covalently bonded to any structure, membrane, solid component, or other portion of platelets, cryopreserved platelets, or platelet derivatives.
  • an MRI agent complex can comprise an MRI agent, and a moiety to mask the toxic effect of the MRI agent.
  • an MRI agent complex can comprise an MRI agent and a moiety that can effectuate bonding of the MRI agent to platelets, cryopreserved platelets, or platelet derivatives.
  • an MRI agent complex comprises an MRI agent, a chelator, and a linker.
  • an MRI agent complex can comprise an MRI agent and a linker.
  • an MRI agent complex can comprise an MRI agent and a chelator.
  • a chelator can be any chelator known in the art to chelate an MRI agent.
  • a chelator can be selected from the group consisting of dodecane tetra acetic acid (DOTA), diethylenetriaminepentaacetic acid (DTPA), 4-Carbox -5,8,11- tris(carboxymethyl)-l-phenyl-2-oxa- 5,8,1 l-triazatridecan-13-oic acid (BOPTA), Ethylenediaminetetraacetic acid (ED'T'A), and l,4,7,10-tetraazacyclododecane-l,4,7-tetracetic acid (DO3A).
  • DOTA dodecane tetra acetic acid
  • DTPA diethylenetriaminepentaacetic acid
  • BOPTA 4-Carbox -5,8,11- tris(carboxymethyl)-l-phenyl-2-oxa- 5,8,1 l-triazatridecan-13-
  • a means for chelating ions such as cations, for example divalent cations, is any of the chelators listed in the preceding sentence.
  • a chelator is associated with an MRI agent for example through a non-covalent ionic interaction, and/or through a bond other than a covalent bond, such as an ionic bond.
  • Chelators can be associated with an MRI agent, and in illustrative embodiments, the chelator is covalently attached to a platelet, cryopreserved platelet, or platelet derivative, or in the case where the MRI agent is loaded onto and/or into platelets using a CPP rather than by an MRI agent-complex.
  • any type of chelator that reduces the toxicity of an MRI agent can be used in an MRI agent-complex as well as for CPP loading of MRI agent onto and/or into platelets.
  • a linker can be any linker known in the art that can elfectuate covalent bonding for example of a chelator, with a protein, for example a protein on the surface of a platelet, cryopreserved platelet or platelet derivative.
  • an MRI agent complex herein comprises a linker covalently attached to a chelator, which is associated with an MRI agent, for example through an ionic interaction.
  • an MRI agent complex is covalently attached to a platelet, a cryopreserved platelet, or a platelet derivative via a chelator, which is associated with an MRI agent, for example through an ionic interaction, such as an ionic bond.
  • a linker can be selected from the group consisting of a compound having sulfhydryl reactive groups, such as maleimides and haloacetyl derivatives, amine reactive groups, such as isothiocyanates, succinimidyl esters, and sulfonyl halides, and carbodiimide reactive groups, such as carboxyl and amino groups.
  • a linker is a compound having an amine reactive group, such as, succinimidyl ester, such as, N-Hydroxysuccinimide (NHS) ester.
  • an MRI agent complex comprises MRI agent such as, gadolinium, a chelator such as, DOTA, and a linker such as, NHS.
  • MRI agent such as, gadolinium
  • a chelator such as, DOTA
  • NHS linker
  • gadolinium is chelated by DOTA and DOTA is conjugated with, for example covalently bonded to NHS.
  • the NHS linker selectively reacts with primary' aliphatic amine groups on proteins.
  • NHS linker is released in the reaction, and the chelator is covalently bonded to proteins on the surface of platelets via a stable amide linkage.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent complex comprising an MRI agent, a chelator, and a linker, to form MRI agent-loaded platelets; and (c) cry opreserving or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives.
  • the MRI agent is associated with the chelator, and the chelator is covalently linked to the surface of the platelets.
  • the MRI agent is associated with a surface of the cryopreserved platelets or the platelet derivatives.
  • the MRI agent is associated with the external surface of the cryopreserved platelets or the platelet derivatives.
  • the MRI agent is associated with the surface of the cryopreserved platelets or the surface of the platelet derivatives via the chelator.
  • the chelator is covalently attached to the surface of the cryopreserved platelets or the surface of the platelet derivatives.
  • the linker is covalently bonded to the chelator in the MRI agent complex.
  • the MRI agent is associated with the chelator through an ionic interaction.
  • contacting the platelets with MRI agent complex such that the MRI agent complex is covalently bound to the platelets, to form MRI agent-loaded platelets is done in the presence of a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.
  • a method can comprise of steps that make use of lyophilized or cryopreserved platelets as a starting material for loading.
  • MRI agents are contrast agents are used to improve the visibility of internal body structures in magnetic resonance imaging (MRI).
  • MRI agents as per the present invention can be any MRI agent that are known in the art.
  • an MRI agent is selected from the group consisting of a superparamagnetic contrast agent, a diamagnetic agent, or combinations thereof.
  • an MRI agent can be a superparamagnetic contrast agent selected from the group consisting of Gd(III), Fe(III), Mn(II and III), Cr(III), Cu(II), Dy(III), Tb(III and IV), Ho(III), Er(III), Pr(IIT) and Eu(II and III).
  • an MRI agent can be selected from the group consisting of metal ions with atomic numbers 21-29, 39-47, or 57-83.
  • an MRI agent is a gadolinium-based compound.
  • an MRI agent can include gadolinium.
  • an MRI agent is Gd(III) that can be coupled with a chelator such as, DOTA.
  • MRI agents arc widely used to increase the contrast difference between normal and abnormal tissues.
  • MRI agents may be categorized according to magnetic properties, chemical composition, the presence or absence of metal atoms, route of administration, effect on the magnetic resonance image, and biodistribution.
  • Certain aspects provided herein are or include MRI agent-loaded platelets.
  • the MRI agent-loaded platelets can be MRI agent-loaded ciyopreserved platelets.
  • the MRI agent-loaded platelets can be MRI agent-loaded platelet derivatives.
  • Certain aspects provided herein are or include MRI agent-loaded freeze-dried platelet derivatives (FDPDs).
  • FDPDs MRI agent-loaded freeze-dried platelet derivatives
  • TFF processing methods as disclosed herein can be used to process the platelets prior to loading or after loading with imaging agent(s) such as MRI agent(s).
  • the lyophilization or cryopreservation methods as disclosed herein can be used to process the platelets prior to loading or after loading with imaging agent(s) such as MRI agent(s).
  • imaging agent-loaded (MRI agent-loaded) FDPDs can retain one, two, three or all of the properties of FDPDs as disclosed herein.
  • imaging agent-loaded (e.g MRI agent-loaded) platelet derivatives e.g. MRI agent-loaded) FDPDs, or imaging-agent loaded (e.g. MRI agent- loaded) cryopreserved platelets are capable of retaining the properties of FDPDs that were present before loading with imaging agent (e.g. MRI agent) as disclosed elsewhere herein.
  • imaging agent e.g. MRI agent
  • MRI agent-loaded platelet derivatives or MRI agent-loaded FDPDs in some embodiments, have a higher propensity to co-aggregate in the presence of fresh platelets and an agonist, while having a reduced propensity to aggregate in the absence of fresh platelets and an agonist, compared to the propensity of fresh platelets to aggregate under these conditions.
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets at a concentration of about 4.8xl0 3 particle s/pL) as described herein can generate a thrombin peak height (TPH) of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) when in the presence of a reagent containing tissue factor (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM or 10 pM) and optionally phospholipids.
  • TPH thrombin peak height
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets can have a potency of at least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5) thrombin generation potency units (TGPU) per 10 6 particles.
  • TGPU thrombin generation potency units
  • platelets or platelet derivatives e.g., FDPDs
  • platelets or platelet derivatives can have a potency of between 1.2 and 2.5 TPGU per 10 6 particles (e.g., between 1.2 and 2.0.
  • MRI agent- loaded FDPDs can have a potency of at least 1.5, 2, 2,5, 3, or 4 TGPU per 10 6 particles.
  • MRI agent-loaded FDPDs can have a potency in the range of 1.5 to 10, 2 to 10, 2 to 9, 2 to 7, 2 to 6, 1 to 6, or 3 to 6 TGPU per 10 6 particles.
  • MRI agent-loaded cryopreserved platelets can have a potency that is higher than MRI agent-loaded FDPDs.
  • MRI agent-loaded cryopreserved platelets can have a potency of at least 5, 6, 7, 8, 9, or 10 TGPU per 10 6 particles. In some embodiments, MRI agent-loaded cryopreserved platelets can have a potency in the range of 5 to 25, 5 to 22, 5 to 20, 6 to 25, 7 to 25, or 7 to 16 TGPU per 10 6 particles.
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets when at a concentration of at least 70xl0 3 particles/pL can result in a T-TAS occlusion time (e.g., time to reach kPa of 80) of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes), for example, in platclct-rcduccd citrated whole blood.
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein can have a percent thrombin-induced trapping of at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) in the presence of thrombin.
  • platelets or platelet derivatives as described herein can have a percent thrombin-induced trapping of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%) in the presence of thrombin.
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein can have the presence of thrombospondin (TSP-1) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • TSP-1 thrombospondin
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein can have the presence of von Willebrand factor (vWF) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • vWF von Willebrand factor
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein are not able to show an increase in the platelet activation markers on them as compared to the level of the platelet activation markers which were present prior to the exposure with the agonist.
  • the platelet derivatives as described herein show an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of an agonist.
  • MRI agent-loaded platelet derivatives, or MRI agent-loaded FDPDs as described herein are surrounded by a compromised plasma membrane.
  • the platelet derivatives lack an integrated membrane around them.
  • the membrane surrounding such platelet derivatives e.g. FDPDs
  • the membrane surrounding such platelet derivatives comprises pores that are larger than pores observed on living cells.
  • MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein can have a particle size (e.g., diameter, max dimension) of at least about 0.5 pm (e.g., at least about at least about 0.6 pm, at least about 0.7 pm, at least about 0.8 pm, at least about 0.9 pm, at least about 1.0 pm, at least about 1.2 pm, at least about 1.5 pm, at least about 2.0 pm, at least about 2.5 pm, or at least about 5.0 pm).
  • 0.5 pm e.g., at least about at least about 0.6 pm, at least about 0.7 pm, at least about 0.8 pm, at least about 0.9 pm, at least about 1.0 pm, at least about 1.2 pm, at least about 1.5 pm, at least about 2.0 pm, at least about 2.5 pm, or at least about 5.0 pm.
  • the particle size is less than about 5.0 pm (e.g., less than about 2.5 pm, less than about 2.0 pm, less than about 1.5 pm, less than about 1.0 pm, less than about 0.9 pm, less than about 0.8 pm, less than about 0.7 pm, less than about 0.6 pm, less than about 0.5 pm, less than about 0.4 pm, or less than about 0.3 pm). In some embodiments, the particle size is from about 0.5 pm to about 5.0 pm (e.g., from about 0.5 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • a composition comprising MRI agent-loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets as described herein can have a microparticle content that contributes to less than about 5.0% (e.g., less than about 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, or 0.5%) of the total scattering intensity of all particles from about 1 nm to about 60,000 nm in radius in the composition.
  • MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes may shield the MRI agent from exposure in circulation, thereby reducing or eliminating systemic toxicity (e g. cardiotoxicity) associated with the MRI agent.
  • MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent- loaded thrombosomes may also protect the MRI agent from metabolic degradation or inactivation.
  • MRI agent delivery with MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes may therefore be advantageous in treatment of diseases such as cancer, since MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes facilitate targeting of cancer cells while mitigating systemic side effects.
  • MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes may be used in any therapeutic setting in which expedited healing process is required or advantageous.
  • Flow cytometry can be used to obtain a relative quantification of loading efficiency by measuring the mean fluorescence intensity of the MRI agent in the MRI agent-loaded platelets. Platelets can be evaluated for functionality by adenosine diphosphate (ADP), collagen, arachidonic acid, thrombin receptor activating peptide (TRAP), and/or any other platelet agonist known in the art for stimulation post-loading.
  • ADP adenos
  • the MRI agent-loaded platelets are lyophilized. In some embodiments, the MRI agent-loaded platelets are cryopreserved.
  • MRI agent-loaded platelets such as MRI agent-loaded platelet derivatives retain the loaded MRI agent upon rehydration and release the MRI agent or the MRI agent complex upon stimulation by endogenous platelet activators.
  • MRI agent-loaded cryopreserved platelets as disclosed herein retain the loaded MRI agent upon thawing and release the MRI agent or the MRI agent complex upon stimulation by endogenous platelet activators.
  • the dried platelets (such as MRI agent-loaded freeze-dried platelets) retain the loaded MRI agent upon rehydration and release the MRI agent upon stimulation by endogenous platelet activators. In some embodiments, at least about 10%, such as at least about 20%, such as at least about 30% of the MRI agent is retained. In some embodiments, from about 10% to about 30%, or from about 20% to about 30% of the MRI agent is retained. In some embodiments, more than 30% of the MRI agent is retained.
  • MRI agent-loaded cryopreserved platelets upon thawing form thawed MRI agent-loaded platelets, such that the thawed MRI agent-loaded platelets retain at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more of the loaded MRI agent upon thawing.
  • the thawed MRI agent-loaded platelets retain 5%-90%, 5%-80%, 5%-70%, 5-60%, 10-80%, 10-75%, 10-60%, 15-90%, 20-90%, or 25-90% of the loaded MRI agent upon thawing.
  • the MRI agent-loaded cryopreserved platelets retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more of the MRI agent loaded on platelets before the step of cryopreserving.
  • the MRI agent-loaded cryoprcscrvcd platelets retain 5%-90%, 5%-80%, 5%-70%, 5-60%, 10-80%, 10-75%, 10-60%, 15-90%, 20-90%, or 25-90% of the MRI agent loaded on platelets before the step of cryopreservmg.
  • MRI agent-loaded platelet derivatives upon rehydrating form rehydrated MRI agent-loaded platelet derivatives, such that the rehydrated MRI agent-loaded platelet derivatives retain at least 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50% or more of the loaded MRI agent upon rehydrating.
  • the rehydrated MRI agent-loaded platelet derivatives retain 5%-90%, 5%-80%, 5%-70%, 5-60%, 10-80%, 10-75%, 10-60%, 15-90%, 20-90%, or 25-90% of the loaded MRI agent upon rehydrating.
  • the MRI agent-loaded platelet derivatives retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more of the MRI agent loaded on platelets before the step of lyophilizing. In some embodiments, the MRI agent-loaded platelet derivatives retain 5%-90%, 5%- 80%, 5%-70%, 5-60%, 10-80%, 10-75%, 10-60%, 15-90%, 20-90%, or 25-90% of the MRI agent loaded on platelets before the step of lyophilizing.
  • MRI agent-loaded cryopreserved platelets can have higher retention of MRI agent as compared to MRI agent-loaded platelet derivatives such as FDPDs.
  • Any suitable MRI agent can be loaded in a platelet.
  • any agent suitable for magnetic resonance imaging can be loaded into a platelet.
  • the MRI agent can include Gadolinium.
  • Gadolinium (Gd (III)) can be coupled with a chelator.
  • the chelator can be DOTA (tetrxetan).
  • the MRI agent can be a complex of Gd and DOTA (e.g., Gd-DOTA).
  • the MRI agent can be a nanoparticle. Any suitable nanoparticle (e.g., magnetic nanoparticle) can be loaded into the platelet. In some embodiments, the nanoparticle is a FeOa nanoparticle.
  • the nanoparticle can be about 15 nm to about 100 nm in diameter. In some embodiments, the nanoparticle can be about 20 nm to about 90 nm in diameter. In some embodiments, the nanoparticle can be about 30 nm to about 80 nm in diameter. In some embodiments, the nanoparticle can be about 40 nm to about 70 nm in diameter. In some embodiments, the nanoparticle can be about 50 nm to about 60 nm in diameter. In some embodiments, the nanoparticle can be about 20 nm to about 30 nm in diameter.
  • the nanoparticles can be at a concentration of about 1 x IO' 20 to about 1 x 10' 14 particles/mL. In some embodiments, the nanoparticles can be at a concentration of about 1 x 10' 19 to about 1 x 10' 15 particles/mL. In some embodiments, the nanoparticles can be at a concentration of about 1 x 10' 18 to about 1 x 10' 16 particles/mL.
  • the MRI agent loaded platelets can be coupled (e.g., conjugated) with a cell penetrating peptide.
  • Cell penetrating peptides are peptides that can facilitate cellular uptake of various cargo (e.g., nucleic acid, protein, metabolites, lipids, nanoparticles, metals, etc.).
  • cargo e.g., nucleic acid, protein, metabolites, lipids, nanoparticles, metals, etc.
  • cell penetrating peptides can cross a cellular membrane by direct penetration in the membrane, endocytosis mediated entry , or translocation through the formation of a transitory structure, although additional mechanisms are known.
  • the HIV Tat protein is an example of a cell penetrating peptide.
  • the Tat protein includes betw een 86 and 101 amino acids depending on the subtype. Tat is a regulatory protein that enhances the viral transcription efficiency. Tat also contains a protein transduction domain which functions as a cell-penetrating domain allowing Tat to cross cellular membranes.
  • an MRI agent can be coupled with any functional fragment of an HIV Tat protein.
  • the full-length of HIV-TAT protein is as set forth in SEQ ID NO: 1.
  • an MRI agent can be coupled with a portion/functional fragment of an HIV Tat protein.
  • an MRI agent can be coupled with a portion of the Tat protein: L- Tat49-57 as described in Mishra, R., et. al., Cell-Penetrating Peptides and Peptide Nucleic Acid-Coupled MRI Contrast Agents: Evaluation of Cellular Delivery and Target Binding, Bioconjugate Chem. 20, 1860-1868 (2009).
  • the MRI agent can be coupled with a lipophilic moiety.
  • a lipophilic moiety include a lipid coated nanoparticle or a liposome.
  • the MRI agent can be coupled with a cyclodextrin cage.
  • the one or more other components that are loaded in the platelets include Prostaglandin El.
  • the one or more other components that are loaded in the platelets do not include Prostaglandin El.
  • the one or more other components that are loaded in the platelets include a glycoprotein Ilb/IIIa inhibitor (GP Ilb/IIIa).
  • GP Ilb/IIIa inhibitors include GR144053, eptifibatide, ethylenediaminetetraacetic acid (EDTA), abciximab, tirofiban.
  • EDTA ethylenediaminetetraacetic acid
  • the one or more other components that are loaded in the platelets include GR144053.
  • the one or more other components that are loaded in the platelets do not include GR144053.
  • the one or more other components that are loaded in the platelets include eptifibatide.
  • the one or more other components that are loaded in the platelets do not include eptifibatide.
  • MRI agent-loaded platelets MRI agent-loaded platelets
  • MRI agent-loaded platelet derivatives MRI agent-loaded FDPDs
  • MRI agent-loaded cryopreserved platelets respectively.
  • a composition comprising MRI agent-loaded platelets, MRI agent- loaded platelet derivatives, MRI agent-loaded FDPDs, or MRI agent-loaded cryopreserved platelets comprising MRI agent coupled to a cell penetrating peptide (CPP).
  • CPP cell penetrating peptide
  • MRI agent coupled to a CPP can further comprise a chelator.
  • a chelator can be any chelator that is described elsewhere in the specification.
  • the chelator is DOTA
  • the MRI agent is gadolinium
  • the CPP is a TAT peptide that is elfective at penetrating a cell.
  • one non-limiting exemplary method of preparing FDPDs loaded with an MRI agent is as follows: Prepare the MRI agent (e.g., FITC-CPP- Ga-DOTA or FITC-labeled nanoparticles) in aqueous bulfer at room temperature. Incubate the FITC- CPP-Ga-DOTA or FITC-labeled nanoparticles with platelets up to 3 hours at 37°C on a rocker with low frequency agitation. Transfected platelets may be lyophilized to create FDPDs with an MRI agent. Fluorescently labeled FITC-CPP-Ga-DOTA or FITC-labeled nanoparticles can be detected via flow cytometry and visualized using fluorescence microscopy.
  • the MRI agent e.g., FITC-CPP- Ga-DOTA or FITC-labeled nanoparticles
  • Cell penetrating peptides are peptides that can facilitate cellular uptake of various cargo (e.g., nucleic acid, protein, metabolites, lipids, nanoparticles, metals, etc.). They are included in some of the aspects and embodiments herein typically to facilitate uptake of imaging agents, which in illustrative embodiments are MRI agents.
  • Cargo can be coupled (e.g., conjugated) to a cell penetrating peptide either covalently or non-covalently.
  • a cell penetrating peptide conjugated to cargo can transport the cargo across a cellular membrane, generally via endocytosis, however other mechanisms are known in the art.
  • CPPs are typically between 5 and 30, and in some illustrative embodiments between 10 and 30 amino acids in length, or arc oligomers thereof that can include for example, 2 tandem copies or between 2 and 10, 9, 8, 7, 6, 5, 4, or 3 tandem repeats of the CPP, that can optionally be separated by a 1-3 amino acid peptide linker, that are capable of crossing the cytoplasmic membrane efficiently.
  • a CPP included in any aspect or embodiment herein can be any peptide having 5 to 150, 5 and 100, 5 to 75, 50 to 50, or 5 to 30 amino acids that are capable of crossing the cytoplasmic membrane.
  • a CPP is any of the peptides that are disclosed in Kersemans et al 2008 (Kersemans, Veerle et al. “Cell penetrating peptides for in vivo molecular imaging applications.” Current pharmaceutical design vol. 14,24 (2008): 2415-47) incorporated in its entirety herein by reference.
  • CPP can be a protein-derived CPP, a synthetic CPP, or a mixed/chimeric CPP.
  • a protein-derived CPP is derived from a naturally occurring protein such as, but not limiting to, TAT protein, and penetratin.
  • a synthetic CPP such as, but not limiting to polyarginines can be a CPP that is developed by known techniques, such as, phage display method.
  • a mixed or a chimeric CPP can be a CPP which is a combination of naturally occurring (protein derived) CPP, and synthetic CPP, such as transportan CPP, or can be a combination of the N-terminal fragment of the neuropeptide gelanin and the membrane-interacting wasp venom peptide, mastoparan.
  • a CPP can be considered as any peptide that is capable of penetrating a cell membrane without the involvement of energy-dependent processes.
  • a CPP in illustrative embodiments is a peptide that is capable of, has the property of, and/or is adapted to penetrate a cell membrane without the involvement of energy-dependent processes.
  • a CPP can have at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids and in illustrative embodiments less than or equal to 75 amino acids.
  • a CPP can have 3 to 75, 4 to 65, 5 to 50, 5 to 40, 5 to 30, 10 to 75, 10 to 65, 10 to 50, 10 to 40, or 10 to 30 amino acids.
  • a CPP can be a positively charged peptide (cationic peptide).
  • a cationic CPP can comprise multiple lysine and/or arginine residues.
  • a cationic CPP can comprise at least 4, 5, 6, 7, 8, 9, or 10 lysine and/or arginine residues.
  • a cationic CPP comprises between 4 and 15, 14, 13, 12, 11, or 10 lysine and/or arginine residues.
  • a cationic CPP can comprise a nuclear localization sequence (NLS).
  • a CPP can be an amphipathic CPP that can comprise alternating regions of hydrophilic and hydrophobic amino acids, and the amphipathic CPP can have a resulting charge that can be positive, negative, or neutral.
  • an amphipathic CPP can be proline-rich amphipathic peptide.
  • a CPP can be a hydrophobic CPP having hydrophobic properties, one or more hydrophobic domains, and comprising hydrophobic residues.
  • proteins such as, as a stretch of between 2, 3, 4, 5, or 6 but not limiting to, alanine, leucine, isoleucine, phenylalanine, tryptophan, methionine, and tyrosine.
  • a CPP can be any of the CPPs or a functional fragment of any of the CPPs that has been disclosed in the publication - Bbhmova, E et al. “Cellpenetrating peptides: a useful tool for the delivery of various cargoes into cells.” Physiological research vol. 67, Suppl 2 (2016): S267-S279 (Bbhmova et al 2018), incorporated in its entirety by reference herein, and the publication Bbhmova et al 2018 incorporated in its entirety herein by reference.
  • a CPP can be any one of the CPPs or a functional fragment of any of the CPPs that has been disclosed in the publication - Ramaker, E et al. “Cell penetrating peptides: a comparative transport analysis for 474 sequence motifs” Drug Delivery, 25:1, 928-937 (2016) (Ramaker et al 2018), incorporated in its entirety by reference herein.
  • a protein-derived CPP is selected from the group consisting of Pep- 1 , penetratin, TAT peptide (e. g. amino acid residues 49-57 of SEQ ID NO: 1) (amino acid numbering is with respect to the full-length HIV-TAT peptide as set forth in SEQ ID NO: 1), TAT peptide (e.g.
  • a protein-derived CPP is penetratin. In certain illustrative embodiments, a CPP can be any functional fragment of penetratin peptide. In certain illustrative embodiments, a protein-derived CPP is TAT peptide (e.g. comprising, consisting essentially of, or consisting of amino acid residues 49-57 of SEQ ID NO:1). In certain illustrative embodiments, a protein-derived CPP is TAT peptide (e.g. comprising, consisting essentially of, or consisting of amino acid residues 48-60 of SEQ ID NO: 1). In certain illustrative embodiments, a CPP can be any functional fragment of TAT peptide.
  • a protein-derived CPP can be any peptide disclosed in the publication Kersemans et al 2008.
  • a synthetic and/or mixed and/or chimeric CPP is selected from the group consisting of transportan, polyarginine CPPs, poly-d-arginine, KLAL peptide/model amphipathic peptide (MAP), KALA model amphipathic peptide, modeled Tat peptide, Loligomer, b- sheet-forming peptide, retro-inverso forms of established CPPs, W/R penetratin, MPG, Pep-1, Signal- scqucncc-bascd peptides (I), Signal-scqucncc-bascd peptides (II), Carbamate 9, PTD-4, PTD-5, RSV- A9, CTP-512, and U2AF.
  • a synthetic and/or mixed CPP is selected from the group consisting of transportan, polyarginine CPPs, poly-d-arg
  • a CPP can comprise a functional fragment of any of the CPPs disclosed herein including those in any of the cited references incorporated herein.
  • a functional fragment can be any fragment of a CPP that retains the ability to penetrate a cell membrane and deliver a cargo molecule inside the cell, which in illustrative embodiments can include an imaging agent such as an MRI agent.
  • a CPP can be, comprise, consistent essentially of, or consist of any one or more of the peptides as set forth in amino acid residues 49-57 of SEQ ID NO: 1, amino acid resides 48-60 of SEQ ID NO:1, or tire peptide whose sequence is provided in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO:
  • a CPP can comprise, consist essentially of, or consist of any peptide that is at least 75%, 80%, 85%, 90%, 92%, 95%, 97.5%, 99%, or 99.5% identical to any of the foregoing sequences or a functional fragment thereof.
  • a CPP can be any peptide that is at least 75%, 80%, 85%, 90%, 92%, 95%, 97.5%, 99%, or 99.5% identical to any peptide or its functional fragments as set forth in SEQ ID NO: 1 to SEQ ID NO: 87.
  • a CPP can be any one of the peptides as set forth in SEQ ID NO: 2 to 7, or functional fragments thereof. In some illustrative embodiments, a CPP can be any one of the peptides as set forth in SEQ ID NO: 8 to 11, or functional fragments thereof.
  • a CPP in certain illustrative embodiments herein, is a functional fragment of HIV-TAT protein (SEQ ID NO: 1). In some embodiments, a CPP as per the present disclosure can be derived from the peptide as set forth in SEQ ID NO: 1. In some embodiments, a CPP as per the present disclosure can be a functional fragment of the peptide as set forth in SEQ ID NO: 1.
  • a CPP as per the present disclosure can be a functional fragment having at least 3, 4, 5, 6, 7, 8, 9, 10 amino acids of the peptide as set forth in SEQ ID NO: 1.
  • the functional fragment can be derived from SEQ ID NO: 1 and identified using mutational techniques.
  • a CPP can be any peptide that is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 95%, 97.5%, 99%, or 99.5% identical to a consecutive stretch of at least 10 amino acids or the entire peptide as set forth in SEQ ID NO: 1.
  • a CPP can form a complex/linked with an MRI agent/imaging agent that needs to be delivered to a cell.
  • a CPP linked with an imaging agent such as an MRI agent
  • a CPP-MRI/Imaging agent complex can have a size of at most 500 nm, 450 nm, 400 run, 350 run, 300 nm, 250 run, or 200 run.
  • a CPP can be any means provided herein, typically any peptide means provided herein, for penetrating a cell membrane for delivering a cargo molecule inside the cell, for example a platelet or platelet derivative.
  • the cargo can comprise an imaging agent, in illustrative embodiments an MRI agent.
  • Such means is typically any one or more peptides consisting of SEQ ID NOs: 2 to 87 with oligomeric numbers as provided in the Exemplary CPPs Table.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent- loaded platelet derivatives.
  • a method can comprise of steps that make use of lyophilized or cryopreserved platelets as a starting material for loading.
  • the “Exemplary CPPs Table” below discloses a non-limiting set of CPPs that can be included in any of the aspects or embodiments of the present disclosure.
  • the CPPs include certain fragments of SEQ ID NO: 1 as indicated herein, and SEQ ID Nos:22-87 with oligomeric numbers indicated in the Table if indicated after the sequence.
  • a process for preparing MRI agent-loaded platelets comprising: (a) providing platelets; and (b) treating the platelets with an MRI agent, to form MRI agent-loaded platelets.
  • MRI agent-loaded platelet derivatives comprising: a) providing platelets; (b) treating the platelets with an MRI agent, to form MRI agent-loaded platelets; and (c) lyophilizing the MRI agent-loaded platelets, to form MRI agent- loaded platelet derivatives.
  • MRI agent-loaded platelet derivatives can be MRI agent-loaded FDPDs.
  • a process as disclosed herein can further comprise a step of rehydrating the MRI agent-loaded platelet derivatives.
  • a process for preparing MRI agent-loaded cryopreserved platelets comprising: a) providing platelets; (b) treating the platelets with an MRI agent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent-loaded platelets, to form MRI agent-loaded cryopreserved platelets.
  • a process as disclosed herein can further comprise a step of thawing the MRI agent-loaded cryopreserved platelets.
  • an MRI agent is present in a complex, referred herein as MRI agent complex as defined elsewhere in the present disclosure.
  • an MRI agent is present in the form of MRI agent complex
  • the MRI agent complex is covalently bonded to platelets.
  • an MRI agent is coupled to a cell penetrating peptide.
  • the CPP functions as a means to penetrate a cell membrane and facilitate uptake of MRI agent by the cells/platelets.
  • platelets are isolated prior to treating (e.g., contacting) the platelets with an MRI agent.
  • the methods for preparing an MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; and step (b) treating the platelets with an MRI agent coupled to a cell penetrating peptide and with a loading buffer comprising a salt, a base, a loading agent, and optionally ethanol, to form the MRI agent-loaded platelets.
  • the methods for preparing an MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; and step (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide and with a loading buffer comprising a salt, a base, a loading agent, and optionally ethanol, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) treating the platelets with an MRI agent coupled with a cell penetrating peptide to form a first composition; and step (c) treating the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) contacting the platelets with an MRI agent coupled with a cell penetrating peptide to form a first composition; and step (c) contacting the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the MRI agent-loaded platelets.
  • suitable organic solvents include, but are not limited to alcohols, esters, ketones, ethers, halogenated solvents, hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixtures thereof.
  • suitable organic solvents includes, but are not limited to methanol, ethanol, n-propanol, isopropanol, acetic acid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropyl ether (IPE), tert- butyl methyl ether, dioxane (e.g., 1,4-dioxane), acetonitrile, propionitrile, methylene chloride, chloroform, toluene, anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane, dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethylacetamide, and combinations thereof.
  • IPE isopropyl ether
  • dioxane
  • the methods for preparing MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) treating the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) treating the first composition with an MRI agent coupled to a cell penetrating peptide, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) isolating platelets, for example in a liquid medium; step (b) contacting the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) contacting the first composition with an MRI agent coupled to a cell penetrating peptide, to form the MRI agent-loaded platelets.
  • isolating platelets includes isolating platelets from blood.
  • platelets are donor-derived platelets.
  • platelets are obtained by a process that includes an apheresis step.
  • platelets are fresh platelets.
  • platelets are stored platelets.
  • platelets are derived in vitro. In some embodiments, platelets are derived or prepared in a culture prior to treating the platelets with an MRI agent. In some embodiments, preparing the platelets includes deriving or growing the platelets from a culture of megakaryocytes. In some embodiments, preparing the platelets includes deriving or growing the platelets (or megakaryocytes) from a culture of human pluripotent stem cells (PCSs), including embryonic stem cells (ESCs) and/or induced pluripotent stem cells (iPSCs).
  • PCSs human pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; and step (b) treating the platelets with an MRI agent reagent, and with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; and step (b) contacting the platelets with an MRI agent reagent, and with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent- loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; step (b) treating the platelets with an MRI agent to form a first composition; and step (c) treating the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; step (b) contacting the platelets with an MRI agent to form a first composition; and step (c) contacting the first composition with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; step (b) treating the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) treating the first composition with an MRI agent, to form the MRI agent-loaded platelets.
  • the methods for preparing MRI agent-loaded platelets includes: step (a) providing platelets; step (b) contacting the platelets with a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and step (c) contacting the first composition with an MRI agent, to form the MRI agent-loaded platelets.
  • the method for preparing MRI agent-loaded platelets comprises: a) isolating platelets, for example in a liquid medium; and b) treating the platelets with an MRI agent and with a loading buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise treating the platelets with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) isolating platelets, for example in a liquid medium; and b) contacting the platelets with an MRI agent and with a loading buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise contacting the platelets with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) isolating platelets, for example in a liquid medium; b) treating the platelets with an MRI agent to form a first composition; and c) treating the first composition with a buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise treating the platelets with an organic solvent such as ethanol and the method does not comprise treating the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) isolating platelets, for example in a liquid medium; b) contacting the platelets with an MRI agent to form a first composition; and c) contacting the first composition with a buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise contacting the platelets with an organic solvent such as ethanol and the method does not comprise treating the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a)isolating platelets, for example in a liquid medium; b) c)treating the platelets with a buffer comprising a salt, a base, and a loading agent, to form a first composition; and d) treating the first composition with an MRI agent, to form the MRI agent-loaded platelets.
  • the method for preparing MRI agent-loaded platelets comprises: a) isolating platelets, for example in a liquid medium; b) contacting the platelets with a buffer comprising a salt, a base, and a loading agent, to form a first composition; and c) contacting the first composition with an MRI agent, to form the MRI agent-loaded platelets.
  • the method does not comprise contacting the platelets with an organic solvent such as ethanol and the method does not comprise contacting the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; and b) treating the platelets with an MRI agent-loaded and with a loading buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise treating the platelets with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; and b) contacting the platelets with an MRI agent-loaded and with a loading buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise contacting the platelets with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; b) treating the platelets with an MRI agent to fonn a first composition; and c) treating the first composition with a buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise treating the platelets with an organic solvent such as ethanol and the method does not comprise treating the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; b) contacting the platelets with an MRI agent to form a first composition; and c) contacting the first composition with a buffer comprising a salt, a base, and a loading agent, to form the MRI agent-loaded platelets, wherein the method does not comprise contacting the platelets with an organic solvent such as ethanol and the method does not comprise contacting the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; b) treating the platelets with a buffer comprising a salt, a base, and a loading agent, to form a first composition; and c) treating the first composition with an MRI agent, to form the MRI agent-loaded platelets.
  • the method does not comprise treating the platelets with an organic solvent such as ethanol and the method does not comprise treating the first composition with an organic solvent such as ethanol.
  • the method for preparing MRI agent-loaded platelets comprises: a) providing platelets; b) contacting the platelets with a buffer comprising a salt, a base, and a loading agent, to form a first composition; and c) contacting the first composition with an MRI agent, to form the MRI agent-loaded platelets, wherein the method does not comprise contacting the platelets with an organic solvent such as ethanol and the method does not comprise contacting the first composition with an organic solvent such as ethanol.
  • the loading agent is a saccharide.
  • the saccharide is a monosaccharide.
  • the saccharide is a disaccharide.
  • the saccharide is a non-reducing disaccharide.
  • the saccharide is sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose.
  • the loading agent is a starch.
  • MRI agent is any agent that is suitable for magnetic resonance imaging described herein or known in the art.
  • an MRI agent loaded into platelets is modified.
  • an MRI agent can be modified to increase its stability during the platelet loading process, while the MRI agent is loaded into the platelet, and/or after the MRI agent’s release from a platelet
  • the modified MRI agent’s stability is increased with little or no adverse effect on its activity.
  • the modified MRI agent can have at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the activity of the corresponding unmodified MRI agent.
  • the modified MRI agent has 100% (or more) of the activity of the corresponding unmodified MRI agent.
  • the MRI agent is stabilized by one or more of a stabilizing oligonucleotide (see, e.g., U.S. Application Publication No. 2018/0311176), a backbone/side chain modification (e.g., a 2- sugar modification such as a 2’-fluor, methoxy, or amine substitution, or a 2’-thio (-SH), 2’-azido (- N3), or 2’-hydroxymethyl (-CH2OH) modification), an unnatural nucleic acid substitution (e.g., an S- glycerol, cyclohexenyl, and/or threose nucleic acid substitution, an L-nucleic acid substitution, a locked nucleic acid (LNA) modification (e.g., the ribose moiety of an LNAnucleotide is modified with an extra bridge connecting the 2’ oxygen and 4’ carbon), conjug
  • a stabilizing oligonucleotide see, e.g
  • an MRI agent loaded into platelets is modified to include an imaging agent.
  • an MRI agent can be modified with an imaging agent in order to image the MRI agent loaded platelet in vivo.
  • an MRI agent can be modified with two or more imaging agents (e.g., any two or more of the imaging agents described herein).
  • an MRI agent loaded into platelets is modified with a radioactive metal ion, a paramagnetic metal ion, a gamma-emitting radioactive halogen, a positron-emitting radioactive non-metal, a hyperpolarized NMR-activc nucleus, a reporter suitable for in vivo optical imaging, or a bcta-cmittcr suitable for intravascular detection.
  • a radioactive metal ion can include, but is not limited to, positron emitters such as 54 Cu, 48 V, 52 Fe, 55 Co, 94 Tc or 68 Ga; or gamma-emitters such as
  • a paramagnetic metal ion can include, but is not limited to Gd(III), a Mn(II), a Cu(II), a Cr(III), a Fe(III), a Co(II), a Er(II), a Ni(II), a Eu(III) or a Dy(III), an element comprising an Fe element, a neodymium iron oxide (NdFeO3) or a dysprosium iron oxide (DyFeO3).
  • a paramagnetic metal ion can be chelated to a polypeptide or a monocry stallinc nanoparticle.
  • a gamma-emitting radioactive halogen can include, but is not limited to 123 I 131 I or 77 Br.
  • a positron-emitting radioactive non-metal can include, but is not limited to "C, 13 N, 15 O, 17 F, 18 F, 75 Br, 76 Br or 124 I.
  • a hyperpolarized NMR-active nucleus can include, but is not limited to 13 C, 15 N, 19 F, 29 Si and 31 P.
  • a reporter suitable for in vivo optical imaging can include, but is not limited to any moiety capable of detection either directly or indirectly in an optical imaging procedure.
  • the reporter suitable for in vivo optical imaging can be a light scatterer (e.g., a colored or uncolored particle), a light absorber or a light emitter.
  • the reporter can be any reporter that interacts with light in the electromagnetic spectrum with wavelengths from the ultraviolet to the near infrared.
  • organic chromophoric and fluorophoric reporters include groups having an extensive delocalized electron system, e.g.
  • cyanines merocyanines, indocyanines, phthalocyanines, naphthalocyanines, triphenylmethines, porphyrins, pyrilium dyes, thiapyrilium dyes, squarylium dyes, croconium dyes, azulenium dyes, indoanilines, benzophenoxazinium dyes, benzothiaphenothiazinium dyes, anthraquinones, naptho quinones, indathrenes, phthaloylacridones, trisphenoquinones, azo dyes, intramolecular and intermolecular charge-transfer dyes and dye complexes, tropones, tetrazines, b/s (dithiolene) complexes, brs(benzene-dithiolate) complexes, iodoaniline dyes, b/stS.O-dithiolene) complexes.
  • the reporter can be, but is not limited to a fluorescent, a bioluminescent, or chemiluminescent polypeptide.
  • a fluorescent or chemiluminescent polypeptide is a green florescent protein (GFP), a modified GFP to have different absorption/emission properties, a luciferase, an aequorin, an obelin, a mnemiopsin, a berovin, or a phenanthridinium ester.
  • GFP green florescent protein
  • a reporter can be, but is not limited to rare earth metals (e.g., europium, samarium, terbium, or dysprosium), or fluorescent nanocrystals (e.g., quantum dots).
  • a reporter may be a chromophore that can include, but is not limited to fluorescein, sulforhodamine 101 (Texas Red), rhodamine B, rhodamine 6G, rhodamine 19, indocyanine green, Cy2, Cy3, Cy3.5, Cy5, Cy5.5, Cy7, Marina Blue, Pacific Blue, Oregon Green 88, Oregon Green 514, tctramcthylrhodaminc, and Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 555, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 647, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, and Alexa Fluor 750.
  • a beta-emitter can include, but is not limited to radio metals 67 Cu, 89 Sr, 90 Y, 153 Sm, 185 Re, 188 Re or 192 Ir, and non-metals 32 P, 33 P, 38 S, 38 C1, 39 C1, 82 Br and 83 Br.
  • an MRI agent loaded into platelets can be associated with gold or other equivalent metal particles (such as nanoparticles).
  • a metal particle system can include, but is not limited to gold nanoparticles (e.g., NanogoldTM).
  • an MRI agent loaded into platelets that is modified with an imaging agent is imaged using an imaging unit.
  • the imaging unit can be configmed to image the MRI agent loaded platelets in vivo based on an expected property (e.g., optical property from the imaging agent) to be characterized.
  • imaging techniques in vivo imaging using an imaging unit
  • CAT computer assisted tomography
  • MRS magnetic resonance spectroscopy
  • MRI magnetic resonance imaging
  • PET positron emission tomography
  • SPECT single-photon emission computed tomography
  • bioluminescence imaging BL1
  • the MRI agent may be loaded in a liquid medium that may be modified to change the proportion of media components or to exchange components for similar products, or to add components necessary for a given application.
  • the loading buffer and/or the liquid medium include one or more of a) water or a saline solution, b) one or more additional salts, or c) a base.
  • the loading buffer, and/or the liquid medium may include one or more of a) DMSO, b) one or more salts, or d) a base.
  • the loading agent is loaded into the platelets in the presence of an aqueous medium.
  • the loading agent is loaded in the presence of a medium comprising DMSO.
  • one embodiment of the methods herein includes treating (e.g., contacting) platelets with an MRI agent coupled with a cell penetrating peptide and with an aqueous loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • one embodiment of the methods herein includes treating (e.g., contacting) platelets with an MRI agent coupled with a cell penetrating peptide and with a loading buffer comprising DMSO and comprising a salt, a base, a loading agent, and optionally ethanol, to form the MRI agent-loaded platelets.
  • the loading buffer and/or the liquid medium include one or more salts selected from phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products, or that is known to be useful in drying platelets, or any combination of two or more of these.
  • these salts are present in the composition at an amount that is about the same as is found in whole blood.
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium for different durations at or at different temperatures from about 15-45 °C, or about 22°C.
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium at a temperature from about 18-42 °C, about 20-40 °C, about 22-37 °C, or about 16 °C, about 18 °C, about 20 °C, about 22 °C, about 24 °C, about 26 °C, about 28 °C, about 30 °C, about 32 °C, about 34 °C, about 36 °C, about 37 °C, about 39 °C, about 41 °C, about 43 °C, or about 45 °C for at least about 5 minutes (mins) (e.g., at least about 20 mins, about 30 mins, about 1 horn (hr), about 2 hrs
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium at a temperature from about 18-42 °C, about 20-40 °C, about 22-37 °C, or about 16 °C, about 18 °C, about 20 °C, about 22 °C, about 24 °C, about 26 °C, about 28 °C, about 30 °C, about 32 °C, about 34 °C, about 36 °C, about 37 °C, about 39 °C, about 41 °C, about 43 °C, or about 45 °C for no more than about 48 hrs (e.g., no more than about 20 mins, about 30 mins, about 1 horn (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30
  • the MRI agent- loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium from about 10 mins to about 48 hours (e.g., from about 20 mins to about 36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs, from about 2 hrs to about 16 horns, from about 10 mins to about 24 hours, from about 20 mins to about 12 hours, from about 30 mins to about 10 hrs, or from about 1 hr to about 6 hrs. .
  • 10 mins to about 48 hours e.g., from about 20 mins to about 36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs, from about 2 hrs to about 16 horns, from about 10 mins to about 24 hours, from about 20 mins to about 12 hours, from about 30 mins to about 10 hrs, or from about 1 hr to about 6 hrs.
  • the platelets are at a concentration from about 1,000 platelets/pl to about 10,000,000 platelets/pl. In some embodiments, the platelets are at a concentration from about 50,000 platelets/pl to about 4,000,000 platelets/pl. In some embodiments, the platelets are at a concentration from about 100,000 platelets/pl to about 300,000,000 platelets/ pl. In some embodiments, the platelets arc at a concentration from about 1,000,000 to about 2,000,000. In some embodiments, the platelets are at a concentration of about 200,000,000 platelets/ pl. [00179] In some embodiments, the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium for different durations.
  • the step of incubating the platelets to load one or more MRI agent(s) includes incubating the platelets for a time suitable for loading, as long as the time, taken in conjunction with the temperature, is sufficient for the MRI agent to come into contact with the platelets and, preferably, be incorporated, at least to some extent, into the platelets.
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium for at least about 5 minutes (mins) (e.g., at least about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs. about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, about 42 hrs,, about 48 hrs, or at least about 48 hrs.
  • mins e.g., at least about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs. about 20 hrs, about 24 hrs, about 30 hrs,
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium for no more than about 48 hrs (e.g., no more than about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, or no more than about 42 hrs).
  • 48 hrs e.g., no more than about 20 mins, about 30 mins, about 1 hour (hr), about 2 hrs, about 3 hrs, about 4 hrs, about 5 hrs, about 6 hrs, about 7 hrs, about 8 hrs, about 9 hrs, about 10 hrs, about 12 hrs, about 16 hrs, about 20 hrs, about 24 hrs, about 30 hrs, about 36 hrs, or no more than
  • the MRI agent- loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium from about 10 mins to about 48 hours (e.g., from about 20 mins to about 36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs, from about 2 hrs to about 16 hours, from about 10 mins to about 24 hours, from about 20 mins to about 12 hours, from about 30 mins to about 10 hrs, or from about 1 hr to about 6 hrs.
  • 10 mins to about 48 hours e.g., from about 20 mins to about 36 hrs, from about 30 mins to about 24 hrs, from about 1 hr to about 20 hrs, from about 2 hrs to about 16 hours, from about 10 mins to about 24 hours, from about 20 mins to about 12 hours, from about 30 mins to about 10 hrs, or from about 1 hr to about 6 hrs.
  • the MRI agent-loaded platelets are prepared by incubating the platelets with the MRI agent in the liquid medium at different temperatures.
  • the step of incubating the platelets to load one or more MRI agent(s) includes incubating the platelets with the MRI agent in the liquid medium at a temperature that, when selected in conjunction with the amount of time allotted for loading, is suitable for loading.
  • the platelets with the MRI agent in the liquid medium are incubated at a suitable temperature (e.g., a temperature above freezing) for at least a sufficient time for the MRI agent to come into contact with the platelets.
  • incubation is conducted at 22°C.
  • incubation is performed at 4 °C to 45 °C, such as 15 °C to 42 °C.
  • incubation is performed from about 18-42 °C, about 20-40 °C, about 22-37 °C, or about 16 °C, about 18 °C, about 20 °C, about 22 °C, about 24 °C, about 26 °C, about 28 °C, about 30 °C, about 32 °C, about 34 °C, about 36 °C, about 37 °C, about 39 °C, about 41 °C, about 43 °C, or about 45 °C for 110 to 130 (e g., 120) minutes and for as long as 24-48 hours.
  • incubation is performed from about 18-42 °C, about 20-40 °C, about 22-37 °C, or about 16 °C, about 18 °C, about 20 °C, about 22 °C, about 24 °C, about 26 °C, about 28 °C, about 30
  • the methods further include acidifying the platelets, or pooled platelets, to a pH of about 6.0 to about 7.4, prior to a treating (e.g., contacting) step disclosed herein.
  • the methods include acidifying the platelets to a pH of about 6.5 to about 6.9.
  • the methods include acidifying the platelets to a pH of about 6.6 to about 6.8.
  • the acidifying includes adding to the pooled platelets a solution comprising Acid Citrate Dextrose.
  • the platelets are isolated prior to a treating (e.g., contacting) step.
  • the methods further include isolating platelets by using centrifugation.
  • the centrifugation occurs at a relative centrifugal force (RCF) of about 800 g to about 2000 g.
  • the centrifugation occurs at relative centrifugal force (RCF) of about 1300 g to about 1800 g.
  • the centrifugation occurs at relative centrifugal force (RCF) of about 1500 g.
  • the centrifugation occurs for about 1 minute to about 60 minutes.
  • the centrifugation occurs for about 10 minutes to about 30 minutes.
  • the centrifugation occurs for about 20 minutes.
  • the platelets are at a concentration from about 1,000 platelets/pl to about 10,000,000 platelets/pl. In some embodiments, the platelets are at a concentration from about 50,000 platelets/pl to about 4,000,000 platelets/pl. In some embodiments, the platelets are at a concentration from about 100,000 platelets/pl to about 300,000,000 platelets/pl. hi some embodiments, the platelets are at a concentration from about 1,000,000 to about 2,000,000. In some embodiments, the platelets are at a concentration of about 2,000,000 platelets/pl.
  • the buffer is a loading buffer comprising the components as listed in Table 5 herein.
  • the loading buffer includes one or more salts, such as phosphate salts, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salt that can be found in blood or blood products.
  • Exemplary salts include sodium chloride (NaCl), potassium chloride (KC1), and combinations thereof.
  • the loading buffer includes from about 0.5 mM to about 100 mM of the one or more salts.
  • the loading buffer includes from about 1 mM to about 100 mM (e.g., about 2 mM to about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 to about 85 mM) about of the one or more salts. In some embodiments, the loading buffer includes about 5 mM, about 75 mM, or about 80 mM of the one or more salts.
  • the loading buffer includes one or more buffers, e.g., N-2- hydroxy ethylpiperazine-N’-2-ethanesulfonic acid (HEPES), and/or sodium-bicarbonate (NaHCCh).
  • the loading buffer includes from about 5 to about 100 mM of the one or more buffers.
  • the loading buffer includes from about 5 to about 50 mM (e.g., from about 5 mM to about 40 mM, from about 8 mM to about 30 mM, about 10 mM to about 25 mM) about of the one or more buffers.
  • the loading buffer includes about 10 mM, about 20 mM, about 25 mM, or about 30 mM of the one or more buffers.
  • the loading buffer includes one or more saccharides, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. In some embodiments, the loading buffer includes from about 10 mM to about 1,000 mM of the one or more saccharides. In some embodiments, the loading buffer includes from about 50 to about 500 mM of the one or more saccharides. In embodiments, one or more saccharides is present in an amount of from 10 mM 10 to 500 mM. In some embodiments, one or more saccharides is present in an amount of from 50 mM to 200 mM. In embodiments, one or more saccharides is present in an amount from 100 mM to 150 mM.
  • saccharides such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. In some embodiments, the loading buffer includes from about 10
  • the loading buffer includes adding an organic solvent, such as ethanol, to the loading solution.
  • the solvent can range from about 0.1 % (v/v) to about 5.0 % (v/v), such as from about 0.3 % (v/v) to about 3.0 % (v/v), or from about 0.5 % (v/v) to about 2 % (v/v).
  • the MRI agent includes one MRI agent. In some embodiments, the MR1 agent includes one or more MRI agents.
  • the methods further include incubating the MRI agent in the presence of the loading buffer prior to the treatment (e.g., contacting) step. In some embodiments, the methods further include incubating the loading buffer and a solution comprising the MRI agent and water at about 37°C using different incubation periods. In some embodiments, the solution includes a concentration of about 0.1 nM to about 150 pM of the MRI agent. In some embodiments, the solution includes a concentration of about 1 nM to about 100 pM of the MRI agent. In some embodiments, the solution includes a concentration of about 10 nM to 50 pM of the MRI agent.
  • the solution includes a concentration of about 500 nM to about 50 pM of the MRI agent. In some embodiments, the solution includes a concentration of about 1 pM to about 30 pM of the MRI agent. In some embodiments, the solution includes a concentration of about 10 pM to about 30 pM of the MRI agent. In some embodiments, the incubation of the MRI agent in the presence of the loading buffer is performed from about 1 minute to about 2 hours. In some embodiments, the incubation is performed at an incubation period of from about 5 minutes to about 1 hour Tn some embodiments, the incubation is performed at an incubation period of from about 10 minutes to about 30 minutes. In some embodiments, the incubation is performed at an incubation period of about 20 minutes.
  • the methods further include incubating the MRI agent in the presence the loading buffer prior to the treatment (e.g., contacting) step.
  • the methods further include mixing the platelets and the coupled cell penetrating peptide (CPP) and MRI agent (e.g., MRI agent-CPP) in the presence of the loading buffer at about room temperature (e g , at about 20°C to about 25°C).
  • CPP coupled cell penetrating peptide
  • MRI agent e.g., MRI agent-CPP
  • Coupled describes attaching (e.g., complexing, conjugating) a cell penetrating peptide to an MRI agent.
  • the coupling of the cell penetrating peptide and the MRI agent can be a covalent or a non-covalent coupling.
  • the incubation is performed at an incubation period of from about 5 minutes to about 12 hours. In some embodiments, the incubation is performed at an incubation period of from about 10 minutes to about 6 hours. In some embodiments, the incubation is performed at an incubation period of from about 15 minutes to about 3 hours. In some embodiments, the incubation is performed at an incubation period of about 2 hours. In some embodiments, the final product includes platelets and the MRI-agent CPP at a volume ratio of 10: 1, with a range in volume ratio of about 1 to about 0.
  • the concentration of MRI agent in the MRI agent-loaded platelets is from about 0.1 nM to about 10 pM. In some embodiments, the concentration of MRI agent in the MRI agent-loaded platelets is from about 1 nM to about 1 pM. In some embodiments, the concentration of MRI agent in the MRI agent-loaded platelets is from about 10 nM to 10 pM. In some embodiments, the concentration of MRI agent in the MRI -loaded platelets is about 100 nM.
  • the methods further include drying the MRI agent-loaded platelets.
  • the drying step includes freeze-drying the MRI agent-loaded platelets.
  • the methods further include rchydrating the MRI agent-loaded platelets obtained from the drying step.
  • MRI agent-loaded platelets are prepared by using any of the variety of methods provided herein.
  • rehydrated MRI agent-loaded platelets are prepared by any one method comprising rehydrating the MRI agent-loaded platelets provided herein.
  • the MRI agent-loaded platelets may be used, for example, in therapeutic applications as disclosed herein. Additionally or alternatively, the MRI agent-loaded platelets may be employed in functional assays. In some embodiments, the MRI agent-loaded platelets are cold stored, cryopreserved, or lyophilized (to produce thrombosomes) prior to use in therapy or in functional assays. In some embodiments, process for loading MRI agent on to platelets as disclosed herein to form MRI agent- loaded platelets/ platelet derivatives/cryopreserved platelets/FDPDs is also applicable for loading imaging agent on to platelets that does not involve prior loading with an MRI agent.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent complex such that the MRI agent complex is covalently bound to the platelets, to form MRI agent-loaded platelets; and (c) cryopreserving or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives.
  • contacting the platelets with MRI agent complex such that the MRI agent complex is covalently bound to the platelets, to form MRI agent-loaded platelets is done in the presence of a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent.
  • a method can comprise steps that make use of lyophilized or cryopreserved platelets as a starting material for loading.
  • a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded platelet derivatives.
  • platelets can be provided by isolating the platelets using TFF method as disclosed herein.
  • a process using the platelets isolated/purified by TFF method and that further undergoes lyophilization after loading with MRI agents are also called as MRI agent-loaded TFF-FDPDs or MRI agent-loaded FDPDs.
  • platelets can be provided by isolating the platelets using a centrifugation based method as disclosed herein.
  • a process using the platelets isolated/purified by centrifugation based method and that further undergoes lyophilization after loading with MRI agents are also called as MRI agent-loaded FDPDs or MRI agent-loaded platelet derivatives.
  • a process using the platelets isolated/purified by centrifugation based method or by TFF method and that further undergoes cryopreservation after loading with MRI agents are also called as MRI agent-loaded cryopreserved platelets.
  • platelets that are to be used for loading with MRI agents are isolated/purified using centrifugation based methods.
  • a centrifugation based method can include the steps of: (a) pooling apheresis platelet units to form pooled platelet rich plasma; (b) acidifying the pooled platelet rich plasma to form pooled acidified pooled platelet rich plasma; (c) centrifuging the acidified pooled platelet rich plasma and discarding the supernatant comprising platelet pooled plasma; and (d) resuspending the remaining solution obtained after centrifuging, in a buffer comprising at least one saccharide, at least one salt, and optionally at least one organic solvent.
  • resuspending can also be done in a buffer that further comprises at least one cryoprotectant, or platelets can be incubated with the buffer comprising at least one saccharide, isolated out of that solution using for example, TFF or centrifugation, and resuspended in a buffer comprising a cr oprotectant.
  • cryoprotectants include but are not limiting to, polysugars, Ficoll, DMSO, bovine serum albumin, human serum albumin, dextran, polyvinyl 51ehydrate51e (PVP), starch, hydroxy ethyl starch (HES).
  • polysugar can comprise poly sucrose, Ficoll 70, and Ficoll 400.
  • a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs comprising: (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryopreserved platelets or the rehydrated platelet derivatives with an MRI agent coupled to a cell penetrating peptide, to form the composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives.
  • Imaging agents such as a radioactive metal ion, an MRI agent (such as, but not limiting to, a paramagnetic metal ion, a superparamagnetic metal ion, and a diamagnetic metal ion), a gammaemitting radioactive halogen, a positron-emitting radioactive non-metal, a hyperpolarized NMR-active nucleus, a reporter suitable for in vivo optical imaging, or a beta-emitter suitable for intravascular detection can be of importance in the field of diagnostics and therapy.
  • an MRI agent such as, but not limiting to, a paramagnetic metal ion, a superparamagnetic metal ion, and a diamagnetic metal ion
  • a gammaemitting radioactive halogen such as, but not limiting to, a paramagnetic metal ion, a superparamagnetic metal ion, and a diamagnetic metal ion
  • Platelets, platelet derivatives such as FDPDs, or cryopreserved platelets can be loaded with any of the imaging agents known in the art to form imaging agent-loaded platelets, platelet derivatives, or cryopreserved platelets.
  • a radioactive metal ion can include, but is not limited to, positron emitters such as 54 Cu, 48 V, 52 Fe, 55 Co, 94 Tc or 68 Ga; or gamma-emitters such as 171 Tc, m In, 113 In, or 67 Ga.
  • imaging agent can include radiometal nuclides that serve as diagnostic markers in molecular imaging or can be used in therapeutic settings.
  • an imaging agent can be an Indium isotope, such as, In 3+ .
  • an imaging agent can be an Yttrium isotope, such as, Y 3+ .
  • an imaging agent can be a Lutetium isotope, such as, Lu 3+ .
  • an imaging agent can be a copper isotope, such as, Cu 2+ . Any appropriate imaging agent, such as, radiometal nuclides as disclosed in the publication - Wangler, B., et al.
  • an imaging agent can be present in a complex along with a chelator for loading on to platelets.
  • a chelator acts to reduce the toxicity of an imaging agent that is been loaded on to the platelets.
  • a person of skill in the art can use any chelator known in the art for forming a complex with an imaging agent that further can be loaded onto platelets. Any appropriate chelator as disclosed in Wangler et al 2011 can be used for the purposes of the present invention. In some embodiments, any of chelators as disclosed elsewhere in this invention can be used for forming a complex with an imaging agent.
  • imaging agent-loaded platelets/FDPDs/cryopreserved platelets can be prepared using loading with a CPP as disclosed for MRI agent-loaded platelets/FDPDs/cryopreserved platelets as disclosed elsewhere in this disclosure.
  • a CPP is any type of CPP that is disclosed for MRI agent- loaded platelets/FDPDs/cryopreserved platelets elsewhere in this invention.
  • imaging agent-loaded platelets/FDPDs/cryopreserved platelets can be prepared using an imaging agent-complex that comprises an imaging agent, a chelator for reducing toxicity of the imaging agent, and a linker for covalently binding to a protein molecule on the surface of platelets. Any appropriate linker known in the art can be used for preparing imaging agent-loaded platelets/FDPDs/cryopreserved platelets.
  • a linker can be selected from the group consisting of a compound having sulfhydryl reactive groups, such as maleimides and haloacetyl derivatives, amine reactive groups, such as isothiocyanates, succinimidyl esters, and sulfonyl halides, and carbodiimidc reactive groups, such as carboxyl and amino groups.
  • a linker is a compound having an amine reactive group, such as, succinimidyl ester, such as, N- Hydroxysuccinimide (NHS) ester.
  • any of the aspects or embodiments herein that include MRI agent- loaded platelets/platelet derivatives such as FDPDs/cryopreserved platelets or methods to obtain any of the above products/composition provided herein is a method of delivering MRI agent-loaded platelets/FDPDs/cryopreserved platelets comprising administering an effective amount or a therapeutically effective amount of a composition comprising MRI agent-loaded platelets, MRI agent- loaded cryopreserved platelets or MRI agent-loaded platelet derivatives of any of the aspects or embodiments disclosed herein, or the composition prepared by the process of any of the aspects or embodiments disclosed herein.
  • Delivering of MRI agent-loaded compositions as disclosed herein can allow targeted delivery of the MRI agents to sites of interest.
  • MRI agents can be used to image blood vessels and inflamed or diseased tissue where blood vessels have become compromised (e g., “leaky”), in some illustrative embodiments to detect sites of bleeding.
  • Certain conditions where such delivery methods can be used in the detection or diagnosis, and may aid in the treatment include conditions where damage to a tissue or vessels, any disruption to a tissue or vessels, or any vascular damage occurs.
  • a delivery method as disclosed herein can provide enhanced diagnosis of a disease or a condition, such as cancer (any type of cancers as disclosed herein), stroke, brain injury, embolism, or hemorrhage.
  • provided herein is a method for targeted delivery of an MRI agent to a site or sites of interest in a subject, comprising administering to a subject an effective amount or a therapeutically effective amount of the MRI agent-loaded compositions or products disclosed in any of the aspects or embodiments herein.
  • a method of detecting, diagnosing, or enhancing diagnosis of a disease comprising administering an effective amount or a therapeutically effective amount of a composition comprising MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives of any of the aspects or embodiments disclosed herein, or the composition prepared by the process of any of the aspects or embodiments disclosed herein, and imaging and/or detecting the MRI agent for enhancing diagnosis of a disease.
  • the subject to which such imaging agent-loaded (e.g. MRI agent-loaded) platelet or platelet derivatives are administered can be a subject suspected of having compromised vessels and/or tissues.
  • the disease can be any of the diseases that include compromised vessels or tissues.
  • a method for detecting site of bleeding in a subject comprising: (a) administering an effective amount or a therapeutically effective amount of a composition comprising MRI agent loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives as disclosed in any of the aspects or embodiments herein, or the composition prepared by the process as disclosed in any of the aspects or embodiments herein, to the subject; and (b) detecting/locating/imaging the site of the MRI agent-loaded compositions/products for detecting image blood vessels and inflamed or diseased tissue, or the site of bleeding in the subject.
  • a method for detecting/locating/imaging an MRI agent well known in the art can be used herein for detecting the site of bleeding in a subject after the administration of MRI agent-loaded compositions/products as disclosed herein.
  • detecting the site of bleeding can be performed after at least 30 seconds, 1 minute, 2 minutes, 5 minutes, 7 minutes, 10 minutes, 15 minutes, 20 minutes, or 30 minutes of administering an effective amount or a therapeutically effective amount of MRI agent-loaded compositions/products.
  • detecting the site of bleeding can be performed after 1 minute to 24 hours, 1 minute to 20 hours, 1 minute to 15 horns, 1 minute to 12 hours, 1 minute to 10 hours, 1 minute to 8 hours, 1 minute to 6 hours, 1 minute to 3 hours, 1 minute to 1 hour, 2 minutes to 20 hours, 5 minutes to 15 hours. 10 minutes to 10 hours, 5 minutes to 5 hours, 5 minutes to 1 hour, or 5 minutes to 30 minutes of administering an effective amount or a therapeutically effective amount of MRI agent-loaded compositions/products.
  • a method for imaging compromised blood vessels or inflamed tissues in a subject comprising: (a) administering to a subject an effective amount or a therapeutically effective amount of the MRI agent-loaded compositions or products disclosed in any of the aspects or embodiments herein; and (b) imaging the MRI agent-loaded platelets for imaging the compromised blood vessels or inflamed tissues in the subject.
  • MRI agent-loaded products or compositions can include MRI agent-loaded platelets, MRI agent-loaded platelet derivatives such as FDPDs, or MRI agent-loaded cryopreserved platelets.
  • disease/condition/indication can include types of cancer as disclosed herein.
  • disease/condition/indication can be selected from the group consisting of Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Breast cancer, Gastric cancer, Hodgkin lymphoma, Neuroblastoma, Non - Hodgkin lymphoma, Ovarian cancer, Cervical cancer, Small cell lung cancer, Non-small cell lung cancer (NSCLC), Soft tissue and bone sarcomas, Thyroid cancer, Transitional cell bladder cancer, Wilms tumor Neuroendocrine tumors, Pancreatic cancer, Multiple myeloma, Renal cancer, Glioblastoma Prostate cancer, Sarcoma, Colon cancer, Melanoma, Colitis, Chronic inflammatory demyelinating polyneuropathy, Guillain - Barre syndrome, Immune Thrombocytopenia, Kawasaki disease, Lupus, Multiple Sclerosis, Myasthenia gravis, Myositis, Cirrhosis with refractory ascites, Hep
  • disease/condition/indication can be selected from the group consisting of Von Willebrand disease, Immune thrombocytopenia, Hermansky Pudlak Syndrome (HPS), Chemotherapy induced thrombocytopenia (CIT), Scott syndrome, Evans syndrome, Hematopoietic Stem Cell Transplantation, Fetal and neonatal alloimmune thrombocytopenia, Bernard Soulier syndrome, Acute myeloid leukemia, Glanzmann thrombasthenia, Myelodysplastic syndrome, Hemorrhagic Shock, Coronary thrombosis (myocardial infarction), Ischemic Stroke, Arterial Thromboembolism, Wiskott Aldrich Syndrome, Venous Thromboembolism, MYH9 related disease, Acute Lymphoblastic Lymphoma (ALL), Acute Coronary Syndrome, Chronic Lymphocytic Leukemia (CLL), Acute Promyelocytic Leukemia, Cerebral Venous Sin
  • MRI agent-loaded products/compositions as disclosed herein can be used for enhancing diagnosis of any of the disease/condition/indication as disclosed herein.
  • MRI agent-loaded products/compositions as disclosed herein can be used for delivering MRI agent to a subject having any of the discasc/condition/indication as disclosed herein.
  • MRI agent-loaded products/compositions as disclosed herein can be used for imaging compromised blood vessels or inflamed tissue in a subject having any of the disease/condition/indication as disclosed herein.
  • an effective amount or a therapeutically effective amount of MRI agent-loaded products/compositions disclosed herein can be administered or delivered for a number of applications including for treating to a subject afflicted with any one or combination of indications/diseases as disclosed herein, for detecting site of bleeding in a subject, for imaging compromised blood vessels or inflamed tissue in a subject, for targeted delivery of MRI agent to a subject, or for enhancing diagnosis of a condition/indication in a subject, and the dose/amount of MRI agent-loaded platclcts/FDPDs/cryoprcscrvcd platelets can be in the range of 1.0 x 10 7 to 1.0 x 10 11 particles/kg of the subject, or 1.0 x 10 7 to 1.0 x 10 14 particles/kg of the subject.
  • a dose of a composition comprising MRI agent-loaded platelets, platelet derivatives (e.g., FDPDs), or crvoprcscrvcd platelets can include between about or exactly 1.0 x 10 7 to 1.0 x 10 11 particles /kg of a subject, 1.0 x 10 7 to 1.0 x 10 10 particles/kg of a subject, 1.6 x 10 7 to 1.0 x 10 10 particles (e.g.
  • 1.6 x 10 7 to 5.1 x 10 9 particles/kg of a subject 1.6 x 10 7 to 3.0 x 10 9 particles/kg of a subject, 1.6 x 10 7 to l.O x 10 9 particles/kg of a subject, 1.6 x 10 7 to 5.0 x 10 8 particles/kg of a subject, 1.6 x 10 7 to 1.0 x 10 8 particles/kg of a subject, 1.6 xlO 7 to 5.0 x 10 7 particles/kg of a subject, 5.0 x 10 7 to 1.0 x 10 8 particles/kg of a subject, 1.0 x 10 8 to 5.0 x 10 8 particles/kg of a subject, 5.0 x 10 8 to 1.0 x 10 9 particles/kg of a subject, 1.0 x 10 9 to 5.0 x 10 9 particles/kg of a subject, or 5.0 x 10 9 to 1.0 x 10 10 particles/kg of a subject).
  • a therapeutically effective dose or effective dose or amount of the MRI agent-loaded platelet derivatives in a platelet derivative composition or amount of the MRI agent-loaded cryopreserved platelets in a cryopreserved platelet composition is in the range of 1.0 x 10 7 to 1.0 x 10 14 particles/kg of the subject, 1.6 x 10 7 to 1.0 x 10 14 particles (e.g.
  • FDPDs, FPH, or cryopreserved platelets/kg of subject 1.6 x 10 7 to 8 x 10 13 particles, 1.6 x 10 7 to 5.1 x 10 13 particles, 1.6 x 10 7 to 3.0 x 10 13 particles/kg of a subject, 1.6 x 10 7 to 1.0 x 10 13 particles/kg of a subject, 1.6 x 10 7 to 8.0 x 10 12 particles/kg of a subject, 1.6 x 10 7 to 5.0 x 10 12 particles/kg of a subject, 1 .6 x 10 7 to 3.0 x 10 12 particles/kg of a subject, 1.6 x 10 7 to 1 .0 x 10 12 particles/kg of a subject, 1.6 xlO 7 to 8.0 x 10 11 particles/kg of a subject, 1.6 xlO 7 to 5.0 x 10 11 particles/kg of a subject, 1.6 xlO 7 to 3.0 x 10 11 particles/kg of a subject, 1.6 xlO' to
  • FDPDs FDPDs/kg of a subject
  • 5.0 x 10 8 to 1.0 x 10 14 particles e.g. FDPDs
  • 8.0 x 10 8 to 1.0 x IO 14 particles e.g. FDPDs
  • 1.0 x 10 9 to l.O x 10 14 particles e.g. FDPDs
  • 3.0 x 10 9 to 1.0 x 10 14 particles e.g. FDPDs
  • 5.0 x 10 9 to 1.0 x 10 14 particles e.g. FDPDs
  • 8.0 x 10 9 to 1.0 x 10 14 particles e.g.
  • FDPDs FDPDs/kg of a subject
  • 1.0 x 10 10 to 1.0 x 10 14 particles e.g. FDPDs
  • 3.0 x 10 10 to 1.0 x 10 14 particles e.g. FDPDs
  • 5.0 x IO 10 to 1.0 x 10 14 particles e.g. FDPDs
  • 8.0 x IO 10 to 1.0 x 10 14 particles e.g. FDPDs
  • 1.0 x 10 11 to 1.0 x 10 14 particles e.g. FDPDs
  • 5.0 x 10 11 to 1.0 x 10 14 particles e.g.
  • FDPDs FDPDs/kg of a subject
  • 8.0 x 10 11 to 1.0 x 10 14 particles e.g. FDPDs
  • 1.0 x 10 12 to 1.0 x 10 14 particles e.g. FDPDs
  • 3.0 x 10 12 to 1.0 x 10 14 particles e.g. FDPDs
  • 5.0 x 10 12 to 1.0 x 10 14 particles e.g. FDPDs
  • 8.0 x 10 12 to 1.0 x 10 14 particles e g. FDPDs)/kg of a subject).
  • a person of skill in the art can contemplate the effective dose or a therapeutically effective dose of MRI agent-loaded platelets/FDPDs/cryopreserved platelets that can be required to deliver into a subject can vary based on the utility of MRI agent-loaded products/compositions as per the requirements.
  • the dose can differ for treating a subject having an indication/ disease as compared to administering MRI agent-loaded products/compositions for enhancing diagnosis in a patient.
  • the dose can vary for detecting a site of bleeding in a subject. The need may differ based on the condition of the subject.
  • the effective dosage can be categorized into a) low dosage; b) medium dosage; and c) high dosage.
  • a medicament or a method of treating a subject can have the effective dose as low, medium, or high dosage of MRI agent-loaded platelets/FDPDs/cryopreserved platelets that can broadly range from LO X 10 7 to 1.0 X 10 14 /kg of ln some embodiments, the dose can be in the range of 250 and 5000 TGPU per kg of the subject.
  • administering can be performed until the bleeding potential of the subject is reduced as compared to the bleeding potential before the administering, or until any one of the applications as disclosed herein is achieved, for example, until the detection/diagnosis/imaging or targeted delivery to the site of interest is confirmed. In some embodiments, the administering can be performed until the bleeding stops.
  • the platelets or pooled platelets may be acidified to a pH of about 5.5 to about 8.0 prior to TFF or being diluted with the preparation agent.
  • the method comprises acidifying the platelets to a pH of about 6.5 to about 6.9.
  • the method comprises acidifying the platelets to a pH of about 6.6 to about 6.8.
  • the method comprises acidifying the platelets to a pH of about 6.6 to 7.5.
  • the acidifying comprises adding to the pooled platelets a solution comprising Acid Citrate Dextrose (ACD).
  • ACD Acid Citrate Dextrose
  • the platelets are isolated prior to the step comprising tangential flow filtration (TFF) or being diluted with the preparation agent.
  • the method further comprises isolating platelets by using centrifugation.
  • the centrifugation occurs at a relative centrifugal force (RCF) of about 1000 x g to about 2000 x g.
  • the centrifugation occurs at relative centrifugal force (RCF) of about 1300 x g to about 1800 x g.
  • the centrifugation occurs at relative centrifugal force (RCF) of about 1 00 x g.
  • the centrifugation occurs for about 1 minute to about 60 minutes.
  • the centrifugation occurs for about 10 minutes to about 30 minutes.
  • the centrifugation occurs for about 30 minutes.
  • platelets are isolated, for example in a liquid medium, prior to treating a subject.
  • platelets are donor-derived platelets.
  • platelets are obtained by a process that comprises an apheresis step.
  • platelets are pooled platelets.
  • platelets are pooled from a plurality of donors. Such platelets pooled from a plurality of donors may be also referred herein to as pooled platelets.
  • the donors are more than 5, such as more than 10, such as more than 20, such as more than 50, such as up to about 100 donors.
  • the donors are from about 5 to about 100, such as from about 10 to about 50, such as from about 20 to about 40, such as from about 25 to about 35.
  • Pooled platelets can be used to make any of the compositions described herein.
  • the platelets can be pooled wherein the platelets are donated by human subjects.
  • the donor can be a non-human animal.
  • the donor can be a canine subject.
  • the donor can be an equine subject.
  • the donor can be a feline subject.
  • platelets are derived in vitro. In some embodiments, platelets are derived or prepared in a culture. In some embodiments, preparing the platelets comprises deriving or growing the platelets from a culture of megakaryocytes. In some embodiments, preparing the platelets comprises deriving or growing die platelets (or megakaryocytes) from a culture of human pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and/or induced pluripotent stem cells (iPSCs).
  • PSCs human pluripotent stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • platelets or platelet derivatives are prepared prior to treating a subject as described herein.
  • the platelets or platelet derivatives e.g., thrombosomes
  • the platelets or platelet derivatives are lyophilized.
  • the platelets or platelet derivatives e.g., thrombosomes
  • the platelets or platelet derivatives can be cryopreserved in plasma and DMSO (e.g., 3-9% DMSO (e g., 6% DMSO)).
  • the platelets or platelet derivatives are cryopreserved as described in U.S. Patent Application Publication No. 2020/0046771 Al, published on February 13, 2020, incorporated herein by reference in its entirety.
  • platelets e.g., apheresis platelet, platelets isolated from whole blood, pooled platelets, or a combination thereof
  • a preparation agent comprising a liquid medium at a concentration from 10,000 platelets/pL to 10,000,000 platelets/ JJL, such as 50,000 platelets/ pL to 2,000,000 platelets/pL, such as 100,000 platelets/pL to 500,000 platelets/pL, such as 150,000 platelets/ pL to 300,000 platcIcts/uL. such as 200,000 platelets/pL.
  • the method further comprises drying the platelets or platelet derivatives (e.g., thrombosomes).
  • the drying step comprises lyophilizing the platelets or platelet derivatives (e.g., thrombosomes).
  • the drying step comprises freeze-dry ing the platelets or platelet derivatives (e.g., thrombosomes).
  • the method further comprises rehydrating the platelets or platelet derivatives (e.g., thrombosomes) obtained from the dry ing step.
  • the platelets or platelet derivatives e.g., thrombosomes
  • the platelets or platelet derivatives are cold stored, cryopreserved, or lyophilized (e.g., to produce thrombosomes) prior to use in therapy or in functional assays.
  • any known technique for drying platelets can be used in accordance with the present disclosure, as long as the technique can achieve a final residual moisture content of less than 5%. Preferably , the technique achieves a final residual moisture content of less than 2%, such as 1%, 0.5%, or 0.1%.
  • suitable techniques are freeze-drying (lyophilization) and spray-drying.
  • a suitable lyophilization method is presented in Table A. Additional exemplary lyophilization methods can be found in U.S. Patent No. 7,811,558, U.S. Patent No. 8,486,617, and U.S. Patent No. 8,097,403.
  • An exemplary spray -drying method includes: combining nitrogen, as a drying gas, with a loading buffer according to the present disclosure, then introducing the mixture into GEA Mobile Minor spray dryer from GEA Processing Engineering, Inc. (Columbia MD, USA), which has a Two-Fluid Nozzle configuration, spray drying the mixture at an inlet temperature in the range of 150°C to 190°C, an outlet temperature in the range of 65°C to 100°C, an atomic rate in the range of 0.5 to 2.0 bars, an atomic rate in the range of 5 to 13kg/hr, a nitrogen use in the range of 60 to 100 kg/hr, and a run time of 10 to 35 minutes.
  • the final step in spray drying is preferentially collecting the dried mixture.
  • the dried composition in some embodiments is stable for at least six months at temperatures that range from -20°C or lower to 90°C or higher.
  • the step of drying the MRI agent-loaded platelets that are obtained as disclosed herein includes incubating the platelets with a lyophilizing agent.
  • the lyophilizing agent is polysucrose.
  • the ly ophilizing agent is a non-reducing disaccharidc.
  • the methods for preparing MRI agent-loaded platelets further include incubating the MRI agent-loaded platelets with a lyophilizing agent.
  • the lyophilizing agent is a saccharide.
  • the saccharide is a disaccharide, such as a non-reducing disaccharide.
  • the platelets and/or platelet derivatives are incubated with a lyophilizing agent for a sufficient amount of time and at a suitable temperature to incubate the platelets with the lyophilizing agent.
  • suitable lyophilizing agents are saccharides, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, and xylose.
  • suitable lyophilizing agents include serum albumin, dextran, polyvinyl 61ehydrate61e (PVP), starch, and hydroxy ethyl starch (HES).
  • exemplary lyophilizing agents can include a high molecular weight polymer.
  • high molecular weight it is meant a polymer having an average molecular weight of about or above 70 kDa and up to 1,000,000 kDa.
  • Non-limiting examples are polymers of sucrose and epichlorohydrin (e g., polysucrosc).
  • the lyophilizing agent is poly sucrose.
  • any amount of high molecular weight polymer can be used as a lyophilizing agent, it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w/v), such as 3% to 7%, for example 6%.
  • polysucrose is used in the range of 2% to 8%%, or 2.25-7.75%, or 2.5-7.5%, or 2.5-6.5%.
  • the composition comprises 3% polysucrose.
  • the composition comprises 6% poly sucrose.
  • the polysucrose is a cationic form of polysucose.
  • the cationic form of polysucrose is diethylaminoethyl (DEAE)-polysucrose.
  • the poly sucrose is an anionic form of poly sucrose.
  • the anionic form of poly sucrose is carboxymethyl-polysucrose.
  • polysucrose has a molecular weight in the range of 70,000 Da to 400,000 Da.
  • poly sucrose has a molecular weight in the range of 80,000 Da to 350,000 Da, or 100,000 Da to 300,00 Da.
  • polysucrose has a molecular weight in the range of 120,000 Da to 200,000 Da.
  • poly sucrose has a molecular weight of 150,000 Da, or 160,000 Da, or 170,000 Da, or 180,000 Da, 190,000 Da, or 200,000 Da.
  • the process for preparing a composition includes adding an organic solvent, such as ethanol, to the loading solution.
  • the solvent can range from 0.1 % to 5.0 % (v/v).
  • addition of the lyophilizing agent can be the last step prior to dry ing.
  • the lyophilizing agent is added at the same time or before the MR1 agent, the cryoprotectant, or other components of the loading composition.
  • the ly ophilizing agent is added to the loading solution, thoroughly mixed to form a drying solution, dispensed into a drying vessel (e.g., a glass or plastic serum vial, a lyophilization bag), and subjected to conditions that allow for drying of the solution to form a dried composition.
  • a drying vessel e.g., a glass or plastic serum vial, a lyophilization bag
  • An exemplary saccharide for use in the compositions disclosed herein is trehalose. Regardless of the identity of the saccharide, it can be present in the composition in any suitable amount. For example, it can be present in an amount of 1 mM to 1 M. In embodiments, it is present in an amount of from 10 mM 10 to 500 mM. In some embodiments, it is present in an amount of from 20 mM to 200 mM. In some embodiments, it is present in an amount from 40 mM to 100 mM. In various embodiments, the saccharide is present in different specific concentrations within the ranges recited above, and one of skill in the art can immediately understand the various concentrations without the need to specifically recite each herein. Where more than one saccharide is present in the composition, each saccharide can be present in an amount according to the ranges and particular concentrations recited above.
  • the step of incubating the platelets to load them with a cryoprotectant or as a lyophilizing agent includes incubating the platelets for a time suitable for loading, as long as the time, taken in conjunction with the temperature, is sufficient for the cryoprotectant or lyophilizing agent to come into contact with the platelets and, preferably, be incorporated, at least to some extent, into the platelets. In embodiments, incubation is carried out for about 1 minute to about 180 minutes or longer.
  • the step of incubating the platelets to load them with a cryoprotectant or lyophilizing agent includes incubating the platelets and the cryoprotectant at a temperature that, when selected in conjunction with the amount of time allotted for loading, is suitable for loading.
  • the composition is incubated at a temperature above freezing for at least a sufficient time for the cryoprotectant or lyophilizing agent to come into contact with the platelets.
  • incubation is conducted at 37°C.
  • incubation is performed at 20°C to 42°C.
  • incubation is performed at 35°C to 40°C (e.g., 37°C) for 110 to 130 (e.g., 120) minutes.
  • the bag is a gas-permeable bag configured to allow gases to pass through at least a portion or all portions of the bag during the processing.
  • the gas- permeable bag can allow for the exchange of gas within the interior of the bag with atmospheric gas present in the surrounding environment.
  • the gas-permeable bag can be permeable to gases, such as oxygen, nitrogen, water, air, hydrogen, and carbon dioxide, allowing gas exchange to occur in the compositions provided herein.
  • the gas-permeable bag allows for the removal of some of the carbon dioxide present within an interior of the bag by allowing the carbon dioxide to permeate through its wall.
  • the release of carbon dioxide from the bag can be advantageous to maintaining a desired pH level of the composition contained within the bag.
  • the container of the process herein is a gas-permeable container that is closed or sealed.
  • the container is a container that is closed or sealed and a portion of which is gas-permeable.
  • the surface area of a gas- permeable portion of a closed or sealed container (e.g., bag) relative to the volume of the product being contained in the container (hereinafter referred to as the “SA/V ratio”) can be adjusted to improve pH maintenance of the compositions provided herein.
  • the SA/V ratio of the container can be at least about 2.0 cm 2 /mL (e.g., at least about 2.1 cm 2 /mL, at least about 2.2 cm 2 /mL, at least about 2.3 cm 2 /mL, at least about 2.4 cm 2 /mL, at least about 2.5 cm 2 /mL, at least about 2.6 cm 2 /mL, at least about 2.7 cm 2 /mL, at least about 2.8 cm 2 /mL, at least about 2.9 cm 2 /mL, at least about 3.0 cm 2 /mL, at least about 3.
  • 2.0 cm 2 /mL e.g., at least about 2.1 cm 2 /mL, at least about 2.2 cm 2 /mL, at least about 2.3 cm 2 /mL, at least about 2.4 cm 2 /mL, at least about 2.5 cm 2 /mL, at least about 2.6 cm 2 /mL, at least about 2.7 cm 2 /mL, at least about 2.8 cm 2
  • the SA/V ratio of the container can be at most about 10.0 cm 2 /mL (e.g., at most about 9.9 cm 2 /mL.
  • the SA/V ratio of the container can range from about 2.0 to about 10.0 cm 2 /mL (e.g., from about 2.1 cm 2 /mLto about 9.9 cm 2 /mL, from about 2.2 cm 2 /mLto about 9.8 cm 2 /mL, from about 2.3 cm 2 /mL to about 9.7 cm 2 /mL, from about 2.4 cm 2 /mL to about 9.6 cm 2 /mL, from about 2.5 cm 2 /mL to about 9.5 cm 2 /mL, from about 2.6 cm 2 /mL to about 9.4 cm 2 /mL, from about 2.7 cm 2 /mL to about 9.3 cm 2 /mL, from about 2.8 cm 2 /mL to about 9.2 cm 2 /mL, from about 2.9 cm 2 /mL to about 9.
  • Gas-permeable closed containers e.g., bags
  • the gas-permeable bag can be made of one or more polymers including fluoropolymers (such as polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers), polyolefins (such as low-density polyethylene (LDPE), high-density polyethylene (HDPE)), fluorinated ethylene propylene (FEP), polystyrene, polyvinylchloride (PVC), silicone, and any combinations thereof.
  • fluoropolymers such as polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers
  • polyolefins such as low-density polyethylene (LDPE), high-density polyethylene (HDPE)
  • FEP fluorinated ethylene propylene
  • PVC polyvinylchloride
  • silicone silicone
  • the lyophilizing agent as disclosed herein may be a high molecular weight polymer.
  • high molecular weight it is meant a polymer having an average molecular weight of about or above 70 kDa and up to 1,000,000 kDa
  • Non-limiting examples are polymers of sucrose and epichlorohydrin (polysucrose).
  • any amount of high molecular weight polymer can be used, it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w/v), such as 3% to 7%, for example 6%.
  • the loading buffer includes an organic solvent, such as an alcohol (e.g., ethanol).
  • an organic solvent such as an alcohol (e.g., ethanol).
  • the amount of solvent can range from 0. 1 % to 5.0 % (v/v).
  • the MRI agent-loaded platelets prepared as disclosed herein have a storage stability that is at least about equal to that of the platelets prior to the loading of the MRI agent.
  • the loading bulfer may be any buffer that is non -toxic to the platelets and provides adequate buffering capacity to the solution at the temperatures at which the solution will be exposed during the process provided herein.
  • the buffer may include any of the known biologically compatible buffers available commercially, such as phosphate buffers, such as phosphate buffered saline (PBS), bicarbonate/carbonic acid, such as sodium-bicarbonate buffer, N-2-hydroxyethylpiperazine-N’-2- ethane sulfonic acid (HEPES), and tris-based buffers, such as tris-buffered saline (TBS).
  • PBS phosphate buffered saline
  • bicarbonate/carbonic acid such as sodium-bicarbonate buffer
  • tris-based buffers such as tris-buffered saline (TBS).
  • buffers propane- 1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic; maleic; 2,2- dimethylsuccinic; 3,3-dimethylglutaric; bis(2- hydroxy ethyl) imino- tris(hydroxymethy l)-methane (BIS-TRIS); benzenehexacarboxylic (mellitic); N- (2- acetamido)imino-diacetic acid (ADA); butane-l,2,3,4-tetracarboxylic; pyrophosphoric; 1,1- cyclopentanediacetic (3,3 tetramethylene -glutaric acid); piperazine- l,4-bis-(2 -ethanesulfonic acid) (PIPES); N-(2-acetamido )-2- amnoethanesulfonic acid (ACES); 1,1-cyclohe
  • the method can include an initial dilution step, for example, a starting material (e.g., an unprocessed blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)) can be diluted with a preparation agent (e.g., any of the preparation agents described herein) to form a diluted starting material.
  • a preparation agent e.g., any of the preparation agents described herein
  • the initial dilution step can include dilution with a preparation agent with a mass of preparation agent equal to at least about 10% of the mass of the starting material (e.g., at least about 15%, 25%, 50%, 75%, 100%, 150%, or 200% of the mass of the starting material.
  • an initial dilution step can be carried out using the TFF apparatus.
  • the method can include concentrating (e.g., concentrating platelets) (e.g., concentrating a starting material or a diluted starting material) to form a concentrated platelet composition.
  • concentrated can include concentrating to a about 1000 x 10 3 to about 4000 x 10 3 platelets/ pL (e.g., about 1000 x 10 3 to about 2000 x 10 3 , about 2000 x 10 3 to about 3000 x 10 3 , or about 4000 x 10 3 platelets/ L).
  • a concentration step can be carried out using the TFF apparatus.
  • the concentration of platelets or platelet derivatives can be determined by any appropriate method.
  • a counter can be used to quantitate concentration of blood cells in suspension using impedance (e.g., a Beckman Coulter AcT 10 or an AcT diff 2).
  • TFF can include diafiltering (sometimes called “washing”) of a starting material, a diluted starting material, a concentrated platelet composition, or a combination thereof.
  • diafiltering can include washing with at least 2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) diavolumes.
  • TFF can include buffer exchange.
  • a buffer can be used in TFF.
  • a buffer can be any appropriate buffer.
  • the buffer can be a preparation agent (e.g., any of the preparation agents described herein).
  • the buffer can be the same preparation agent as was used for dilution.
  • a buffer can be a different preparation than was used for dilution.
  • a buffer can Include a lyophilizing agent, including a buffering agent, a base, a loading agent, optionally a salt, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof.
  • a buffering agent can be any appropriate buffering agent.
  • a buffering agent can be HEPES ((4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid).
  • Abase can be any appropriate base. In some embodiments, a base can be sodium bicarbonate.
  • a saccharide can be a monosaccharide.
  • a loading agent can be a saccharide. In some embodiments, a saccharide can include sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, a monosaccharide can be trehalose In some embodiments, tire loading agent can include polysucrose.
  • a salt can be any appropriate salt. In some embodiments, a salt can be selected from the group consisting of sodium chloride (NaCl), potassium chloride (KC1), or a combination thereof.
  • a membrane with a pore size of about 0.1 pm to about 1 pm (e.g., about 0. 1 pm to about 1 pm, about 0. 1 pm to about 0.5 pm, about 0.2 to about 0.45 pm, about 0.45 to about 1 pm, about 0.1 un. about 0.2 un, about 0.45 pun, about 0.65 pun, or about 1 pun) can be used in TFF.
  • a membrane can be made from any appropriate material.
  • a membrane can be a hydrophilic membrane.
  • a membrane can be a hydrophobic membrane.
  • a membrane with a nominal molecular weight cutoff (NMWCO) of at least about 100 kDa e.g., at least about 200, 300 kDa, 500 kDa, or 1000 kDa
  • the TFF can be performed with any appropriate pore size within the range of 0. 1 pm to 1.0 pm with the aim of reducing the microparticles content in the composition and increasing the content of platelet derivatives in the composition.
  • a skilled artisan can appreciate the required optimization of the pore size in order to retain the platelet derivatives and allow the microparticles to pass through the membrane.
  • the pore size in illustrative embodiments is such that the microparticles pass through the membrane allowing the TFF-treated composition to have less than 5% microparticles.
  • the pore size in illustrative embodiments is such that a maximum of platelet derivatives gets retained in the process allowing the TFF-treated composition to have a concentration of the platelet derivatives in the range of 100 x 103 to 20,000 x 103.
  • the pore size during the TFF process can be exploited to obtain a higher concentration of platelet derivatives in the platelet derivative composition such that a person administering the platelet derivatives to a subject in need has to rehydrate/reconstitute fewer vials, therefore, being efficient with respect to time and effort during the process of preparing such platelet derivatives for a downstream procedure, for example a method of treating provided herein.
  • TFF can be performed at any appropriate temperature.
  • TFF can be performed at a temperature of about 20 °C to about 37 °C (e.g., about 20 °C to about 25 °C, about 20 °C to about 30 °C, about 25 °C to about 30 °C, about 30 °C to about 35 °C, about 30 °C to about 37 °C, about 25 °C to about 35 °C, or about 25 °C to about 37 °C).
  • TFF can be carried out at a How rate (e.g., a circulating flow rate) of about 100 mFmin to about 800 mFmin (e.g., about 100 to about 200 ml/min, about 100 to about 400 ml/min, about 100 to about 600 mFmin, about 200 to about 400 ml/min, about 200 to about 600 ml/min, about 200 to about 800 ml/min, about 400 to about 600 ml/min, about 400 to about 800 ml/min, about 600 to about 800 ml/min, about 100 ml/min, about 200 ml/min, about 300 ml/min, about 400 ml/min, about 500 ml/min, about 600 ml/min, about 700 ml/min, or about 800 ml/min).
  • a How rate e.g., a circulating flow rate
  • TFF can be performed until a particular endpoint is reached, forming a TFF-treated composition.
  • An endpoint can be any appropriate endpoint.
  • an endpoint can be a percentage of residual plasma (e.g., less than or equal to about 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of residual plasma).
  • an endpoint can be a relative absorbance at 280 nm (A280).
  • an endpoint can be an A280 (e.g., using a path length of 0.5 cm) that is less than or equal to about 50% (e.g., less than or equal to about 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the A280 (e.g., using a path length of 0.5 cm) prior to TFF (e.g., of a starting material or of a diluted starting material).
  • an instrument to measure A280 can be configured as follows: a 0.5cm gap flow cell can be attached to the filtrate line of the TFF system.
  • the flow cell can be connected to a photometer with fiber optics cables attached to each side of the flow cell (light source cable and light detector cable).
  • the flow cell can be made with a silica glass lens on each side of the fiber optic cables.
  • the protein concentration in the aqueous medium can also be measured in absolute terms.
  • the protein concentration in the aqueous medium is less than or equal to 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or 1%, or 0.1%, or 0.01%. In some exemplary embodiments, the protein concentration is less than 3% or 4%. In some embodiments, the protein concentration is in the range of 0.01-15%, or 0.1-15%, or 1- 15%, or 1-10%, or 0.01-10%, or 3-12%, or 5-10% in the TFF-treated composition. In some embodiments, an endpoint can be an absolute A280 (e.g., using a path length of 0.5 cm).
  • an endpoint can be an A280 that is less than or equal to 2.50 AU, 2.40 AU, 2.30 AU, 2.20 AU, 2.10 AU, 2.0 AU, 1.90 AU, 1.80 AU, or 1.70 AU (e.g., less than or equal to 1.66, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 AU) (e.g., using a path length of 0.5 cm).
  • a percentage of residual plasma, a relative A280, or an A280 can be determined based on the aqueous medium of a composition comprising platelets and an aqueous medium.
  • an endpoint can be a platelet concentration, as TFF can include concentration or dilution of a sample (e g., using a preparation agent).
  • an endpoint can be a platelet concentration of at least about 2000 x 103 platelets/pL (e.g., at least about 2050 x 103, 2100 x 103, 2150 x 103, 2200 x 103, 2250 x 103, 2300 x 103, 2350 x 103, 2400 x 103, 2450 x 103, or 2500 x 103 platelets/ pL).
  • an endpoint can be a platelet concentration of about 1000 x 103 to about 2500 platclcts/
  • an endpoint can be a concentration of platelets in the TFF-treated composition are at least 100 x 103 platelets/pL, 200 x 103 platelets/ pL, 400 x 103 platelets/pL, 1000 x 103 platelels/pL, 1250 x 103 platelets/pL, 1500 x 103 plate lets/pL, 1750 x 103 platelets/pL, 2000 x 103 platelets/pL, 2250 x 103 platelets/pL, 2500 x 103 plate lets/pL, 2750 x 103 platelets/pL, 3000 x 103 platclets/pL. 3250 x 103 platelets/pL.
  • 3500 x 103 plate lets/pL, 3750 x 103 platelets/pL, 4000 x 103 platelets/pL, 4250 x 103 platelets/pL, 4500 x 103 platelets/pL, 4750 x 103 platelets/pL, 5000 x 103 platelets/pL, 5250 x 103 platelets/pL, 5500 x 103 platelets/pL, 5750 x 103 platelets/pL, 6000 x 103 platelets/pL, 7000 x 103 plate Icts/pL. 8000 x 103 plate lets/pL, 9000 x 103 plate Icts/pL. 10,000 x 103 platelets/pL, 11,000 x
  • 103 platclcts/pL 12,000 x 103 platelets/ pL, 13,000 x 103 platclcts/pL, 14,000 x 103 platclcts/pL, 15,000 x 103 platelets/pL, 16,000 x 103 platelets/pL, 17,000 x 103 platelets/pL, 18,000 x 103 platelets/pL, 19,000 x 103 platelets/pL, 20,000 x 103 platelets/pL.
  • the platelets or platelet derivatives in the TFF-treated composition is in the range of 100 x 103 - 20,000 x 103 platelets/pL, or 1000 x 103 - 20,000 x 103 platelets/pL, or 1000 x 103 - 10,000 x 103 platelets/pL, or 500 x 103 - 5,000 x 103 platelets/pL, or 1000 x 103 - 5,000 x 103 platelets/pL, or 2000 x 103 - 8,000 x 103 platelets/pL, or 10,000 x 103 - 20,000 x 103 platelets/pL, or 15,000 x 103 - 20,000 x 103 platelets/pL.
  • an endpoint can include more than one criterion (e.g., a percentage of residual plasma and a platelet concentration, a relative A280 and a platelet concentration, or an absolute A280 and a platelet concentration).
  • a TFF-treated composition is subsequently lyophilized, optionally with a thermal treatment step, to form a final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)).
  • a final blood product e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • a TFF-treated composition can be considered to be a final blood product.
  • a blood product for example a blood product that includes platelets and/or platelet derivatives
  • a blood product can be prepared using centrifugation of a blood product (e.g., an unprocessed blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)), or a partially processed blood product (e.g., a blood product that has undergone TFF)), for example to isolate platelets or platelet derivatives away from some, most, virtually all, or all liquid blood components.
  • an unprocessed blood product e.g., donor apheresis material (e.g., pooled donor apheresis material)
  • a partially processed blood product e.g., a blood product that has undergone TFF
  • methods provided herein that include providing, cry opreserving, and/or lyophilizing platelets include centrifugation of suspensions such as blood or a platelet-containing fraction thereof, that include platelets, for example to isolate the platelets ftom some, most, virtually all, or all soluble components in the suspension.
  • a blood product for example a blood product that includes platelets and/or platelet derivatives
  • a blood product can be prepared without centrifugation of a blood product (e.g., an unprocessed blood product (e.g., donor apheresis material), or a partially processed blood product (e.g., a blood product that has undergone TFF)), for example to isolate platelets or platelet derivatives away from some, most, virtually all, or all liquid blood components.
  • a blood product e.g., an unprocessed blood product (e.g., donor apheresis material), or a partially processed blood product (e.g., a blood product that has undergone TFF)
  • methods provided herein that include providing, cry opreserving, and/or lyophilizing platelets include using methods other than centrifugation, for example TFF, to process suspensions such as blood or a platelet-containing fraction thereof, that include platelets, for example to isolate the platelets from some, most, virtually all, or all soluble components in the suspension.
  • Centrifugation can include any appropriate steps, typically such that platelets are pelleted and can be isolated away from/enriched from soluble components of a plateletcontaining suspension such as blood or a fraction thereof, or a buffered solution comprising platelets or platelet derivatives.
  • centrifugation can include a slow acceleration, a slow deceleration, or a combination thereof.
  • centrifugation can include centrifugation at about 1400 x g to about 1550 x g (e.g., about 1400 to about 1450 x g, about 1450 to about 1500 x g, or 1500 to about 1550 x g, about 1400 x g, about 1410 x g, about 1430 x g, about 1450 x g, about 1470 x g, about 1490 x g, about 1500 x g, about 1510 x g, about 1530 x g, or about 1550 x g).
  • centrifugation can include centrifugation at about 1400 x g to about 1550 x g (e.g., about 1400 to about 1450 x g, about 1450 to about 1500 x g, or 1500 to about 1550 x g, about 1400 x g, about 1410 x g, about 1430 x g, about 1450 x g, about 1470 x g,
  • the duration of centrifugation can be about 10 min to about 30 min, about 15 min to about 30 min, about 10 to about 20 min, about 20 to about 30 min, about 10 min, about 20 min, about 30 min, 10 min to 30 min, 15 min to 30 min, 10 to 20 min, 20 to 30 min, 10 min, 15 min, 20 min, or 30 min).
  • a final blood product can be prepared using both TFF and centrifugation (e.g., TFF followed by centrifugation or centrifugation followed by TFF).
  • compositions prepared by any of the methods described herein are compositions prepared by any of the methods described herein.
  • a composition as described herein can be analyzed at multiple points during processing.
  • a starting material e.g., donor apheresis material (e.g., pooled donor apheresis material)
  • antibody content e.g., HLA or HNA antibody content
  • a starting material e.g., donor apheresis material (e.g., pooled donor apheresis material)
  • protein concentration e.g., by absorbance at 280 nm (e.g., using a path length of 0.5 cm)).
  • a composition in an intermediate step of processing e g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of an unprocessed blood product
  • antibody content e.g., HLA or HNA antibody content
  • the antibody content (e.g., HLA or HNA antibody content) of a blood product in an intermediate step of processing can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of the starting material.
  • a final blood product e.g., (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • antibody content e.g, HLA or HNA antibody content
  • a final blood product can be a composition that includes platelets and an aqueous medium.
  • the antibody content (e.g., HLA or HNA antibody content) of a final blood product e.g., (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • a final blood product e.g., can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of the starting material.
  • a final blood product can have no detectable level of an antibody selected from the group consisting of HLA Class I antibodies, HLA Class II antibodies, and HNA antibodies.
  • the aqueous medium of a composition as described herein can be analyzed as described herein.
  • a composition as described herein can be analyzed at multiple points during processing.
  • donor apheresis plasma can be analyzed for antibody content (e.g., HLA or HNA antibody content).
  • donor apheresis plasma can be analyzed for protein concentration (e.g.. by absorbance at 280 run).
  • a composition in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of an unprocessed blood product
  • antibody content e.g., HLA or HNA antibody content
  • the antibody content (e.g., HLA or HNA antibody content) of a blood product in an intermediate step of processing can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of donor apheresis plasma.
  • a final blood product e.g., (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • antibody content e.g., HLA or HNA antibody content
  • a final blood product can be a composition that includes platelets and an aqueous medium.
  • the antibody content (e.g., HLA or HNA antibody content) of a final blood product e.g., (e g., platelets, cryopreserved platelets, freeze-dried platelets (e g., thrombosomes)
  • a final blood product e.g., can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of donor apheresis plasma.
  • a final blood product can have no detectable level of an antibody selected from the group consisting of HLA Class I antibodies, HLA Class II antibodies, and HNA antibodies.
  • the aqueous medium of a composition as described herein can be analyzed as described herein.
  • the protein concentration of a blood product can be measured by any appropriate method. In some embodiments, the protein concentration of a blood product can be measured using absorbance at 280 nm.
  • the antibody content (e.g., HLA or HNA antibody content) of a blood product can be measured by any appropriate method.
  • a FLOWPRATM Screening or a LABScreen Multi test kits from One Lambda, Thermo Fisher Scientific can be used as a method of HLA detection.
  • Raw materials can be tested prior to the TFF or centrifugation processes to determine a baseline level of class I and II antibodies for Human Leukocyte Antigen (HLA) and Human Neutrophil Antigens (HNA). Testing can be repeated after processing by centriftigation or TFF to measure the removal of HLA and HNA. Additional testing points can be performed throughout the TFF procedure to maintain in-process control. Post-lyophilization and annealing, random samples can be selected from a batch and qualitative HLA/HNA antibody testing can be performed to ensure reduction and compliance with current FDA testing and acceptance requirements.
  • HLA Human Leukocyte Antigen
  • HNA Human Neutrophil Antigens
  • the antibody content (e.g., HLA or HNA antibody content) of two blood products can be compared by determining the percentage of beads positive for a marker (e.g., HLA or HNA coated beads bound to HLA or HNA antibodies, respectively). Any appropriate comparative method can be used.
  • the antibody content of two blood products can be compared using a method as described herein. In some embodiments, such a method can be carried out as follows. An aliquot of plasma (e.g., about 1 mL) platelet-poor plasma can be obtained.
  • an aliquot of filtered (e.g., using a 0.2 pm filter) platelet-poor plasma (PPP) (e.g., about 1 mL) can be obtained.
  • PPP platelet-poor plasma
  • Beads coated with Class I HLA and/or beads coated with Class II HLA can be added to the plasma (e g., about 5 pL of each type of bead to about 20 pL of PPP) to form a mixture of PPP and beads.
  • the mixture of PPP and beads can be vortexed.
  • the mixture of PPP and beads can be incubated to form an incubated mixture. Any appropriate incubation conditions can be used.
  • incubation can occur for a time (e.g., for about 30 minutes) at a temperature (e.g., at room temperature) with other conditions (e.g., in the dark) to form an incubated mixture.
  • incubation can include agitation (e.g., gentle rocking).
  • the beads in the incubated mixture can be washed using any appropriate conditions.
  • the beads in the incubated mixture can be washed with a wash buffer. Washed beads can be separated from the incubated mixture by any appropriate method.
  • the washed beads can be separated by centrifugation (e.g., at 9,000 x g for 2 minutes) to obtain pelleted beads.
  • the washing step can be repeated.
  • the beads can be resuspended to form a bead solution.
  • An antibody e.g., an antibody that will bind to the assayed antibody content (e.g., HLA or HNA antibody content)
  • a detectable moiety can be added to the bead solution (e.g., an odgG conjugated to a fluorescent reporter, such as FITC).
  • the antibody can be incubated with the bead solution under any appropriate conditions.
  • the antibody can be incubated for a time (e.g., for about 30 minutes) at a temperature (e.g, at room temperature) with other conditions (e g., in the dark) to form labeled beads.
  • Labeled beads can be washed to remove unbound antibody conjugated to a detectable moiety.
  • the labeled beads can be washed using any appropriate conditions.
  • the labeled beads can be washed with a wash buffer. Washed labeled beads can be separated by any appropriate method.
  • the washed labeled beads can be separated by centrifugation (e.g., at 9,000 g for 2 minutes) to obtain pelleted labeled beads.
  • the washing step can be repeated.
  • Labeled beads can be detected by any appropriate method.
  • labeled beads can be detected by flow cytometry.
  • detection can include measurement of the percentage of beads that are positive for the detectable moiety as compared to a negative control.
  • a negative control can be prepared as above, using a PPP sample that is known to be negative for antibodies (e.g. HLA Class 1, HLA Class 11, or HNA antibodies).
  • a blood product e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • a starting material e.g., donor apheresis material
  • a starting material can be analy zed for protein concentration (e.g., by absorbance at 280 nm).
  • a blood product in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of a starting material
  • protein concentration reduced to less than or equal to 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • positive beads e.g., HLA or HNA coated beads
  • a blood product in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of a starting material
  • protein concentration reduced to less than or equal to 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • positive beads e.g., HLA or HNA coated beads
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a blood product in an intermediate step of processing can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the percent of positive beads from a starting material.
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a blood product in an intermediate step of processing can be less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the total amount of beads.
  • 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • a final blood product e.g., (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • a final blood product e.g., can be analyzed to determine the percent of positive beads (e.g., HLA or HNA coated beads).
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a final blood product can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the percent of positive beads from a starting material.
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a final blood product can be less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the total amount of beads.
  • the aqueous medium of a composition as described herein can be analyzed as described herein.
  • a blood product e g , platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • donor apheresis plasma can be analyzed to determine the percent of positive beads (e.g., HLA or HNA coated beads).
  • donor apheresis plasma can be analyzed for protein concentration (e.g., by absorbance at 280 nm).
  • a blood product in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of a starting material
  • protein concentration reduced to less than or equal to 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • positive beads e.g., HLA or HNA coated beads
  • a blood product in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of a starting material
  • protein concentration reduced to less than or equal to 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • positive beads e.g., HLA or HNA coated beads
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a blood product in an intermediate step of processing can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the percent of positive beads from donor apheresis plasma.
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a blood product in an intermediate step of processing can be less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the total amount of beads.
  • 75% e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less
  • a final blood product e.g., (e g., platelets, cryopreserved platelets, freeze-dried platelets (e g., thrombosomes)
  • a final blood product e.g., (e g., platelets, cryopreserved platelets, freeze-dried platelets (e g., thrombosomes)
  • positive beads e.g., HLA or HNA coated beads
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a final blood product can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the percent of positive beads from donor apheresis material.
  • the percent of positive beads (e.g., HLA or HNA coated beads) from a final blood product can be less than or equal to 75% (e g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the total amount of beads.
  • the aqueous medium of a composition as described herein can be analyzed as described herein.
  • a percentage of positive beads can be determined using any appropriate method.
  • positive beads can be determined compared to a negative control sample.
  • a negative control sample can be any appropriate negative control sample.
  • a negative control sample can be used to determine positivity gating such that less than a certain percentage (e.g., between about 0.01% and about 1% (e.g., about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, about 0.5% to about 1%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, or about 1%)) of the negative control sample is present within the positivity gate.
  • a negative control sample can be a buffer (e g., PBS). In some embodiments, a negative control sample can be a synthetic plasma composition. In some embodiments, a negative control sample can be a blood product known to be negative for the assayed antibodies (e g., HLA or HNA antibodies).
  • a buffer e g., PBS
  • a negative control sample can be a synthetic plasma composition.
  • a negative control sample can be a blood product known to be negative for the assayed antibodies (e g., HLA or HNA antibodies).
  • Also provided herein is a method of reducing the percentage of an antibody (e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody) in a composition (e.g., a blood product) comprising platelets, the method comprising filtering the composition by tangential flow filtration.
  • a method of reducing the amount of an antibody (e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody) in a composition (e.g., a blood product) comprising platelets the method comprising filtering the composition by tangential flow filtration.
  • Also provided herein is a method of reducing the percentage of beads positive for an antibody (e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody) in a composition (e.g., a blood product) comprising platelets, the method comprising filtering the composition by tangential flow filtration.
  • a method of reducing the percentage of an antibody e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody
  • a composition e.g., a blood product
  • the method comprising filtering the composition by centrifugation.
  • Also provided herein is a method of reducing the amount of an antibody (e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody) in a composition (e.g., a blood product) comprising platelets, the method comprising filtering the composition by centrifugation.
  • a method of reducing the percentage of beads positive for an antibody e.g., a HLA antibody (e.g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody
  • a composition e.g., a blood product
  • the method comprising filtering the composition by centrifugation.
  • the amount of an antibody in a composition (e.g., a blood product) can be reduced to below a reference level.
  • a reference level can be any appropriate reference level.
  • the percentage of beads positive an antibody e.g., a HLA antibody (e g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody
  • a composition e.g., a blood product
  • the percentage of beads positive an antibody e.g., a HLA antibody (e g., a HLA Class I antibody or a HLA Class II antibody) or a HNA antibody
  • a percentage of beads positive for an antibody can be reduced by any appropriate amount.
  • a percentage of beads positive for an antibody can be reduced by at least 5% (e.g., reduced by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the blood product before undergoing any of the methods described herein.
  • a composition as described herein can undergo any appropriate additional processing steps.
  • a composition as described herein can be freeze- dried.
  • freeze-dried platelets can be thermally treated (e.g., at about 80 °C for about 24 horns).
  • a composition can be cryopreserved or freeze-dried.
  • a first composition e.g., a composition comprising platelets and an aqueous medium as described herein
  • a mixture can be treated with a mixture.
  • a mixture can include a lyophilizing agent, including a base, a loading agent, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-mcthyl pyrrolidone, dimcthylacctamidc (DMAC), or combinations thereof, to form a second composition comprising platelets.
  • a loading agent can be a saccharide.
  • a saccharide can be a monosaccharide.
  • a saccharide can be sucrose, maltose, trehalose, glucose (e.g., dextrose), mamrose, or xylose.
  • the loading agent can be polysucrose.
  • a first composition or a second composition can be dried.
  • a first composition or a second composition can be dried with a cryoprotectant.
  • a cryoprotectant can include a saccharide, optionally a base, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof to form a third composition.
  • a cryoprotectant can be polysucrose.
  • a first composition or a second composition can be freeze-dried.
  • a first composition or a second composition can be freeze-dried with a cryoprotectant.
  • a cryoprotectant can include a saccharide, optionally a base, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof to form a fourth composition.
  • organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol,
  • freeze-drying can occur at a temperature of about -40 °C to about 5 °C. In some embodiments, freeze-drying can occur over a gradient (e.g., about -40 °C to about 5 °C). In some embodiments, a secondary drying step can be carried out (e.g., at about 20 °C to about 40 °C).
  • blood products e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • blood products e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)
  • the percentage of beads positive for an antibody selected from the group consisting of HLA Class I antibodies, HLA Class II antibodies, and HNA antibodies, as determined for a composition as described herein by flow cytometry using beads coated with Class I HLAs, Class II HLAs, or HNAs, respectively, is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) as compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof.
  • the percentage of beads positive for HLA Class I antibodies, as determined for a composition as described herein by flow cytometry using beads coated with Class I HLAs is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) as compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof.
  • the percentage of beads positive for HLA Class II antibodies, as determined for a composition as described herein by flow cytometry using beads coated with Class II HLAs is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) as compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof.
  • the percentage of beads positive for HNA antibodies, as determined for a composition as described herein by flow cytometry using beads coated with HNAs is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) as compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof.
  • a lyophilizing agent can be the last step prior to drying.
  • the lyophilizing agent can be added at the same time or before other components of the composition, such as a salt, a buffer, optionally a cryoprotectant, or other components.
  • the lyophilizing agent is added to a preparation agent, thoroughly mixed to form a drying solution, dispensed into a drying vessel (e.g., a glass or plastic serum vial, a lyophilization bag), and subjected to conditions that allow for drying of a TFF-treated composition to form a dried composition.
  • a drying vessel e.g., a glass or plastic serum vial, a lyophilization bag
  • dried platelets or platelet derivatives can undergo heat treatment. Heating can be performed at a temperature above about 25°C (e.g., greater than about 40°C, 50°C, 60°C, 70°C, 80°C or higher). In some embodiments, heating is conducted between about 70°C and about 85°C (e.g., between about 75°C and about 85°C, or at about 75°C or 80 °C). The temperature for heating can be selected in conjunction with the length of time that heating is to be performed. Although any suitable time can be used, typically, the lyophilized platelets are heated for at least 1 hour, but not more than 36 hours.
  • heating is performed for at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 20 hours, at least 24 hours, or at least 30 hours.
  • the lyophilized platelets can be heated for 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours.
  • Non-limiting exemplary combinations include: heating the dried platelets or platelet derivatives (e.g., thrombosomes) for at least 30 minutes at a temperature higher than 30°C; heating the dried platelets or platelet derivatives (e.g., thrombosomes) for at least 10 hours at a temperature higher than 50°C; heating the dried platelets or platelet derivatives (e.g., thrombosomes) for at least 18 hours at a temperature higher than 75°C; and heating the dried platelets or platelet derivatives (e g., thrombosomes) for 24 hours at 80°C.
  • heating can be performed in sealed container, such as a capped vial.
  • a sealed container be subjected to a vacuum prior to heating.
  • the heat treatment step particularly in the presence of a cryoprotectant such as albumin or poly sucrose, has been found to improve the stability and shelf-life of the freeze-dried platelets. Indeed, advantageous results have been obtained with the particular combination of serum albumin or polysucrose and a post-lyophilization heat treatment step, as compared to those cryoprotectants without a heat treatment step.
  • a cryoprotectant e.g., sucrose
  • can be present in any appropriate amount e.g. about 3% to about 10% by mass or by volume of the platelets or platelet derivatives (e.g., thrombosomes).
  • compositions comprising platelets or platelet derivatives can be rehydrated with water (e.g., sterile water for injection) over about 10 minutes at about room temperature.
  • water e.g., sterile water for injection
  • the rehydration volume is about equal to the volume used to fdl each vial of thrombosomes prior to drying.
  • the platelets or platelet derivatives (e.g., thrombosomes) prepared as disclosed herein have a storage stability that is at least about equal to that of the platelets prior to the preparation.
  • the method further comprises cry opreserving the platelets or platelet derivatives prior to administering the platelets or platelet derivatives (e g., with a preparation agent, e g., a preparation agent described herein).
  • a preparation agent e g., a preparation agent described herein.
  • the method further comprises drying a composition comprising platelets or platelet derivatives, (e.g., with a preparation agent e.g., a preparation agent described herein) prior to administering the platelets or platelet derivatives (e.g., thrombosomes).
  • the method may further comprise heating the composition following the dry ing step.
  • the method may further comprise rehydrating the composition following the freeze-drying step or the heating step.
  • the method further comprises freeze-drying a composition comprising platelets or platelet derivatives (e.g., with a preparation agent e.g., a preparation agent described herein) prior to administering the platelets or platelet derivatives (e.g., thrombosomes)
  • the method may further comprise heating the composition following the freeze-drying step.
  • the method may further comprise rchydrating the composition following the freeze-drying step or the heating step.
  • the method further comprises cold storing the platelets, platelet derivatives, or the thrombosomes prior to administering the platelets, platelet derivatives, or thrombosomes (e.g., with a preparation agent, e.g., a preparation agent described herein).
  • a preparation agent e.g., a preparation agent described herein.
  • Storing conditions include, for example, standard room temperature storing (e.g., storing at a temperature ranging from about 20 to about 30 °C) or cold storing (e.g., storing at a temperature ranging from about 1 to about 10°C).
  • the method further comprises cryopreserving, freeze-drying, thawing, rehydrating, and combinations thereof, a composition comprising platelets or platelet derivatives (e.g., thrombosomes) (e.g., with a preparation agent e.g., a preparation agent described herein) prior to administering the platelets or platelet derivatives (e.g., thrombosomes).
  • the method further comprises drying (e.g., freeze-drying) a composition comprising platelets or platelet derivatives (e.g., with a preparation agent e.g., a preparation agent described herein) (e.g., to form thrombosomes) prior to administering the platelets or platelet derivatives (e.g., thrombosomes).
  • the method may further comprise rehydrating the composition obtained from the drying step.
  • a method for preparing a composition comprising platelets or platelet derivatives (e.g., thrombosomes).
  • the method can include diluting a starting material comprising platelets with an approximately equal weight ( ⁇ 10%) of a preparation agent (e.g., Buffer A, as provided in Example 1), concentrating the platelets to about 2250 x 1 3 cells/pL ( ⁇ 250 x 103) and then washed with 2-4 diavolumes (DV) (e g., about 2 diavolumes) of the preparation agent to form a TFF-treated composition.
  • the residual plasma percentage can be less than about 15% relative plasma (as determined by plasma protein content).
  • the concentration of the cells in the TFF-treated composition is not about 2000 x 103 cells/
  • the cells can be diluted with the preparation agent or can be concentrated to fall within this range.
  • the method can further include lyophilizing the TFF-treated composition and subsequently treating the lyophilized composition comprising platelets or platelet derivatives (e.g., thrombosomes) at about 80 °C for about 24 hours.
  • the method can further include a pathogen reduction step, for example, before diluting the starting material. [00280] Aggregation exhibited by platelet derivatives
  • Platelet derivative compositions which in certain illustrative embodiments herein are FDPD compositions, comprise a population of platelet derivatives (e.g. FDPDs) having a reduced propensity to aggregate under aggregation conditions comprising an agonist but no fresh platelets, and in illustrative embodiments in the absence of divalent cations, compared to the propensity of fresh platelets and/or activated to aggregate under these conditions.
  • FDPDs platelet derivatives having a reduced propensity to aggregate under aggregation conditions comprising an agonist but no fresh platelets, and in illustrative embodiments in the absence of divalent cations, compared to the propensity of fresh platelets and/or activated to aggregate under these conditions.
  • Platelet derivatives e.g., FDPDs as described herein in illustrative embodiments, display a reduced propensity to aggregate under aggregation conditions comprising an agonist but no fresh platelets, compared to the propensity of fresh platelets and/or activated platelets to aggregate under these conditions.
  • FDPDs have the ability to increase clotting and aggregation of platelets in in vitro and in vivo assays, in the presence of anti-thrombotic agents such as anti-coagulants and anti-platelet agents, under conditions where such anti-thrombotic agents reduce clotting and/or aggregation, including in the presence of two of such agents.
  • aggregation of platelet derivatives is different from co-aggregation in that aggregation conditions typically do not include fresh platelets, whereas co-aggregation conditions include fresh platelets.
  • Exemplary aggregation and co-aggregation conditions are provided in the Examples herein.
  • the platelet derivatives as described herein have a higher propensity to co-aggregate in the presence of fresh platelets and an agonist, while having a reduced propensity to aggregate in the absence of fresh platelets and an agonist, compared to the propensity of fresh platelets to aggregate under these conditions.
  • a platelet derivative composition comprises a population of platelet derivatives having a reduced propensity to aggregate, wherein no more than 2%, 3%, 4%, 5%, 7.5%, 10%, 12.5%, 15%, 17.5%, 20%, 22.5%, or 25% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, in illustrative embodiments no fresh platelets.
  • the population of platelet derivatives aggregate in the range of 2-30%, 5-25%, 10- 30%, 10-25%, or 12.5-25% of the platelet derivatives under aggregation conditions comprising an agonist but no platelets, in illustrative embodiments no fresh platelets.
  • exemplary aggregation conditions and related methods include treating FDPD sample preparations at room temperature with an agonist at a final agonist concentration of 20 pM ADP, 0.5 mg/mL arachidonic acid, 10 ug/mL collagen, 200 pM epinephrine, Img/mL ristocetin, and 10 pM TRAP-6 and measured by LTAfor example 5 minutes after agonist addition to the FDPD sample, which can be compared to LTA measurements of the sample prior to agonist addition.
  • Platelet derivatives exhibit presence of Surface markers
  • Platelets or platelet derivatives e.g., FDPDs
  • the presence of cell surface markers can be determined using any appropriate method.
  • the presence of cell surface markers can be determined using binding proteins (e.g., antibodies) specific for one or more cell surface markers and flow cytometry (e.g., as a percent positivity, e.g., using approximately 2.7x105 FDPDs/ pL; and about 4.8 pL of an anti-CD41 antibody, about 3.3 pL of an anti-CD42 antibody, about 1.3 pL of annexin V, or about 2.4 pL of an anti-CD62 antibody).
  • binding proteins e.g., antibodies
  • flow cytometry e.g., as a percent positivity, e.g., using approximately 2.7x105 FDPDs/ pL; and about 4.8 pL of an anti-CD41 antibody, about 3.3 pL of an anti-CD42 antibody, about 1.3
  • Non-limiting examples of cell-surface markers include CD41 (also called glycoprotein ilb or GPIIb, which can be assayed using e.g., an anti-CD41 antibody), CD42 (which can be assayed using, e.g., an anti-CD42 antibody), CD62 (also called CD62P or P-selectin, which can be assayed using, e.g., an anti-CD62 antibody), phosphatidylserine (which can be assayed using, e.g., annexin V (AV)), and CD47 (which is used in self-recognition; absence of this marker, in some cases, can lead to phagocytosis).
  • CD41 also called glycoprotein ilb or GPIIb
  • CD42 which can be assayed using, e.g., an anti-CD42 antibody
  • CD62 also called CD62P or P-selectin, which can be assayed using, e.g., an anti-
  • the percent positivity of any cell surface marker can be any appropriate percent positivity.
  • populations of platelet derivatives e.g., FDPDs
  • populations of platelet derivatives can have an average CD41 percent positivity of at least 55% (e.g., at least 60%, at least 65%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).
  • at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.5-25 pm, 0.5-12.5 pm, or 0.5-2.5 pm in diameter.
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.4-2.8 pm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.3-3 pm.
  • platelets or platelet derivatives can have an average CD42 percent positivity of at least 65% (e.g., at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).
  • at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.5-2.5 pm in diameter by flow cytometry.
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.4-2.8 pm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.3-3 pm in diameter by flow cytometry.
  • platelets or platelet derivatives such as those prepared by methods described herein, can have an average CD62 percent positivity of at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%).
  • at least 10% e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%).
  • At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that arc positive for CD 62 have a size in the range of 0.5-2. pm in diameter by flow cytometry. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 62 have a size in the range of 0.4-2.8 pm.
  • At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 62 have a size in the range of 0.3-3 pm.
  • platelets or platelet derivatives can have an average annexin V positivity of at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%. at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%).
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% platelet derivatives that are positive for annexin V have a size in the range of 0.5-2.5 pm in diameter by flow cytometry. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for annexin V have a size in the range of 0.4-2.8 pm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for annexin V have a size in the range of 0.3-3 pm.
  • the platelet derivative composition comprises a population of platelet derivatives comprising CD61-positive platelet derivatives, wherein less than 15%, 10%, 7.5, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD61-positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm, which in certain illustrative embodiments are less than 0.5 pm. In some illustrative embodiments, the microparticles have a diameter of less than 0.5 pm.
  • Platelet derivatives exhibit thrombin generation potency
  • Platelets or platelet derivatives as described herein can be capable of generating thrombin, for example, when in the presence of a reagent containing tissue factor and phospholipids.
  • platelets or platelet derivatives e.g., FDPDs
  • a thrombin peak height TPH
  • platelets or platelet derivatives e.g., FDPDs
  • TPH thrombin peak height
  • platelets or platelet derivatives (e.g., FDPDs) (e.g., at a concentration of about 4.8x103 particle s/pL) as described herein can generate a TPH of about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM) when in the presence of a reagent containing tissue factor and (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM or 10 pM) and optionally phospholipids.
  • a reagent containing tissue factor and e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 p
  • platelets or platelet derivatives (e.g., FDPDs) (e.g., at a concentration of about 4.8x103 particles/pL) as described herein can generate a TPH of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) when in the presence of PRP Reagent (cat# TS30.00 from Thrombinoscope), for example, using conditions comprising 20 pL of PRP Reagent and 80 pL of a composition comprising about 4.8 x 103 particles/pL of platelets or platelet derivatives (e.g., FDPDs).
  • PRP Reagent catalog TS30.00 from Thrombinoscope
  • platelets or platelet derivatives (e.g., FDPDs) (e.g., at a concentration of about 4.8x103 particles/pL) as described herein can generate a TPH of about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM) when in the presence of PRP Reagent (cat# TS30.00 from Thrombinoscope), for example, using conditions comprising 20 pL of PRP Reagent and 80 pL of a composition comprising about 4.8 x 103 partilces/pL of platelets or platelet derivatives (e.g., FDPDs).
  • PRP Reagent catalog TS30.00 from Thrombinoscope
  • Platelets or Platelet derivatives as described herein can be capable of generating thrombin, for example, when in the presence of a reagent containing tissue factor and phospholipids.
  • platelets or platelet derivatives e.g., FDPDs
  • TGPU thrombin generation potency units
  • platelets or platelet derivatives can have a potency of between 1.2 and 2.5 TPGU per 106 particles (e.g., between 1.2 and 2.0, between 1.3 and 1.5, between 1.5 and 2.25, between 1.5 and 2.0, between 1.5 and 1.75, between 1.75 and 2.5, between 2.0 and 2.5, or between 2.25 and 2.5 TPGU per 106 particles).
  • Potency Coefficient Calculated Calibrator Activity (IU)/ Effective Calibrator Activity (nM).
  • the calibrator activity can be based on a WHO international thrombin standard.
  • Platelets or platelet derivatives e.g., FDPDs as described herein can be capable of clotting, as determined, for example, by using a total thrombus -formation analysis system (T-TAS®).
  • platelets or platelet derivatives as described herein when at a concentration of at least 70x103 particles/pL (e.g., at least 73 xl03, 100 xl03, 150 xl03, 173 xl03, 200 xl03, 250 xl03, or 255 xl03 particlcs/pL) can result in a T-TAS occlusion time (e.g., time to reach kPa of 80) of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes), for example, in platelet-reduced citrated whole blood.
  • T-TAS occlusion time e.g., time to reach kPa of 80
  • platelets or platelet derivatives as described herein when at a concentration of at least 70x103 particles/pL (e.g., at least 73 x!03, 100 x!03, 150 x!03, 173 xl03, 200 xl03, 250 xl03, or 255 xl03 parlicles/pL) can result in an area under the curve (AUC) of at least 1300 (e.g., at least 1380, 1400, 1500, 1600, or 1700), for example, in platelet-reduced citrated whole blood.
  • AUC area under the curve
  • Platelets or platelet derivatives as described herein can be capable of thrombin- induced trapping in the presence of thrombin.
  • platelets or platelet derivatives as described herein can have a percent thrombin-induced trapping of at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) in the presence of thrombin.
  • platelets or platelet derivatives as described herein can have a percent thrombin-induced trapping of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%) in the presence of thrombin.
  • Thrombin-induced trapping can be determined by any appropriate method, for example, light transmission aggregometry. Without being bound by any particular theory, it is believed that the thrombin-induced trapping is a result of the interaction of fibrinogen present on the surface of the platelet derivatives with thrombin.
  • Platelets or platelet derivatives as described herein can be capable of coaggregating, for example, in the presence of an aggregation agonist, and fresh platelets.
  • aggregation agonists include, collagen, epinephrine, ristocetin, arachidonic acid, adenosine di-phosphate, and thrombin receptor associated protein (TRAP).
  • platelets or platelet derivatives as described herein can have a percent co-aggregation of at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) in the presence of an aggregation agonist, and fresh platelets.
  • platelets or platelet derivatives as described herein can have a percent co-aggregation of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%) in the presence of an aggregation agonist.
  • Percent co-aggregation can be determined by any appropriate method, for example, light transmission aggregometry.
  • Platelet derivatives exhibit presence of thrombospondin (TSP) on the surface
  • Thrombospondin is a glycoprotein secreted from the a-granules of platelets upon activation. In the presence of divalent cations, the secreted protein binds to the surface of the activated platelets and is responsible for the endogenous lectin-like activity associated with activated platelets.
  • the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is greater than that presence on the surface of resting platelets, activated platelets, or lyophilized fixed platelets.
  • the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets. In some embodiments, the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is more than 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • TSP-1 thrombospondin
  • the platelet derivatives when analyzed for the binding of antithrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, or 100 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the resting platelets.
  • MFI mean fluorescent intensity
  • the platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, or 40 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • the platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit 10-800 folds, 20-800 folds, 100-700 folds, 150-700 folds, 200-700 folds, or 250-500 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the resting platelets.
  • TSP anti-thrombospondin
  • the platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, or 40 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the active platelets.
  • TSP anti-thrombospondin
  • the platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit 2-40 folds, 5-40 folds, 5-35 folds, 10- 35 folds, or 10-30 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the active platelets.
  • TSP anti-thrombospondin
  • Platelet derivatives exhibit the presence of Von Willebrand factor on the surface
  • Von Willebrand factor is a multimeric glycoprotein that plays a major role in blood coagulation.
  • vWF serves as a bridging molecule that promotes platelet binding to sub-endothelium and other platelets, thereby promoting platelet adherence and aggregation.
  • vWF also binds to collagens to facilitate clot formation at sites of injury.
  • the platelet derivatives as described herein have the presence of von Willebrand factor (vWF) on their surface at a level that is greater than that on the surface of resting platelets, activated platelets, or lyophilized fixed platelets.
  • the platelet derivatives have the presence of von Willebrand factor (vWF) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • vWF von Willebrand factor
  • the platelet derivatives when analyzed for the binding of anti- von Willebrand factor (vWF) antibody to the platelet derivatives using flow cytometry exhibits at least 1.5 folds, 2 folds, or 3 folds, or 4 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-vWF antibody to the resting platelets, or lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • the platelet derivatives when analyzed for the binding of anti-von Willebrand factor (vWF) antibody to the platelet derivatives using flow cytometry exhibits 2-4 folds, or 2.5-3.5 higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-vWF antibody to the resting platelets, or lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • Platelet derivatives exhibit an inability to increase expression of a platelet activation marker
  • the platelet derivatives as described herein are activated to a maximum extent such that in the presence of an agonist, the platelet derivatives are not able to show an increase in the platelet activation markers on them as compared to the level of the platelet activation markers which were present prior to the exposure with the agonist.
  • the platelet derivatives as described herein show an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of an agonist.
  • the agonist is selected from the group consisting of collagen, epinephrine, ristocetin, arachidonic acid, adenosine di-phosphate, and thrombin receptor associated protein (TRAP).
  • the platelet activation marker is selected from the group consisting of Annexin V, and CD 62.
  • the platelet derivatives as described herein show an inability to increase expression of Annexin V in the presence of TRAP. An increased amount of the platelet activation markers on the platelets indicates the state of activeness of the platelets. However, in some embodiments, the platelet derivatives as described herein are not able to increase the amount of the platelet activation markers on them even in the presence of an agonist.
  • This property indicates that the platelet derivatives as described herein are activated to a maximum extent.
  • the property can be beneficial where maximum activation of platelets is required, because the platelet derivatives as described herein is able to show a state of maximum activation in the absence of an agonist.
  • Platelet derivatives in illustrative embodiments FDPDs, in further illustrative aspects and embodiments herein are surrounded by a compromised plasma membrane.
  • the platelet derivatives lack an integrated membrane around them.
  • the membrane surrounding such platelet derivatives e.g. FDPDs
  • the membrane surrounding such platelet derivatives comprises pores that are larger than pores observed on living cells.
  • such platelet derivatives e.g. FDPDs
  • a compromised membrane can be identified through a platelet derivative’s inability to retain more than 50% of lactate dehydrogenase (LDH) as compared to fresh platelets, or cold stored platelets, or cryopreserved platelets.
  • the platelet derivatives are incapable of retaining more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of lactate dehydrogenase as compared to lactate dehydrogenase retained in fresh platelets, or cold stored platelets, or cryopreserved platelets.
  • the platelet derivatives exhibit an increased permeability to antibodies.
  • the antibodies can be IgG antibodies.
  • the increased permeability can be identified by targeting IgG antibodies against a stable intracellular antigen.
  • One non-limiting type of stable intracellular antigen is [3 tubulin.
  • the compromised membrane of the platelet derivatives can also be determined by flow cytometry studies.
  • Platelet or platelet derivatives as described herein can retain some metabolic activity, for example, as evidenced by lactate dehydrogenase (LDH) ac ti x i ty .
  • LDH lactate dehydrogenase
  • platelets or platelet derivatives as described herein can retain at least about 10% (e.g., at least about 12%, 15%, 20%, 25%, 30%, 35%, 40%. or 45%) of the LDH activity of donor apheresis platelets.
  • Platelet derivatives herein have been observed to have numerous surprising properties, as disclosed in further detail herein. It will be understood, as illustrated in the Examples provided herein, that although platelet derivatives in some aspects and embodiments are in a solid, such as a powder form, the properties of such platelet derivatives can be identified, confirmed, and/or measured when a composition comprising such platelet derivatives is in liquid form.
  • the platelets or platelet derivatives e.g., FDPDs
  • a particle size e.g., diameter, max dimension
  • at least about 0.5 pm e.g., at least about at least about 0.6 pm, at least about 0.7 pm, at least about 0.8 pm, at least about 0.9 pm, at least about 1.0 pm, at least about 1.2 pm, at least about 1.5 pm, at least about 2.0 pm, at least about 2.5 pm, or at least about 5.0 pm.
  • the particle size is less than about 5.0 pm (e.g., less than about 2.5 pm, less than about 2.0 pm, less than about 1.5 pm, less than about 1.0 pm, less than about 0.9 pm, less than about 0.8 pm, less than about 0.7 pm. less than about 0.6 pm, less than about 0.5 pm, less than about 0.4 pm, or less than about 0.3 pm). In some embodiments, the particle size is from about 0.5 pm to about 5.0 pm (e.g., from about 0.5 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1 0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • platelets or platelet derivatives e.g, FDPDs
  • At most 99% e.g., at most about 95%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, or at most about 50%
  • the platelets or platelet derivatives are in the range of about 0.5 pm to about 5.0 pm (e.g., from about 0.5 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85, about 70% to about 80%) of the platelets or platelet derivatives (e.g., FDPDs) are in the range of about 0.5 pm to about 5.0 pm (e.g., from about 0.5 pm to about 4.0 pm, from about 0.5 pm to about 2.5 pm, from about 0.6 pm to about 2.0 pm, from about 0.7 pm to about 1.0 pm, from about 0.5 pm to about 0.9 pm, or from about 0.6 pm to about 0.8 pm).
  • a microparticle can be a particle having a particle size (e.g., diameter, max dimension) of less than about 0.5 pm (less than about 0.45 pm or 0.4 pm) In some cases, a microparticle can be a particle having a particle size of about 0.01 pm to about 0.5 pm (e.g., about 0.02 pm to about 0.5 pm).
  • a particle size e.g., diameter, max dimension
  • a microparticle can be a particle having a particle size of about 0.01 pm to about 0.5 pm (e.g., about 0.02 pm to about 0.5 pm).
  • Compositions comprising platelets or platelet derivatives (e.g., FDPDs), such as those prepared according to methods described herein, can have a microparticle content that contributes to less than about 5.0% (e.g., less than about 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, or 0.5%) of the total scattering intensity of all particles from about 1 nm to about 60,000 nm in radius in the composition.
  • FDPDs platelet derivatives
  • the platelet derivative composition comprises a population of platelet derivatives comprising CD41-positive platelet derivatives, wherein less than 15%, 10%, 7.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD41-positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm, which in certain illustrative embodiments are less than 0.5 pm.
  • the platelet derivative composition comprises a population of platelet derivatives comprising CD42-positive platelet derivatives, wherein less than 15%, 10%, 7.5%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD42 -positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm. 0.2 pm, or 0.1 pm, which in certain illustrative embodiments are less than 0.5 pm.
  • the platelet derivative composition comprises a population of platelet derivatives comprising CD61-positive platelet derivatives, wherein less than 15%, 10%, 7.5, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD61-positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm, which in certain illustrative embodiments are less than 0.5 pm. In some illustrative embodiments, the microparticles have a diameter of less than 0.5 pm.
  • the diameter of the microparticles is determined after rehydrating the platelet derivative composition with an appropriate solution.
  • the amount of solution for rchydrating the platelet derivative composition is equal to the amount of buffer or preparation agent present at the step of freeze-drying.
  • a content of microparticles “by scattering intensity” refers to the microparticle content based on the scattering intensity of all particles from about 1 nm to about 60,000 nm in radius in the composition.
  • the microparticle content can be measured by any appropriate method, for example, by dynamic light scattering (DLS).
  • the viscosity of a sample used for DLS can be at about 1.060 cP (or adjusted to be so), as this is the approximate viscosity of plasma.
  • the platelet derivative composition as per any aspects, or embodiments comprises a population of platelet derivatives, and microparticles, wherein the numerical ratio of platelet derivatives to the microparticles is at least 90:1, 91:1, 92:1, 93: 1, 94:1, 95:1, 96:1, 97:1, 98:1, or 99:1.
  • the platelet derivatives have a diameter in the range of 0.5-2.5 pm, and the microparticles have a diameter less than 0.5 pm.
  • Platelets or platelet derivatives as described herein can have cell surface markers.
  • the presence of cell surface markers can be determined using any appropriate method.
  • the presence of cell surface markers can be determined using binding proteins (e.g., antibodies) specific for one or more cell surface markers and flow cytometry' (e.g., as a percent positivity, e.g., using approximately 2.7x105 FDPDs/ pL; and about 4.8 pL of an anti-CD41 antibody, about 3.3 pL of an anti-CD42 antibody, about 1.3 pL of annexin V, or about 2.4 pL of an anti-CD62 antibody).
  • binding proteins e.g., antibodies
  • flow cytometry' e.g., as a percent positivity, e.g., using approximately 2.7x105 FDPDs/ pL; and about 4.8 pL of an anti-CD41 antibody, about 3.3 pL of an anti-CD42 antibody, about 1.3 pL of annexin V,
  • Non-limiting examples of cell-surface markers include CD41 (also called glycoprotein ilb or GPIIb, which can be assayed using e.g., an anti-CD41 antibody), CD42 (which can be assayed using, e.g., an anti-CD42 antibody), CD62 (also called CD62P or P-selectin, which can be assayed using, e.g., an anti-CD62 antibody), phosphatidylserine (which can be assayed using, e.g., annexin V (AV)), and CD47 (which is used in self-recognition; absence of this marker, in some cases, can lead to phagocytosis).
  • CD41 also called glycoprotein ilb or GPIIb
  • CD42 which can be assayed using, e.g., an anti-CD42 antibody
  • CD62 also called CD62P or P-selectin, which can be assayed using, e.g., an anti-
  • the percent positivity of any cell surface marker can be any appropriate percent positivity.
  • platelets or platelet derivatives e.g., FDPDs
  • FDPDs platelet derivatives
  • the percent positivity of any cell surface marker can be any appropriate percent positivity.
  • platelets or platelet derivatives e.g., FDPDs
  • at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.5-2.5 pm in diameter by flow cytometry.
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.4-2.8 pm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 41 have a size in the range of 0.3-3 pm.
  • platelets or platelet derivatives can have an average CD42 percent positivity of at least 65% (e.g., at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%).
  • at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.5-2.5 pm in diameter by flow cytometry.
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.4-2.8 pm. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 42 have a size in the range of 0.3-3 pm.
  • platelets or platelet derivatives such as those prepared by methods described herein, can have an average CD62 percent positivity of at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%).
  • at least 10% e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%).
  • At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 62 have a size in the range of 0.5-2.5 pm in diameter by flow cytometry. In some embodiments, at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 62 have a size in the range of 0.4-2.8 pm.
  • At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for CD 62 have a size in the range of 0.3-3 pm.
  • platelets or platelet derivatives such as those prepared by methods described herein, can have an average annexin V positivity of at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%).
  • At least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% platelet derivatives that are positive for annexin V have a size in the range of 0.5-2.5 pm in diameter by flow cytometry. In some embodiments, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% platelet derivatives that are positive for annexin V have a size in the range of 0.4-2.8 pm.
  • the platelet derivatives as described herein are activated to a maximum extent such that in the presence of an agonist, the platelet derivatives are not able to show an increase in the platelet activation markers on them as compared to the level of the platelet activation markers which were present prior to the exposure with the agonist.
  • the platelet derivatives as described herein show an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of an agonist.
  • the agonist is selected from the group consisting of collagen, epinephrine, ristocetin, arachidonic acid, adenosine di-phosphate, and thrombin receptor associated protein (TRAP).
  • the platelet activation marker is selected from the group consisting of Annexin V, and CD 62.
  • the platelet derivatives as described herein show an inability to increase expression of Annexin V in the presence of TRAP.
  • an increased amount of the platelet activation markers on the platelets indicates the state of activeness of the platelets.
  • the platelet derivatives as described herein are not able to increase the amount of the platelet activation markers on them even in the presence of an agonist. This property indicates that the platelet derivatives as described herein are activated to a maximinn extent. In some embodiments, the property can be beneficial where maximum activation of platelets is required, because the platelet derivatives as described herein is able to show a state of maximum activation in the absence of an agonist.
  • platelets or platelet derivatives e.g., FDPDs
  • FDPDs platelet derivatives
  • Platelet derivatives are dry platelet derivatives, or dry platelet derived particles.
  • dry platelet derivatives are freeze-dried (i.e., lyophilized) platelets or platelet derivatives.
  • dry platelet derivatives are thrombosomes.
  • Dry platelet derivatives are typically in the form of a platelet derivative powder. The dry platelet derivative powder when rehydrated typically form a rehydrated platelet derivative composition comprising particles.
  • compositions comprising a population of platelet derivatives, dry platelet derivatives, platelet derivative powder, or rehydrated platelet derivatives can be characterized by the presence of CD41 on or in at least 55%, 60%, 65% or higher platelet derivatives in the population.
  • compositions comprising a population of platelet derivatives, dry platelet derivatives, platelet derivative powder, or rehydrated platelet derivatives can be characterized by the presence of CD42 on or in at least 55%, 60%, 65% or higher platelet derivatives in the population.
  • dry platelet derivative particles herein can have at least one property selected from: (a) high expression of P-selectin (CD62P), for example, at least 2 fold higher than platelets, for example, apheresis platelets, or at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, or higher platelet derivative particles are positive for CD62; (b) high expression of phosphatidyl serine (PS), for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or higher than the expression on platelets, for example, apheresis platelets, or at least 25%, 30%, 40%, 50%, 60%, 70%, or higher platelet derivative particles are positive for phosphatidyl serine; (c) high expression of von Willebrand Factor (vWF), for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10 fold or higher than the expression on platelets, for example, apheresis platelets; (d) high expression of fibrinogen, for example, at least 2, 3, 4, 5, 6, 7, 8,
  • the method can include an initial dilution step, for example, a starting material (e.g., an unprocessed blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)) can be diluted with a preparation agent (e.g., any of the preparation agents described herein) to form a diluted starting material.
  • a preparation agent e.g., any of the preparation agents described herein
  • the initial dilution step can include dilution with a preparation agent with a mass of preparation agent equal to at least about 10% of the mass of the starting material (e.g., at least about 15%, 25%, 50%, 75%, 100%, 150%, or 200% of the mass of the starting material.
  • an initial dilution step can be carried out using the TFF apparatus.
  • the method can include concentrating (e.g., concentrating platelets) (e.g., concentrating a starting material or a diluted starting material) to form a concentrated platelet composition.
  • concentrated can include concentrating to a about 1000 x 103 to about 4000 x 103 platelets/ pL (e.g., about 1000 x 103 to about 2000 x 103, about 2000 x 103 to about 3000 x 103, or about 4000 x 103 platelets/pL).
  • a concentration step can be carried out using the TFF apparatus.
  • the concentration of platelets or platelet derivatives can be determined by any appropriate method.
  • a counter can be used to quantitate concentration of blood cells in suspension using impedance (e.g., a Beckman Coulter AcT 10 or an AcT diff 2).
  • TFF can include diafiltering (sometimes called “washing”) of a starting material, a diluted starting material, a concentrated platelet composition, or a combination thereof.
  • diafiltering can include washing with at least 2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10, or more) diavolumes.
  • TFF can include buffer exchange.
  • a buffer can be used in TFF.
  • a buffer can be any appropriate buffer, fn some embodiments, the buffer can be a preparation agent (e.g., any of the preparation agents described herein). In some embodiments, the buffer can be the same preparation agent as was used for dilution.
  • a buffer can include a lyophilizing agent, including a buffering agent, a base, a loading agent, optionally a salt, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof.
  • a buffering agent can be any appropriate buffering agent.
  • a buffering agent can be HEPES ((4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid).
  • Abase can be any appropriate base. In some embodiments, a base can be sodium bicarbonate.
  • a saccharide can be a monosaccharide.
  • a loading agent can be a saccharide. In some embodiments, a saccharide can include sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, a monosaccharide can be trehalose. In some embodiments, the loading agent can include polysucrose.
  • a salt can be any appropriate salt. In some embodiments, a salt can be selected from the group consisting of sodium chloride (NaCI), potassium chloride (KC1), or a combination thereof.
  • a membrane with a pore size of about 0.1 pm to about 1 pm (e.g., about 0.1 pm to about 1 pm, about 0.1 pm to about 0.5 pm, about 0.2 to about 0.45 pm, about 0.45 to about 1 pm, about 0.1 pm, about 0.2 pm, about 0.45 pm, about 0.65 pm, or about 1 pm) can be used in TFF.
  • a membrane can be made from any appropriate material.
  • a membrane can be a hydrophilic membrane.
  • a membrane can be a hydrophobic membrane.
  • a membrane with a nominal molecular weight cutoff (NMWCO) of at least about 100 kDa e.g., at least about 200, 300 kDa, 500 kDa, or 1000 kDa
  • the TFF can be performed with any appropriate pore size within the range of 0.1 pm to 1.0 pm with the aim of reducing the microparticles content in the composition and increasing the content of platelet derivatives in the composition.
  • a skilled artisan can appreciate the required optimization of the pore size in order to retain96ehy draff Te‘" derivativ”s “nd allow the microparticle” t“ pas” f ’rough the membrane.
  • the pore”si“e in illustrative embodiments is such that the microparticles pass through the membrane allowing the TFF-treated composition to have less than 5% microparticles.
  • the pore size in illustrative embodiments is such that a maximum of platelet derivatives gets retained in the process allowing the TFF-treated composition to have a concentration of the platelet derivatives in the range of 100 x 103 to 20,000 x 103.
  • the pore size during the TFF process can be exploited to obtain a higher concentration of platelet derivatives in the platelet derivative composition such that a person administering the platelet derivatives to a subject in need has to rehydrate/reconstitute fewer vials, therefore, being efficient with respect to time and effort during the process of preparing such platelet derivatives for a downstream procedure, for example a method of treating provided herein.
  • TFF can be performed at any appropriate temperature.
  • TFF can be performed at a temperature of about 20 °C to about 37 °C (e.g., about 20 °C to about 25 °C, about 20 °C to about 30 °C, about 25 °C to about 30 °C, about 30 °C to about 35 °C, about 30 °C to about 37 °C, about 25 °C to about 35 °C, or about 25 °C to about 37 °C).
  • TFF can be carried out at a flow rate (e.g., a circulating flow rate) of about 100 ml/min to about 800 mFmin (e.g., about 100 to about 200 ml/min, about 100 to about 400 ml/min, about 100 to about 600 ml/min, about 200 to about 400 ml/min, about 200 to about 600 ml/min, about 200 to about 800 ml/min, about 400 to about 600 ml/min, about 400 to about 800 ml/min, about 600 to about 800 ml/min, about 100 ml/min, about 200 ml/min, about 300 ml/min, about 400 ml/min, about 500 ml/min, about 600 ml/min, about 700 ml/min, or about 800 ml/min).
  • a flow rate e.g., a circulating flow rate
  • TFF can be performed until a particular endpoint is reached, forming a TFF-treated composition.
  • An endpoint can be any appropriate endpoint.
  • an endpoint can be a percentage of residual plasma (e.g., less than or equal to about 50%, 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of residual plasma).
  • an endpoint can be a relative absorbance at 280 nm (A280).
  • an endpoint can be an A280 (e.g., using a path length of 0.5 cm) that is less than or equal to about 50% (e.g., less than or equal to about 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the A280 (e.g., using a path length of 0.5 cm) prior to TFF (e.g., of a starting material or of a diluted starting material).
  • an instrument to measure A280 can be configured as follows: a 0.5cm gap flow cell can be attached to the filtrate line of the TFF system.
  • the flow cell can be connected to a photometer with fiber optics cables attached to each side of the flow cell (light source cable and light detector cable).
  • the flow cell can be made with a silica glass lens on each side of the fiber optic cables.
  • the protein concentration in the aqueous medium can also be measured in absolute terms.
  • the protein concentration in the aqueous medium is less than or equal to 15%, or 14%, or 13%, or 12%, or 11%, or 10%, or 9%, or 8%, or 7%, or 6%, or 5%, or 4%, or 3%, or 2%, or
  • the protein concentration is less than 3% or
  • the protein concentration is in the range of 0.01-15%, or 0.1-15%, or 1-
  • an endpoint can be an absolute A280 (e.g., using a path length of 0.5 cm).
  • an endpoint can be an A280 that is less than or equal to 2.50 AU, 2.40 AU, 2.30 AU, 2.20 AU, 2.10 AU, 2.0 AU, 1.90 AU, 1.80 AU, or 1.70 AU (e.g., less than or equal to 1.66, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1,
  • a percentage of residual plasma, a relative A280, or an A280 can be determined based on the aqueous medium of a composition comprising platelets and an aqueous medium. In some embodiments, a percentage of residual plasma can be determined based on a known correlation to an A280.
  • an endpoint can be a platelet concentration, as TFF can include concentration or dilution of a sample (e g., using a preparation agent).
  • an endpoint can be a platelet concentration of at least about 2000 x 103 platelets/pL (e.g., at least about 2050 x 103, 2100 x 103, 2150 x 103, 2200 x 103, 2250 x 103, 2300 x 103, 2350 x 103, 2400 x 103, 2450 x 103, or 2500 x 103 platelets/pL).
  • 2000 x 103 platelets/pL e.g., at least about 2050 x 103, 2100 x 103, 2150 x 103, 2200 x 103, 2250 x 103, 2300 x 103, 2350 x 103, 2400 x 103, 2450 x 103, or 2500 x 103 platelets/pL.
  • an endpoint can be a platelet concentration of about 1000 x 103 to about 2500 platelets/pL (e.g., about 1000 x 103 to about 2000 x 103, about 1500 x 103 to about 2300 x 103, or about 1700 x 1 3 to about 2300 x 103 platelets/pL).
  • an endpoint can be a concentration of platelets in the TFF-treated composition are at least 100 x 103 plate Icts/pL.
  • the platelets or platelet derivatives in the TFF-treated composition is in the range of 100 x 103 - 20,000 x 103 platelets/pL, or 1000 x 103 - 20,000 x 103 platelets/pL, or 1000 x 103 - 10,000 x 103 platelets/pL, or 500 x 103 - 5,000 x 103 platelets/pL, or 1000 x 103 - 5,000 x 103 platelets/pL, or 2000 x 103 - 8,000 x 103 platelets/pL, or 10,000 x 103 - 20,000 x 103 platelets/qL, or 15,000 x 103 - 20,000 x 103 platelets/pL.
  • an endpoint can include more than one criterion (e.g., a percentage of residual plasma and a platelet concentration, a relative A280 and a platelet concentration, or an absolute A280 and a platelet concentration).
  • more than one criterion e.g., a percentage of residual plasma and a platelet concentration, a relative A280 and a platelet concentration, or an absolute A280 and a platelet concentration.
  • a TFF-treated composition is subsequently lyophilized, optionally with a thermal treatment step, to form a final blood product (e g., platelets, cryopreserved platelets, FDPDs.
  • a final blood product e g., platelets, cryopreserved platelets, FDPDs.
  • a TFF-treated composition can be considered to be a final blood product.
  • a final blood product can be prepared using both TFF and centrifugation (e.g., TFF followed by centrifugation or centrifugation followed by TFF).
  • compositions prepared by any of the methods described herein are compositions prepared by any of the methods described herein.
  • a composition as described herein can be analyzed at multiple points during processing.
  • a starting material e.g., donor apheresis material (e.g., pooled donor apheresis material)
  • antibody content e.g., HLA or HNA antibody content
  • a starting material e.g., donor apheresis material (e.g., pooled donor apheresis material)
  • protein concentration e.g., by absorbance at 280 run (e.g., using a path length of 0.5 cm)
  • a composition in an intermediate step of processing e.g., when protein concentration reduced to less than or equal to 75% (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less) of the protein concentration of an unprocessed blood product
  • antibody content e.g., HLA or HNA antibody content
  • the antibody content (e.g., HLA or HNA antibody content) of a blood product in an intermediate step of processing can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of the starting material.
  • a final blood product e.g., (e.g., platelets, cryopreserved platelets, FDPDs can be analyzed for antibody content (e.g., HLA or HNA antibody content).
  • a final blood product can be a composition that includes platelets and an aqueous medium.
  • the antibody content (e.g., HLA or HNA antibody content) of a final blood product e.g., (e.g., platelets, cryopreserved platelets, FDPDs can be at least 5% reduced (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more, reduced) compared to the antibody content of the starting material.
  • a final blood product can have no detectable level of an antibody selected from the group consisting of HLA Class I antibodies, HLA Class II antibodies, and HNA antibodies.
  • the aqueous medium of a composition as described herein can be analyzed as described herein.
  • any individual embodiment recited below or in this full disclosure can be combined with any aspect recited below or in this full disclosure where it is an additional element that can be added to an aspect or because it is a narrower element for an element already present in an aspect.
  • Such combinations are sometimes provided as non-limiting exemplary combinations and/or are discussed more specifically in other sections of this detailed description.
  • a composition comprising MRI agent-loaded cryopreserved platelets, wherein the MRI agent-loaded cryopreserved platelets comprise an MRI agent complex covalently bonded to the surface of the cryopreserved platelets.
  • the MRI agent complex comprises an MRI agent, and a chelator.
  • the MRI agent in the MRI agent-loaded cryopreserved platelets, is associated in a non-covalent bonding with the chelator, and the chelator is covalently bonded to the platelets, typically to protein molecules on the surface of the platelets.
  • the MRI agent-loaded cryopreserved platelets are capable of retaining at least 2%, 5%, 7.5%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40% of the loaded MRI agent upon thawing.
  • a composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the platelet derivatives.
  • the MRI agent complex comprises an MRI agent, and a chelator.
  • the MRI agent-loaded platelet derivatives are surrounded by a compromised plasma membrane.
  • at least 25%, 30%, 35%, 40%, 45%, or 50% of the MRI agent-loaded platelet derivatives are CD 41- positive platelet derivatives.
  • the MRI agent-loaded platelet derivatives are capable of retaining at least 2%, 5%, 7.5%, 10%, 12%, 15%, 20%, 25%, 30%, 35%, 40% of the loaded MRI agent upon rehydrating.
  • the MRI agent in the MRI agent-loaded platelet derivatives, is associated in a non-covalent bonding with the chelator, and the chelator is covalently bonded to the platelets, typically to protein molecules on the surface of the platelets.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets comprising:
  • MRI agent complex that is contacted to the platelets ty pically comprises an MRI agent, a chelator, and a linker.
  • the MRI agent-loaded platelets, or the MRI agent-loaded cryopreserved platelets comprise MRI agent complex covalently bonded to the surface of the MRI agent-loaded platelets, or the MRI agent-loaded cryopreserved platelets.
  • the MRI agent complex that is covalently bonded to the MRI agent-loaded platelets or the MRI agent-loaded cryopreserved platelets comprises the MRI agent, and the chelator.
  • a method for preparing a composition comprising MRI agent-loaded platelet derivatives in a powder comprising:
  • MRI agent complex that is contacted to the platelets typically comprises an MRI agent, a chelator, and a linker.
  • the MRI agent-loaded platelets, or the MRI agent-loaded platelet derivatives comprise MRI agent complex covalently bonded to the surface of the MRI agent-loaded platelets, or the MRI agent-loaded platelet derivatives.
  • the MRI agent complex that is covalently bonded to the MRI agent- loaded platelets or the MRI agent-loaded platelet derivatives comprises the MRI agent, and the chelator.
  • a composition comprising MRI agent-loaded, freeze-dried platelet derivatives, the method comprising:
  • the method further comprises heating the composition comprising MRI agent-loaded, freeze-dried platelet derivatives in a powder form at a temperature in the range of 60°C to 90°C for at least 1 hour to not more than 36 hours to thermally treat the MRI agent-loaded, freeze-dried platelet derivatives.
  • a platelet derivative composition in the form of a powder, comprising a population of MRI-agent loaded platelet derivatives having a reduced propensity to aggregate, such that no more than 25%, and in non-limiting illustrative embodiments, no more than 10%, of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and wherein the platelet derivatives are capable of generating thrombin, and in certain embodiments have a potency of at least 0.5, 1.0, and in non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • a platelet derivative composition in the form of a powder comprising a population of MRI-agent loaded platelet derivatives having a reduced propensity to aggregate, wherein no more than 25%, and in non-limiting illustrative embodiments, no more than 10%, of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets; and having one or more, tw o or more, or all of the following characteristics of a super-activated platelet selected from: a. the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; b.
  • TSP thrombospondin
  • vWF von Willebrand factor
  • illustrative or target platelet derivatives are CD41 positive and/or CD42 positive.
  • Platelets derivatives e.g., thrombosomes
  • dry platelet derivatives or dry platelet derived particles.
  • dry platelet derivatives are typically present in a dried substance that includes other components (e.g.., saccharides such as, for example, trehalose and/or polysucrose) present along with the platelet derivatives when they were dried.
  • less than 5% of the particles are microparticles having a diameter of less than 0.5 pm.
  • at least 90% of the particles therein are at least 0.5 pm in diameter.
  • between 75% and 95% of the platelet derivatives or particles therein are CD41 positive, between 75% and 95% of the platelet derivatives or particles therein are CD42 positive, and less than 5% of the CD 41 -positive platelet derivatives or particles therein are microparticles having a diameter of less than 0.5 pm. It will be understood that in such percent calculations, particles are only intended to cover those that can be detected for example by the instrument (e.g., flow cytometer) used to detect CD41 or CD42 or any surface marker.
  • the platelet derivatives are FPHs. In some examples of such illustrative embodiments, the platelet derivatives have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • FDPD compositions are compositions that include illustrative or target platelet derivatives, wherein at least 50% of the platelet derivatives are CD 41-positive platelet derivatives, wherein less than 15%, 10%, or in further, non-limiting illustrative embodiments less than 5% of the CD 41- positive platelet derivatives are microparticles having a diameter of less than 0.5 pm, and typically such compositions have the ability to generate thrombin in an in vitro thrombin generation assay and/or have the ability to occlude a collagen-coated microchannel in vitro.
  • the platelet derivatives in such compositions have a potency of at least 0.5, 1.0 and in further, non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • the illustrative or target platelet derivatives are between 0.5 and 2.5 pm in diameter by flow cytometry or betw een 0.5 and 25.0 pm in diameter by dynamic light scattering.
  • a platelet derivative composition in the form of a powder comprising a population of MRI -agent loaded platelet derivatives comprising CD 41 -positive platelet derivatives, wherein the population comprises platelet derivatives having a reduced propensity to aggregate such that no more than 25%, and in non-limiting illustrative embodiments, no more than 10%, of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, wherein the platelet derivatives have an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of the agonist, wherein the platelet derivatives are capable of generating thrombin, such that, for example, in illustrative embodiments the platelet derivatives are capable of generating thrombin, such that, for example, the platelet derivatives have a potency of at least 0.5, 1.0, or in non-limiting illustr
  • a platelet derivative composition in the form of a powder comprising a population of MRI -agent loaded platelet derivatives having a reduced propensity to aggregate, such that no more than 25%, and in non-limiting illustrative embodiments, no more than 10%, of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and further having one or both of: the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; and the presence of von Willebrand factor (vWF) on their surface at a level that is greater than on the surface of resting platelets.
  • TSP thrombospondin
  • vWF von Willebrand factor
  • a platelet derivative composition in the form of a powder comprising a population of MRI -agent loaded platelet derivatives comprising CD41 -positive platelet derivatives, wherein less than 15%, and in certain non-limiting illustrative embodiments less than 5% of the CD41-positivc platelet derivatives arc microparticles having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments less than 0.5 pm, and comprising platelet derivatives having one or more of, two or more of, three or more of, and in illustrative embodiments all of the following: a reduced propensity to aggregate, in certain embodiments such that no more than 25%, and in illustrative embodiments no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets; an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation
  • a platelet derivative composition in the form of a powder, comprising trehalose in the range of 20-35% by weight, polysucrose in the range of 45-60% by weight, and MRI-agent loaded platelet derivatives in die range of 0.5-20% by weight, wherein the platelet derivative composition comprises a population of platelet derivatives having a reduced propensity to aggregate such that no more than 25%, and in non-limiting illustrative embodiments, no more than 10%, of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and further having one or both of the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; and the presence of von Willebrand factor (vWF) on their surface at a level that is greater than on the surface of resting platelets.
  • TSP thrombospondin
  • vWF von Willebrand factor
  • a plurality of containers each containing a platelet derivative composition in the form of a powder wherein the platelet derivative composition in each container comprises a population of MRI-agent loaded platelet derivatives having a reduced propensity' to aggregate such that no more than 25%, and in non-limiting illustrative embodiments, no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, wherein the platelet derivative compositions in each container are capable of generating thrombin, such that, for example, in illustrative embodiments the platelet derivatives are capable of generating thrombin, such that, for example, the platelet derivatives have a potency of at least 0.5, 1.0, or in non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives, wherein the platelet derivatives have an inability to increase expression of a platelet activation marker in the presence of an
  • Methods are provided herein for preparing platelet derivative compositions.
  • Illustrative embodiments herein comprise loading platelet derivatives in such compositions with MRI agents either before or after preparing the platelet derivatives.
  • a process for preparing a platelet derivative composition comprising performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% polysucrose, wherein said TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF-treated composition comprising 100 x 10 3 to 20,000 x 10 3 platelets/pl in an aqueous medium having less than or equal to 15% plasma protein, and having less than 15%, and in certain non-limiting illustrative embodiments less than 5.0% microparticles by scattering intensity having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments
  • a process for preparing a platelet derivative composition comprising performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% polysucrose, wherein said TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF- treated composition comprising 100 x 10 3 to 20,000 x 10 3 platelets/ pl in an aqueous medium having less than or equal to 15% plasma protein, and having less than 15%, and in certain non-limiting illustrative embodiments less than 5.0% microparticles by scattering intensity having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments less than 0.5 pm; freeze drying the TFF-treated composition comprising platelets in the aqueous medium to form a freeze-dried composition comprising platelet derivatives; and heating the frcczc-
  • a process for preparing a process for preparing a platelet derivative composition comprising performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% polysucrose, wherein said TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF-treated composition comprising 100 x 10 3 to 20,000 x 10 3 platelets/ql in an aqueous medium having less than or equal to 15% plasma protein, and having less than 15%, and in certain non-limiting illustrative embodiments less than 5.0% microparticles by scattering intensity having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments less than 0.5 pm; freeze drying the TFF-treated composition comprising platelets in the aqueous medium to fonn a freeze-dried composition comprising platelet derivatives
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives, comprising:
  • the composition(s) comprises a population of MRI-agent loaded platelet derivatives having a reduced propensity to aggregate.
  • Illustrative embodiments of exemplary aggregation conditions are provided herein. For example, in illustrative embodiments such aggregation conditions comprise an agonist but no platelets are present in the aggregation conditions.
  • the agonist is selected from the group consisting of collagen, epinephrine, ristocetin, arachidonic acid, adenosine diphosphate, and thrombin receptor associated protein (TRAP).
  • the population of platelet derivatives aggregate in the range of 2-30%, 5-25%, 10-30%, 10-25%, or 12.5-25% of the platelet derivatives under aggregation conditions comprising an agonist but no platelets. It can be contemplated that aggregation conditions involve rehydrating the platelet derivative composition in an appropriate amount of water or an appropriate buffer.
  • a platelet derivative composition in the form of a powder comprising MRI-agent loaded platelet derivatives, wherein less than 15%, and in certain non-limiting illustrative embodiments less than 5% of the CD 41-positive platelet derivatives are microparticles, in non-limiting illustrative embodiments having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments less than 0.5 pm, and wherein the platelet derivatives are capable of generating thrombin, such that, for example, the platelet derivatives have a potency of at least 0.5, 1.0, or in non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • At least 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of the MRI-loaded platelet derivatives in the composition are at least 0.5 pm in diameter by scattering intensity. In some embodiments, at least 25, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99% of the MRI-loadcd platelet derivatives in the composition arc between 0.5 pm and 25 pm in diameter by scattering intensity or between 0.5 pm and 2.5 pm in diameter by flow cytometry.
  • a platelet derivative composition in the form of a powder comprising trehalose in the range of 20-35% by weight, polysucrose in the range of 45-60% by weight, and MRl-agent loaded platelet derivatives in die range of 0.5-20% by weight, wherein the platelet derivatives to microparticles have a numerical ratio of at least 95:1 in the platelet derivative composition, and wherein the platelet derivatives are capable of generating thrombin, such that, for example, in illustrative embodiments the platelet derivatives are capable of generating thrombin, such that, for example, the platelet derivatives have a potency of at least 0.5, 1.0, or in non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • a plurality of containers each filled with a platelet derivative composition in the form of a powder wherein the platelet derivative composition comprises trehalose in the range of 20-35% by weight; polysucrose in the range of 45-60% by weight; and MRI -agent loaded platelet derivatives in the range of 0.5-20% by weight, wherein the platelet derivatives are capable of generating thrombin, such that, for example, in illustrative embodiments the platelet derivatives are capable of generating thrombin, such that, for example, the platelet derivatives have a potency of at least 0.5, 1.0, or in non-limiting illustrative embodiments 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives, and a population of platelet derivatives comprising CD41-positive platelet derivatives, wherein less than 15%, and in certain non-limiting illustrative embodiments less than 5% of the CD41 -positive platelet derivatives are micro
  • the method further comprises thawing the MRI agent-loaded cryopreserved platelets to form thawed MRI agent-loaded platelets, and wherein the thawed MRI agent-loaded platelets retain at least 10% of the loaded MRI agent upon thawing.
  • the MRI agent-loaded platelets retain betw een 10% and 50% of the loaded MRI agent after the thawing.
  • the thawed MRI agent-loaded platelets retain at least 20%, at least 30%, at least 40%, or at least 50% of the loaded MRI agent upon thawing.
  • the thawed MRI agent-loaded platelets are capable of releasing the MRI agent upon stimulation by endogenous platelet activators.
  • the MRI agent-loaded cryopreserved platelets retain at least 10% of the MRI agent loaded on platelets before the step of cryopreserving. In some embodiments, the MRI agent-loaded cryopreserved platelets retain at least 20%, at least 30%, at least 40%, or at least 50% of the MRI agent loaded on platelets before the step of cryopreserving.
  • the method further comprises rehydrating the MRI agent- loaded platelet derivatives in the powder to form rehydrated MRI agent-loaded platelet derivatives wherein the rehydrated MRI agent-loaded platelet derivatives, retain at least 10% of the loaded MRI agent upon thawing.
  • the MRI agent-loaded platelet derivatives in the powder retain at least 20%, at least 30%, at least 40%, or at least 50% of the loaded MRI agent upon rehydrating.
  • the rehydrated MRI agent-loaded platelet derivatives retain the loaded MRI agent upon rehydration.
  • the rehydrated MRI agent-loaded platelet derivatives retain at least 10% of the loaded MRI agent upon rehydration. In some embodiments, the rehydrated MRI agent-loaded platelet derivatives retain between 10% and 50% of the loaded MRI agent upon rehydration. In some embodiments, the rehydrated MRI agent-loaded platelet derivatives retain at least 20%, at least 30%, at least 40% or at least 50% of the loaded MRI agent upon rehydration. In some embodiments, the rehydrated MRI agent-loaded platelet derivatives are capable of releasing the MRI agent upon stimulation by endogenous platelet activators.
  • the rehydrated MRI agent-loaded platelet derivatives retain at least 10% of the MRI agent loaded on platelets before the step of lyophilizing. In some embodiments, the rehydrated MRI agent-loaded platelet derivatives retain at least 20%, at least 30%, at least 40% or at least 50% of the MRI agent-loaded platelets before the step of lyophilizing.
  • the MRI agent-loaded platelets, the MRI agent-loaded cryopreserved platelets, or the MRI agent-loaded platelet derivatives comprise MRI agent complex covalently bonded to the platelets, typically to the surface of the platelets.
  • the MRI agent complex comprises MRI agent.
  • the MRI agent complex comprises MRI agent and another moiety that effectuates the association of the MRI agent with the platelets.
  • the MRI agent is associated with the external surface of the cryopreserved platelets or the platelet derivatives or the platelets.
  • the MRI agent is associated with the surface of tire cryopreserved platelets or the surface of tire platelet derivatives via the chelator.
  • the chelator is covalently attached to the surface of the cryopreserved platelets or the surface of the platelet derivatives, or the surface of the platelets.
  • the linker is covalently bonded to the chelator in the MRI agent complex.
  • the MRI agent is associated with the chelator through an ionic interaction.
  • the MRI agent-loaded platelets, the MRI agent-loaded cryopreserved platelets, or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • the MRI agent- loaded platelets, the MRI agent-loaded cryopreserved platelets, or the MRI agent-loaded platelet derivatives do not comprise a CPP. In some embodiments, the MRI agent-loaded platelets, the MRI agent-loaded cryopreserved platelets, or the MRI agent-loaded platelet derivatives further comprises a drug.
  • the MRI-agent loaded platelet derivatives have a potency of at least 1.25, at least 1.5, at least 1.75, at least 2.0, at least 2.25, at least 2.5 thrombin generation potency units (TGPU) per 10 6 particles.
  • the platelet derivatives have a potency in the range of 1.2 to 2.5, 1.2 to 2.0, 1.3 to 1.5, 1.5 to 2.25, 2 to 2.5, or 2.25 to 2.5 TGPU per 10 6 particles.
  • the MRI-agent loaded platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is more than 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is at least 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of activated platelets, or lyophilized fixed platelets. In some embodiments, the platelet derivatives have the presence of thrombospondin (TSP-1) on their surface at a level that is more than 100% higher than on the surface of activated platelets, or lyophilized fixed platelets.
  • TSP-1 thrombospondin
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, 40 folds, 50 folds, 60 folds, 70 folds, 80 folds, 90 folds, or 100 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the resting platelets.
  • MFI mean fluorescent intensity
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, or 40 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti- TSP antibody to the lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit 10-800 folds, 20-800 folds, 100-700 folds, 150-700 folds, 200-700 folds, or 250-500 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the resting platelets.
  • TSP anti-thrombospondin
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti- thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit at least 2 folds, 5 folds, 7 folds, 10 folds, 20 folds, 30 folds, or 40 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the active platelets.
  • MFI mean fluorescent intensity
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti-thrombospondin (TSP) antibody to the platelet derivatives using flow cytometry exhibit 2-40 folds, 5-40 folds, 5-35 folds, 10-35 folds, or 10-30 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-TSP antibody to the active platelets.
  • TSP anti-thrombospondin
  • the MRI-agent loaded platelet derivatives have the presence of von Willebrand factor (vWF) on their surface at a level that is at least 10%, 20%, 25%, 30%, 50%, 60%, 70%, 80%, 90%, or 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • the platelet derivatives have the presence of von Willebrand factor (vWF) on their surface at a level that is more than 100% higher than on the surface of resting platelets, or lyophilized fixed platelets.
  • the MRI- agent loaded platelet derivatives when analyzed for the binding of anti-von Willebrand factor (vWF) antibody to the platelet derivatives using flow cytometry exhibits at least 1.5 folds, 2 folds, or 3 folds, or 4 folds higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-vWF antibody to the resting platelets, or lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • the MRI-agent loaded platelet derivatives when analyzed for the binding of anti-von Willebrand factor (vWF) antibody to the platelet derivatives using flow cytometry exhibits 2-4 folds, or 2.5-3.5 higher mean fluorescent intensity (MFI) in the absence of an agonist as compared to the MFI of binding of anti-vWF antibody to the resting platelets, or lyophilized fixed platelets.
  • MFI mean fluorescent intensity
  • the MRI-agent loaded platelet derivatives have an inability to increase expression of a platelet activation marker in the presence of
  • the platelet activation marker is selected from the group consisting of Annexin V, and CD 62.
  • the MRI-agent loaded platelet activation marker is Annexin V.
  • the platelet activation marker is CD 62.
  • the agonist is selected from the group consisting of collagen, epinephrine, ristocetin, arachidonic acid, adenosine di-phosphate, and thrombin receptor associated protein (TRAP).
  • any of the aspects and embodiments herein that include a composition or in some compositions used in or formed by a process herein comprises MRI-agent loaded platelet derivatives that are positive for at least one platelet activation marker selected from the group consisting of Annexin V, and CD 62.
  • the MRI-agent loaded platelet derivatives are positive for at least one platelet marker selected from the group consisting of CD 41, CD 42, and CD 61.
  • the MRI-agent loaded platelet derivatives are positive for CD 47.
  • the MRI-agent loaded platelet derivatives are positive for Annexin V.
  • the MRI-agent loaded platelet derivatives are positive for Annexin V. In some embodiments, at least 25%, 50%, or 75% of the MRI-agent loaded platelet derivatives in the platelet derivative composition are Annexin V positive. In some embodiments, the platelet derivatives are positive for CD 41. In some embodiments, at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, or 65% of the MRI-agent loaded platelet derivatives in the platelet derivative composition are CD41 positive.
  • At least 90%, 91%, 92%, 93%, 94%, 95% 96%, 97%, 98/%, or 99% of the MRI- agent loaded platelet derivatives that are positive for CD 41 have a size in the range of 0.5-2.5 pm in diameter by flow cytometry. In some exemplary embodiments, at least 95% of the MRI-agent loaded platelet derivatives that are positive for CD 41 have a size in the range of 0.5-2.5 m in diameter by flow cytometry. In some embodiments, the MRI-agent loaded platelet derivatives are positive for CD 42. In some embodiments, at least 65%, 80%, or 90% of the MRI-agent loaded platelet derivatives in the platelet derivative composition are CD42 positive.
  • the MRI-agent loaded platelet derivatives are positive for CD 47. In some embodiments, at least 8%, 10%, 15%, or 20% of the platelet derivatives in the platelet derivative composition are CD47 positive. In some embodiments, the MRI-agent loaded platelet derivatives are positive for CD 62. In some embodiments, at least 10%, 0%, 80%, or 90% of the MRI-agent loaded platelet derivatives in the platelet derivative composition are CD62 positive. In some embodiments, the MRI-agent loaded platelet derivatives in the platelet derivative composition are positive for CD41, CD62, and Annexin V.
  • the MRI-agent loaded platelet derivatives in the platelet derivative composition are at least 50% platelet derivatives are positive for CD41, at least 70% platelet derivatives are positive for CD62, and at least 70% platelet derivatives are positive for Annexin V. In some embodiments herein, the platelet derivatives are FDHs.
  • the MRI-agent loaded platelet derivatives lack an integrated membrane as compared to platelets.
  • the MRI- agent loaded platelet derivatives are surrounded by a compromised plasma membrane.
  • the MRI-agent loaded platelet derivatives are incapable of retaining more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of lactate dehydrogenase as compared to lactate dehydrogenase retained in fresh platelets, or cold stored platelets, or cryopreserved platelets.
  • the MRI-agent loaded platelet derivatives can retain 35%-75%, 40-70%, 45-65%, or 35-50% lactate dehydrogenase as compared to fresh platelets, or cold stored platelets, or cryopreserved platelets.
  • the MRI-agent loaded platelet derivatives exhibit an increased permeability to antibodies.
  • the antibodies can be IgG antibodies.
  • a process for preparing a platelet derivative composition comprising performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% polysucrose, wherein the TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF- treated composition comprising 100 x 10 3 to 20,000 x 10 3 platelets/ pl in an aqueous medium having less than or equal to 15% plasma protein, and having less than 15%, and in certain non-limiting illustrative embodiments less than 5.0% microparticles by scattering intensity having a diameter of less than 1 pm, and in certain non-limiting illustrative embodiments less than 0.5 pm, freeze drying the TFF-treated composition comprising platelets in the aqueous medium to form a freeze-dried composition comprising platelet derivatives; and heating the freeze-dried composition at
  • the composition comprises less than 10%, 7.5%, 7%, 6%, 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1.0%, 0.75%, 0.5%, 0.25%, or 0.1% (by scattering intensity) microparticles.
  • the composition comprises microparticles (by scattering intensity) in the range of 0.01%-10%, 0.01%-7.5%, 0.01%- 5%, 0.1%-10%, 0.1%-5%, l%-10%, l%-5%, 0.01%-4%, -0.1%-4%, l%-4%, 0. l%-3%, or l%-3%.
  • the microparticles have a diameter less than 1 pm. In illustrative embodiments, the microparticles have a diameter less than 0.5 pm.
  • the microparticles have a diameter in the range of 0.01-0.5 pm, 0.1-0.5 pm, or 0.1-0.49 pm, 0.1-0.47 pm, or 0.1-0.45 pm, or 0.1-0.4 pm, or 0.2-0.49 pm, or 0.25-0.49 pm, or 0.3-0.47 pm. In some embodiments, the diameter of the microparticles is measured using flow cytometry.
  • the platelet derivative composition comprises a population of MRI-agent loaded platelet derivatives comprising CD41 -positive platelet derivatives, wherein less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD41- positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm.
  • the platelet derivative composition comprises a population of MRI-agent loaded platelet derivatives comprising CD42-positive platelet derivatives, wherein less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD42 -positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm.
  • the platelet derivative composition comprises a population of MRI-agent loaded platelet derivatives comprising CD61 -positive platelet derivatives, wherein less than 5%, 4.5%, 4%, 3.5%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the CD61-positive platelet derivatives are microparticles having a diameter of less than 1 pm, 0.9 pm, 0.8 pm, 0.7 pm, 0.6 pm, 0.5 pm, 0.4 pm, 0.3 pm, 0.2 pm, or 0.1 pm. In some illustrative embodiments, the microparticles are having a diameter of less than 0.5 pm.
  • diameter of the microparticles is determined after rehydrating the platelet derivative composition with an appropriate solution.
  • the amount of solution for rehydrating the platelet derivative composition is equal to the amount of buffer or preparation agent present at the step of freeze-drying.
  • the diameter of the microparticles is determined by flow cytometry.
  • the amount of microparticles that are less than 0.5 pm in the powder of any two containers chosen from different lots differs by less than 50%, 40%, 30%, 20%, 10%, 5%, 2%, 1%, or 0.5%.
  • composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives comprise a magnetic resonance imaging (MRI) agent coupled to a cell penetrating peptide (CPP).
  • MRI magnetic resonance imaging
  • CPP cell penetrating peptide
  • composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives comprise an MRI agent coupled to a CPP, and the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a dried powder, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the cryopreserved platelets or the platelet derivatives.
  • composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded platelet derivatives comprise a magnetic resonance imaging (MRI) agent coupled to a cell penetrating peptide (CPP) or the MRI agent-loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the platelet derivatives, and wherein the platelet derivatives are freeze-dried platelet derivatives having one, two, three, or more properties as described herein.
  • MRI magnetic resonance imaging
  • CPP cell penetrating peptide
  • composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded platelet derivatives comprise a magnetic resonance imaging (MRI) agent coupled to a cell penetrating peptide (CPP) or the MRI agent-loaded platelet derivatives comprise an MRI agent complex covalently bonded to the surface of the platelet derivatives, and wherein the MRI agent-loaded platelet derivatives do not comprise a drug, and wherein the platelet derivatives are freeze-dried platelet derivatives having one, two, three, or more properties as described herein.
  • MRI magnetic resonance imaging
  • CPP cell penetrating peptide
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent- loaded platelet derivatives.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives comprising: (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryoprcscrvcd platelets or the rehydrated platelet derivatives with an MRI agent coupled to a cell penetrating peptide, to form the composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives, comprising: (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryopreserved platelets or the rehydrated platelet derivatives with an MRI agent coupled to a cell penetrating peptide, to form the composition comprising MRI agent-loaded cryopreserved platelets and MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives in a powder, comprising: (a) providing platelets; (b) contacting the platelets with an MRI agent complex comprising an MRI agent, a chelator, and a linker, to form MRI agent-loaded platelets; and (c) cry opreserving or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives.
  • the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives comprise MRI agent complex covalently bonded to the cryopreserved platelets or to the platelet derivatives, and the covalently bonded MRI agent complex comprises the MRI agent, and the chelator.
  • a method for preparing a composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives comprising: (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryopreserved platelets or the rehydrated platelet derivatives with an MRI agent complex such that the MRI agent complex comprising an MRI agent, a chelator, and a linker , to form the composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives.
  • a process for preparing a composition comprising MRI agent-loaded platelet derivatives as described in any of the aspects herein, the process comprising: (a) performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% polysucrose, wherein said TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF-treated composition comprising 100 x 10 3 to 20,000 x 10 3 platelets/pl in an aqueous medium having less than or equal to 15% plasma protein, and having less than 5.0% microparticles having a diameter less than 0.5 pm, by scattering intensity; (b) contacting the TFF-treated composition with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic
  • a process for preparing a composition comprising MRI agent-loaded platelet derivatives as described in any of the aspects herein, the process comprising: (a) performing tangential flow filtration (TFF) of a platelet composition with a preparation agent having a pH in the range of 5.5 to 8.0 and comprising 0.4 to 35% trehalose and 2% to 8% poly sucrose, wherein said TFF is performed using a 0.3 to 1 micron filter, thereby preparing a TFF-treated composition comprising 100 x 10 3 to 20,000 x 10 3 platclcts/gl in an aqueous medium having less than or equal to 15% plasma protein, and having less than 5.0% microparticles having a diameter less than 0.5 pm, by scattering intensity; (b) freeze drying the TFF-treated composition of step (a) comprising platelets in the aqueous medium to form a freeze-dried composition comprising platelet derivatives; (c) heating the frcczc
  • TFF tangential flow filtration
  • a method of delivering an MRI agent in a subject comprising administering an effective amount of the composition comprising MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives of any of the aspects or embodiments described herein, or the composition prepared by the process of any of the aspects or embodiments described herein.
  • a method for detecting site of bleeding in a subject comprising: (a) administering an effective amount of the composition comprising MRI agent loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives as described in any of the aspects or embodiments herein, or the composition prepared by the process as described in any of the aspects or embodiments herein, to the subject; and (b) detecting the site of the MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives for detecting the site of bleeding in the subject.
  • a method for detecting an MRI agent well known in the art can be used herein for detecting the site of bleeding in a subject after the administration of MRI agent loaded platelets, MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives.
  • composition comprising MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets, or MRI agent-loaded platelet derivatives as described herein, for use in the treatment of a subject having an indication selected from the group consisting of Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Breast cancer, Gastric cancer, Hodgkin lymphoma, Neuroblastoma, -Non - Hodgkin lymphoma, Ovarian cancer, Cervical cancer, Small cell lung cancer, Non-small cell lung cancer (NSCLC), Soft tissue and bone sarcomas, Thyroid cancer, Transitional cell bladder cancer, Wilms tumor Neuroendocrine tumors, Pancreatic cancer, Multiple myeloma, Renal cancer, Glioblastoma Prostate cancer, Sarcoma, Colon cancer, Melanoma, Colitis, Chronic inflammatory demyelinating polyneuropathy, Guil-a
  • composition comprising MRI agent-loaded platelets, MRI agent-loaded cryopreserved platelets, or MRI agent-loaded platelet derivatives as described herein, for use in the treatment of a subject having an indication selected from the group consisting of Von Willebrand disease, Immune thrombocytopenia, Hermansky Pudlak Syndrome (HPS), Chemotherapy induced thrombocytopenia (CIT), Scott syndrome, Evans syndrome, Hematopoietic Stem Cell Transplantation, Fetal and neonatal alloimmune thrombocytopenia, Bernard Soulier syndrome, Acute myeloid leukemia, Glanzmann thrombasthenia, Myelodysplastic syndrome, Hemorrhagic Shock, Coronary thrombosis (myocardial infarction), Ischemic Stroke, Arterial Thromboembolism, Wiskott Aldrich Syndrome, Venous Thromboembolism, MYH9 related
  • a composition or a process or a method comprising MRI agent-loaded platelets, MRI agent-loaded cryoprcscrvcd platelets, or MRI agent-loaded platelet derivatives, such MRI agent loaded platelets, MRI agent-loaded cryopreserved platelets, or MRI agent-loaded platelet derivatives comprise a CPP that is coupled to an MRI agent.
  • a composition comprising MRI agent-loaded platelets/cry opreserved platelets/platelet derivatives do not comprise a drug.
  • a drug can be any drug known in the art that can be used to treat a subject.
  • a drug can be any drug that is or has been listed as an active ingredient in the FDA Orange Book or Purple Book.
  • a composition comprises MRI agent-loaded platelet derivatives that can be freeze-dried platelet derivatives (FDPDs) as described in any of the aspects or embodiments herein having one, two, three, or more properties of FDPDs as described herein.
  • FDPDs freeze-dried platelet derivatives
  • an MRI agent complex comprises an MRI agent, a linker, and a chelator.
  • an MRI agent complex comprises an MRI agent, and a chelator.
  • a composition comprising MRI agent-loaded platelets/cryopreserved platelets/platelet derivatives having an MRI agent complex covalently bonded to the surface do not comprise a drug.
  • a drug can be any drug known in the art that can be used to treat a subject.
  • a composition comprising MRI agent-loaded platelets/cryopreserved platelets/platelet derivatives having an MRI agent complex covalently bonded to the surface do not comprise a CPP.
  • a composition comprising MRI agent-loaded platelet derivatives having an MRI agent complex covalently bonded to the surface can be freeze-dried platelet derivatives as described in any of the aspects or embodiments herein having one, two, three, or more properties of FDPDs as described herein.
  • a chelator can be a well-known chelator in the art.
  • a chelator is selected from the group consisting of dodecane tetra acetic acid (DOT A), diethylenetriaminepentaacetic acid (DTP A), 4- Carboxy-5,8,l l-tris(carboxymethyl)-l-phenyl-2-oxa- 5,8,1 l-triazatridecan-13-oic acid (BOPTA), Ethylenediaminetetraacetic acid (EDTA), and l,4,7,10-tetraazacyclododecane-l,4,7-tetracetic acid (DO3A).
  • a chelator can be a chelator that is known to suppress any toxic effects of an MRI agent.
  • a linker can be any linker molecule/moiety that can effectuate a covalent bonding to protein(s).
  • a linker can be selected from the group consisting of a compound having sulfhydryl reactive groups, such as maleimides and haloacetyl derivatives, amine reactive groups, such as isothiocyanates, succinimidyl esters, and sulfonyl halides, and carbodiimidc reactive groups, such as carboxyl and amino groups.
  • a linker can be NHS ester.
  • the linker moiety is released after effectuating the bonding between the chelator and the primary amine that typically can come from the proteins present on die surface of platelets/cryopreserved platelets/platelet derivatives.
  • an MRI agent can be any contrast agents known in the art that can facilitate imaging when administered to a subject.
  • an MRI agent is selected from the group consisting of a superparamagnetic contrast agent, a diamagnetic agent, or combinations thereof.
  • the superparamagnetic metal ion is selected from the group consisting of Gd(lll), Fe(lll), Mn(ll and 111), Cr(lll), Cu(ll), Dy(lll), Tb(lll and IV), Ho(lll), Er(III), Pr(III) and Eu(II and III).
  • an MRI agent is selected from the group consisting of metal ions with atomic numbers 21-29, 39-47, or 57-83. In illustrative embodiments, an MRI agent is gadolinium.
  • MRI agent-loaded platelets such as MRI agent-loaded cryopreserved platelets, or MRI agent-loaded platelet derivatives
  • such MRI agent loaded platelets, MRI agent-loaded cryopreserved platelets, or MRI agent-loaded platelet derivatives comprise a CPP that is coupled to an MRI agent, wherein a CPP can be any CPP that is known to get across plasma membrane.
  • a CPP can be a protein-derived CPP, a synthetic CPP, or a mixed CPP.
  • a protein-derived CPPs is selected from the group consisting of Pep-1, penetratin, TAT peptide (49-57 amino acids), TAT peptide (48-60 amino acids), calcitonin-derived CPP, nuclear localization sequences, new polybasic CPPs, N-terminal repetitive domain of maize gamma-zein, peptides from gp41 fiision sequence, preS2-TLM, signal-sequence hydrophobic region (SSHR), pVEC, Vpr, VP22, Human integrin b3 signal sequence, gp41 fusion sequence, Caiman crocodylus Ig(v) light chain, hCT derived peptide, Kaposi FGF signal sequences, CPP from pestivirus envelope glycoprotein, CPP derived from the prion protein, Yeast PRP6 (129-144), Phi21 N (12-29), DeltaN (1-22), FHV coat (35-49), BMV Gag (7-25), HT
  • a CPP can be selected from the group consisting of penetratin, calcitonin-derived CPP, nuclear localization sequences, new poly basic CPPs, N -terminal repetitive domain of maize gamma-zein, peptides from gp41 fusion sequence, preS2-TLM, signal-sequence hydrophobic region (SSHR), pVEC, Vpr, VP22, Human integrin b3 signal sequence, gp41 fusion sequence, Caiman crocodylus Ig(v) light chain, hCT derived peptide, Kaposi FGF signal sequences, CPP from pestivirus envelope glycoprotein, CPP derived from the prion protein, Yeast PRP6 (129-144), Phi21 N (12-29), Delta N (1-22), FHV coat (35-49), BMV Gag (7-25), HTLV-II Rex (4-16), HIV-1 Rev (9-20), RSG-1.2, Lambda-N (48-62),
  • synthetic and mixed CPP is selected from the group consisting of transportan, polyarginine CPPs, poly-d-arginine, KLAL peptide/model amphipathic peptide (MAP), KALA model amphipathic peptide, modeled Tat peptide, Loligomer, b-sheet-forming peptide, retro-inverso forms of established CPPs, W/R penetratin, MPG, Pep-1, Signal-sequence-based peptides (I), Signal-sequence-based peptides (II), Carbamate 9, PTD-4, PTD-5, RSV-A9, CTP-512, and U2AF.
  • transportan polyarginine CPPs
  • poly-d-arginine KLAL peptide/model amphipathic peptide (MAP), KALA model amphipathic peptide
  • modeled Tat peptide Loligomer
  • b-sheet-forming peptide retro-inverso forms of established CPPs
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the composition comprises a population of platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets having a reduced propensity to aggregate such that no more than 10% of the platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets in the population aggregate under aggregation conditions comprising an agonist but no platelets.
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the platelet derivatives freeze-dried platelet derivatives/cryopreserved platelets have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets.
  • TGPU thrombin generation potency units
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the platelet derivatives, frcczc-dricd platelet derivatives/cryopreserved platelets are capable of generating thrombin in an in vitro thrombin generation assay (also referred to herein as a thrombin formation assay).
  • any of the aspects or embodiments that describe a composition comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the platelet derivatives, freeze-dried platelet derivatives/cryopreserved platelets are capable of occluding a collagen coated channel in a T-TAS assay.
  • the MRI agent-loaded cryopreserved platelets are capable of forming aggregates such that when at a concentration of 80 X 10 3 particles/pl, a T-TAS occlusion time of less than 30, 28, 25, or in illustrative embodiments less than 23 minutes is achieved.
  • the MRI agent-loaded platelet derivatives arc capable of forming aggregates such that when at a concentration of at 80 X 10 3 particles/pl, a T-TAS occlusion time of less than 25, 22, 20, 18, or in illustrative embodiments less than 15 minutes is achieved.
  • compositions comprising or a proccss/mcthod forming a population of MRI agent-loaded platelet dcrivativcs/frcczc- dried platelet derivatives/cryopreserved platelets, wherein at least 50% of the platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets are CD 41 -positive platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets, wherein less than 5% of the CD 41 -positive platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets are microparticles having a diameter of less than 0.5 pm.
  • any of the aspects or embodiments that describe a composition comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets have one or more characteristics of a super-activated platelet selected from A) the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; B) the presence of von Willebrand factor (vWF) on their surface at a level that is greater than on the surface of resting platelets; and C) an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of an agonist.
  • TSP thrombospondin
  • vWF von Willebrand factor
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives, wherein MRI agent-loaded platelet derivatives/freeze-dried platelet derivatives have a reduced propensity to aggregate such that no more than 10% of the MRI agent-loaded platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and wherein the platelet derivatives have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives.
  • TGPU thrombin generation potency units
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives, wherein less than 5% of the CD 41 -positive platelet derivatives arc microparticles having a diameter of less than 0.5 pm, and wherein the platelet derivatives have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives or are capable of generating thrombin in an in vitro thrombin generation assay or are capable of occluding a collagen coated channel in a T-TAS assay.
  • TGPU thrombin generation potency units
  • compositions comprising or a proccss/mcthod forming a population of MRI agent-loaded platelet dcrivativcs/frcczc- dried platelet derivatives, wherein the composition comprises a population of platelet derivatives having a reduced propensity to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets; and having one or more characteristics of a super-activated platelet selected from A) the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; B) the presence of von Willebrand factor (vWF) on their surface at a level that is greater than on the surface of resting platelets; and C) an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of an an thrombospondin (TSP) on their surface at a level that is
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives, wherein the composition comprises a population of platelet derivatives having a reduced propensity to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, wherein the platelet derivatives have an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of the agonist, wherein the platelet derivatives have a potency of at least 1.5 thrombin generation potency units (TGPU) per 10 6 platelet derivatives or are capable of generating thrombin in an in vitro thrombin generation assay or are capable of occluding a collagen coated channel in a T-TAS assay; and wherein less than 5% of the CD 41 -positive
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives, wherein the composition comprises a population of platelet derivatives having a reduced propensity to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets, and further having one or both of: the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; and the presence of von Willebrand factor (vWF) on their surface at a level that is greater than on the surface of resting platelets.
  • TSP thrombospondin
  • vWF von Willebrand factor
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives, wherein the composition comprises a population of platelet derivatives comprising CD 41-positive platelet derivatives, wherein less than 5% of the CD 41-positive platelet derivatives arc microparticles having a diameter of less than 0.5 pm, and comprising platelet derivatives having: a reduced propensity to aggregate such that no more than 10% of the platelet derivatives in the population aggregate under aggregation conditions comprising an agonist but no platelets; an inability to increase expression of a platelet activation marker in the presence of an agonist as compared to the expression of the platelet activation marker in the absence of the agonist; the presence of thrombospondin (TSP) on their surface at a level that is greater than on the surface of resting platelets; the presence of von Willebrand factor (vWF) on their surface at a
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs wherein an MRI agent in the MRI agent complex or in MRI agent coupled to a CPP, has a concentration in the range of 2 mM to 100 mM.
  • an MRI agent during any step of preparing as per any of the aspects, or embodiments herein has a concentration of at least 0.1 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, 150 mM, 200 mM, 250 mM, 300 mM, 350 mM, 400 mM, 450 mM, 500 mM, or higher.
  • an MRI agent has a concentration in the range of 0.1 to 500 M, 1 to 400 mM, 20 to 400 mM, 50 to 400 mM, 75 to 350 mM, 1 to 350 mM, 2 to 300 mM, 4 to 250 mM, or 5 to 150 mM.
  • an MRI agent complex has a concentration in the range of 5 to 500 pM.
  • an MRI agent complex has a concentration of at least 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 700 pM, 800 pM, or higher.
  • an MRI agent complex has a concentration in the range of 1 to 500 pM, 5 to 500 pM, 10 to 500 pM, 20 to 400 pM, 25 to 300 pM, 30 to 500 pM, or 35 to 250 pM.
  • CPP has a concentration in the range of 5 pM to 500 pM.
  • CPP has a concentration of at least 1 pM, 5 pM, 10 pM, 50 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 700 pM, 800 pM, or higher.
  • CPP has a concentration in the range of 1 to 500 pM, 5 to 500 pM, 10 to 500 pM, 20 to 400 pM, 25 to 300 pM, 30 to 500 pM, or 35 to 250 pM. In some embodiments, CPP has a concentration in the range of 1 to 500 mM, 5 to 500 mM, 10 to 500 mM, 20 to 400 mM, 25 to 300 mM, 30 to 500 mM, or 35 to 250 mM.
  • any of the aspects or embodiments herein that relate to a proccss/mcthod for forming MRI agent-loaded platclcts/platclct dcrivativcs/cryoprcscrvcd plate lets/FDPDs, wherein platelets has a concentration in the range of 1000 plate lets/pl to 300 X 10 6 plate lets/ pl. In some embodiments, platelets has a concentration of at least 1000, 2000, 3000, 5000, 7500, 10 4 , 5 X 10 4 , or higher.
  • platelets has a concentration in the range of 1000 to 500 X 10 6 , 1500 to 500 X 10 6 , 2000 to 500 X 10 5 , 2000 to 1 X 10 6 , 2000 to 500 X 10 4 , 3000 to 1 X 10 6 , 4000 to 50 X 10 5 , or 5000 to 500 X 10 6 .
  • compositions comprising or a process/method forming a population of MRI agent-loaded platelet derivatives/freeze- dried platelet derivatives/cryopreserved platelets, wherein the MRI agent-loaded platelet derivatives/freeze-dried platelet derivatives/cryopreserved platelets have less than 10% crosslinking of platelet membranes via proteins and/or lipids present on the membranes. In some embodiments, have less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1%. In some embodiments, have crosslinking in the range of 0.1-10%, 0.1-2%, 1-10%, 2-10%, 1-8%, 1-7%, 1-5%, or 1-3%.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs wherein contacting the platelets, the cryopreserved platelets, the platelet derivatives, or the TFF-treated composition with the MRI agent coupled to a cell penetrating peptide or with the MRI agent complex is done at a temperature in the range of 15 to 45°C. In some embodiments, contacting is done at a temperature in the range of 10 to 45°C, 15 to 45°C, 20 to 45°C, 25 to 45°C, 30 to 45 °C, or 32 to 42°C.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs wherein contacting the platelets, the cryopreserved platelets, the platelet derivatives, or the TFF-treated composition with an MRI agent coupled to a cell penetrating peptide or with the MRI agent complex is done for a time period in the range of 5 minutes to 48 hours.
  • time period is in the range of 1 minutes to 72 hours, 5 minutes to 60 horns, 5 minutes to 52 hours, 5 minutes to 45 hours, 10 minutes to 40 hours, 10 minutes to 30 horns, 15 minutes to 25 hours, 15 minutes to 15 hours, 15 minutes to 10 hours, 15 minutes to 5 hours, 20 minutes to 3 hours, 20 minutes to 1 hour, or 20 minutes to 45 minutes.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) contacting platelets with an MRI agent complex in the presence of a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, at a temperature in the range of 15-45°C for a time period in the range of 5 minutes to 48 hours, to form MRI agent- loaded platelets such that the MRI agent complex is covalently linked to the platelets in the MRI agent- loaded platelets; and (b) cryoprcscrving the MRI agent-loaded platelets, to form the composition comprising MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets, to form the composition comprising MRI agent-loaded platelet derivatives.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) providing platelets suspended in a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent; (b) contacting an MRI agent with a conjugate having a chelator conjugated, for example covalently bonded, to a linker, to form an MRI agent complex; (c) contacting the platelets with the MRI agent complex at a temperature in the range of 1 -45 °C for a time period in the range of 5 minutes to 48 hours, to form MRI agent-loaded platelets such that the MRI agent complex is covalently linked to the platelets in the MRI agent-loaded platelets; and (d) cryopreserving the MRI agent-loaded platelets, to form the composition comprising MRI agent-loaded cryopreserved platelets or lyophil
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent- loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising the MRI agent-loaded platelet derivatives.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryoprcscrvcd platelets or the rehydrated platelet derivatives with an MRI agent coupled to a cell penetrating peptide, to form the composition comprising MRI agent-loaded cryopreserved platelets or MRI agent-loaded platelet derivatives.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) providing platelets; (b) contacting the platelets with an MRI agent coupled to a cell penetrating peptide, and a buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form MRI agent-loaded platelets; and (c) cry opreserving the MRI agent- loaded platelets to form the composition comprising the MRI agent-loaded cryopreserved platelets or lyophilizing the MRI agent-loaded platelets to form the composition comprising MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryoprcscrvcd platelets or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • any of the aspects or embodiments herein that relate to a process/method for forming MRI agent-loaded platelets/platelet derivatives/cryopreserved platelets/FDPDs include (a) providing cryopreserved platelets or rehydrated platelet derivatives; and (b) contacting the cryopreserved platelets or the rehydrated platelet derivatives with an MRI agent coupled to a cell penetrating peptide, to form the composition comprising MRI agent-loaded cryopreserved platelets and MRI agent-loaded platelet derivatives, wherein the MRI agent-loaded cryopreserved platelets or the MRI agent-loaded platelet derivatives do not comprise a drug.
  • the effective dose of the MRI agent-loaded platelets or platelet derivatives is at least 10 n /kg, for example, at least 10 12 /kg, 10 13 /kg, 10 14 /kg, 10 15 /kg, or at least 10 16 /kg or more.
  • the effective dose of the platelet derivatives is in the range of 1.0 x 10 7 to 1.0 x 10 ie 7kg of the subject, for example, the effective dose is in the range of 1.0 x 10 7 to 1.0 x 10 15 /kg of the subject, 1.0 x 10 7 to 1.0 x 10 14 /kg, 1.0 xlO 7 to 1.0 x 10 13 /kg, 1.0 x 10 7 to 1.0 x 10 12 /kg, 1.6 x 10 7 to 5.1 x 10 n /kg, 1.6 x 10 7 to 3.0 x 10 n /kg, 1.5 x 10 9 to 1.1 x 10 14 /kg, 3.0 x 10 9 to 1 .0 x 10 13 /kg, 3.0 x 10 9 to 1 .0 x 10 12 /kg, 1 x 10 13 to 1 .0 x 10 I4 /kg, or 5.0 x 10 12 to 1 .0 x 10 14 / kg of the subject.
  • provided herein is a method of enhancing diagnosis and treatment of a disease as disclosed herein, comprising administering MRI agent -loaded platelets, MRI agent- loaded platelet derivatives, or MRI agent-loaded thrombosomes as disclosed herein.
  • a method of treating a disease as disclosed herein comprising administering cold stored, room temperature stored, cryopreserved thawed, rehydrated, and/or lyophilized platelets, platelet derivatives, or thrombosomes as disclosed herein.
  • the disease is cancer.
  • the disease is Traumatic Brain injury.
  • the disease is cancer.
  • the disease is Traumatic Brain injury.
  • the disease is stroke. In some embodiments, the disease is an embolism. In some embodiments, the disease is a hemorrhage.
  • methods of preparing MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, MRI agent-loaded cryopreserved platelets, or MRI agent-loaded thrombosomes comprising: contacting platelets, platelet derivatives, or thrombosomes with an MRI agent, and at least one loading agent and optionally one or more plasticizers such as organic solvents, such as organic solvents selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacet
  • organic solvents such as organic solvents selected from the group consisting of ethanol, acetic acid, acetone,
  • the methods of preparing MRI agent-loaded platelets can include contacting the platelets, the platelet derivatives, and/or the thrombosomes with the MRI agent and with one loading agent. In some embodiments, the methods of preparing MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes can include contacting the platelets, the platelet derivatives, or the thrombosomes with the MRI agent and with multiple loading agents.
  • suitable organic solvents include, but are not limited to alcohols, esters, ketones, ethers, halogenated solvents, hydrocarbons, nitriles, glycols, alkyl nitrates, water or mixtures thereof.
  • suitable organic solvents include, but are not limited to methanol, ethanol, n-propanol, isopropanol, acetic acid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropyl ether (IPE), tertbutyl methyl ether, dioxane (e.g., 1,4-dioxane), acetonitrile, propionitrile, methylene chloride, chloroform, toluene, anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane, dimethylformamide, dimethyl sulfoxide, N-methyl pyrrolidone, dimethylacetamide, and combinations thereof.
  • IPE isopropyl ether
  • dioxane
  • organic solvents such as ethanol
  • the organic solvent may open up and/or increase the flexibility of the plasma membrane of the platelets, platelet derivatives, and/or thrombosomes.
  • a method of preparing MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes comprising: contacting platelets, platelet derivatives, or thrombosomes with a MRI agent, and a loading buffer comprising a base, a loading agent, and optionally at least one organic solvent such as an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof, to form the MRI agent-loaded platelets, the MRI agent-loaded platelet derivatives, or the MRI agent-loaded thrombosomes.
  • a loading buffer comprising a base, a loading agent, and optionally at least one organic solvent such as an organic solvent
  • a method of preparing MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes comprising: contacting platelets, platelet derivatives, or thrombosomes with a MRI agent and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or the MRI agent-loaded thrombosomes.
  • a method of preparing MRI-loaded platelets, MRI- loaded platelet derivatives, or MRI-loaded thrombosomes comprising: contacting platelets, platelet derivatives, or thrombosomes with an MRI and with a loading agent and optionally at least one organic solvent to form the MRI-loaded platelets, the MRI-loaded platelet derivatives, or the MRI- loaded thrombosomes.
  • a method of preparing MRI-loaded platelets, the MRI-loaded platelet derivatives, or the MRI-loaded thrombosomes comprising: contacting the platelets, the platelet derivatives, or the thrombosomes with a MRI in the presence of a buffer comprising a salt, a base, a loading agent, and optionally ethanol, to form the MRI-loaded platelets, the MRI-loaded platelet derivatives, or the MRI-loaded thrombosomes.
  • the methods further include drying the MRI-loaded platelets or the MRI-loaded platelet derivatives. In some embodiments, the methods further include freeze-drying the MRI-loaded platelets or the MRI-loaded platelet derivatives. In such embodiments, the methods further include rehydrating the MRI-loaded platelets or the MRI-loaded platelet derivatives obtained from the drying step.
  • the methods that further include drying the MRI-loaded platelets or the MRI-loaded platelet derivatives and rehydrating the MRI-loaded platelets or the MRI-loaded platelet derivatives obtained from the drying step provides rehydrated platelets or the thrombosomes comprising at least 10% of the amount of the MRI prior to loading.
  • the methods do not comprise contacting platelets, platelet derivatives, or thrombosomes with ethanol.
  • the methods do not comprise contacting platelets, platelet derivatives, or thrombosomes with a solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-mcthyl pyrrolidone, dimcthylacctamidc (DMAC), or combinations thereof.
  • a solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-mcthyl pyrrolidone
  • the methods do not comprise contacting platelets, platelet derivatives, or thrombosomes with an organic solvent.
  • the methods do not comprise contacting platelets, platelet derivatives, or thrombosomes with a solvent.
  • the methods comprise contacting platelets, platelet derivatives, or thrombosomes with a solvent, such as an organic solvent, such as organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof, such as ethanol.
  • a solvent such as an organic solvent, such as organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof, such as
  • platelets, platelet derivatives, or thrombosomes are pooled from a plurality of donors. Such platelets, platelet derivatives, and thrombosomes pooled from a plurality of donors may be also referred herein to as pooled platelets, platelet derivatives, or thrombosomes.
  • the donors are more than 5, such as more than 10, such as more than 20, such as more than 50, such as up to about 100 donors.
  • the donors are from about 5 to about 100, such as from about 10 to about 50, such as from about 20 to about 40, such as from about 25 to about 35.
  • the methods of preparing MRI agent-loaded platelets, MRI agent- loaded platelet derivatives, or MRI agent-loaded thrombosomes that include pooling platelets, platelet derivatives, or thrombosomes from a plurality of donors include a viral inactivation step.
  • the methods of preparing MRI agent-loaded platelets, MRI agent- loaded platelet derivatives, or MRI agent-loaded thrombosomes that include pooling platelets, platelet derivatives, or thrombosomes from a plurality of donors do not include a viral inactivation step.
  • the platelets, the platelet derivatives, or the thrombosomes are loaded with the MRI agent in a period of time of about less than 1 minute to 48 horns, such as 5 minutes to 24 hours, such as 20 minutes to 12 hours, such as 30 minutes to 6 horns, such as 1 hour to 3 hours, such as about 2 hours.
  • platelets, platelet derivatives, or thrombosomes are loaded with the MRI agent for a time of about 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 horn, 2 hours, 3 horns, 4 horns, 5 horns, 6 hours, 7 hours, 8 hours, 9 hours 10 horns, 11 horns, 12 horns, 13 horns, 14 horns, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, or longer, or any time period range therein.
  • platelets, platelet derivatives, or thrombosomes are loaded with the MRI agent for a time of less than one minute.
  • a concentration of MRI agent from about 0.1 nM to about 10 pM, such as about 1 nM to about 1 pM, such as about 10 nM to 10 pM, such as about 100 nM is loaded in a period of time of about less than 1 minute to 48 hours, such as 5 minutes to 24 hours, such as 20 minutes to 12 horns, such as 30 minutes to 6 hours, such as 1 hour minutes to 3 hours, such as about 2 hours.
  • MRI agent-loaded platelets arc MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes prepared according to any of the variety of methods disclosed herein.
  • MRI agent-loaded platelet derivatives prepared according to any of the variety of methods disclosed herein.
  • thrombosomes prepared as according to any of the variety of methods disclosed herein.
  • MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes protect the MRI from metabolic degradation or inactivation.
  • MRI-loaded platelet derivatives, or MRI-loaded thrombosomes may therefore be advantageous in diagnosing diseases such as cancer, since MRI-loaded platelets, MRI-loaded platelet derivatives, or MRI-loaded thrombosomes facilitate targeting of cancer cells while mitigating systemic side effects.
  • MRI-loaded platelets, MRI-loaded platelet derivatives, or MRI-loaded thrombosomes may be used in any therapeutic setting in which expedited healing process is required or advantageous.
  • provided herein is a method of enhancing diagnosis of a disease (e.g., any of the variety of diseases disclosed herein), comprising administering any of the variety of MRI agent-loaded platelets, MRI agent-loaded platelet derivatives, or MRI agent-loaded thrombosomes disclosed herein.
  • a method of diagnosing a disease comprising administering cold stored, room temperature stored, cryopreserved thawed, rehydrated, and/or lyophilized platelets, platelet derivatives, or thrombosomes as disclosed herein.
  • the disease is cancer.
  • the disease is Traumatic Brain injury.
  • the disease is stroke.
  • the disease is an embolism.
  • the disease is a hemorrhage.
  • Embodiment 1 is a method of preparing MRI agent-loaded platelets, comprising: treating platelets with an MRI agent coupled to a cell penetrating peptide; and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent- loaded platelets.
  • Embodiment 2 is a method of preparing MRI agent-loaded platelets, comprising: providing platelets; and treating the platelets with an MRI agent coupled to a cell penetrating peptide; and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the MRI agent-loaded platelets.
  • Embodiment 3 is the method of any one of the preceding embodiments, wherein the platelets are treated with the MRI agent coupled to a cell penetrating peptide and with the loading buffer sequentially, in either order.
  • Embodiment 4 is method of preparing MRI agent-loaded platelets, comprising: treating the platelets with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form a first composition; and treating the first composition with an MRI agent coupled to a cell penetrating peptide, to form the MRI agent-loaded platelets.
  • Embodiment 5 is the method of embodiment 1 or 2, wherein the platelets are treated with the MRI agent coupled to the cell penetrating peptide and with the loading buffer concurrently.
  • Embodiment 6 is a method of preparing MRI agent-loaded platelets, comprising: treating the platelets with an MRI agent in the presence of a cell penetrating peptide and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent to form the MRI agent-loaded platelets.
  • Embodiment 7 is the method of any one of the preceding embodiments, wherein the platelets are pooled from a plurality of donors.
  • Embodiment 8 is a method of preparing MRI agent-loaded platelets comprising (A) pooling platelets from a plurality of donors; and treating the platelets from step (A) with an MRI agent coupled to a cell penetrating peptide; and (B) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent -loaded platelets.
  • Embodiment 9 is a method of preparing MRI agent-loaded platelets comprising (A) pooling platelets from a plurality of donors; and (B) treating the platelets from step (A) with an MRI agent coupled to a cell penetrating peptide to form a first composition; and (C) treating the first composition with a loading bulfer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • Embodiment 10 A method of preparing MRI agent-loaded platelets comprising (A) pooling platelets from a plurality of donors; and (B) treating the platelets from step (A) with a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form a first composition; and (C) treating the first composition with an MRI agent coupled to a cell penetrating peptide to form the MRI agent-loaded platelets.
  • a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent
  • Embodiment 11 is a method of preparing MRI agent-loaded platelets comprising pooling platelets from a plurality of donors; and treating the platelets with an MRI agent coupled to a cell penetrating peptide and a loading buffer comprising a salt, a base, a loading agent, and optionally at least one organic solvent, to form the MRI agent-loaded platelets.
  • Embodiment 12 is the method of any one of the preceding embodiments, wherein the loading buffer comprises optionally at least one organic solvent.
  • Embodiment 13 is the method of any one of the preceding embodiments, wherein the loading agent is a monosaccharide or a disaccharide.
  • Embodiment 14 is the method of any one of the preceding embodiments, wherein the loading agent is sucrose, maltose, dextrose, trehalose, glucose, mannose, or xylose.
  • the loading agent is sucrose, maltose, dextrose, trehalose, glucose, mannose, or xylose.
  • Embodiment 15 is the method of any one of the preceding embodiments, wherein the platelets are isolated prior to a treating step.
  • Embodiment 16 is the method of any one of the preceding embodiments, wherein the platelets are selected from the group consisting of fresh platelets, stored platelet, and any combination thereof.
  • Embodiment 17 is the method of any one of the preceding embodiments, wherein the MRI agent comprises Gadolinium.
  • Embodiment 18 is the method of any one of the preceding embodiments, wherein the MRI agent comprises a nanoparticle.
  • Embodiment 19 is the method of any one of the preceding embodiments, wherein the cell penetrating peptide is Tat, or a portion thereof.
  • Embodiment 20 is the method of any one of the preceding embodiments, wherein the platelets are loaded with the MRI agent in a period of time of 1 minute to 48 hours.
  • Embodiment 21 is the method of any one of the preceding embodiments, wherein the one or more organic solvents selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof.
  • the one or more organic solvents selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methyl pyrrolidone, dimethylacetamide (DMAC), or combinations thereof.
  • Embodiment 22 is the method of any one of the preceding embodiments, further comprising cold storing, cryopreserving, freeze-drying, thawing, rehydrating, and combinations thereof the MRI agent-loaded platelets.
  • Embodiment 23 The method of embodiment 22, wherein the drying step comprises freeze- drying the MRI agent-loaded platelets.
  • Embodiment 24 is the method of embodiment 22 or 23, further comprising rehydrating the MRI agent-loaded platelets obtained from the drying step.
  • Embodiment 25 are MRI agent-loaded platelets prepared by the method of any one of the preceding embodiments.
  • Embodiment 26 are MRI agent-loaded platelets prepared by a method comprising rehydrating the MRI agent-loaded platelets of embodiment 25.
  • Embodiment 27 is the method of any one of the preceding embodiments, wherein the method does not comprise treating the platelets with an organic solvent.
  • Embodiment 28 is the method of any one of embodiments 4, 9, or 10, wherein tire method does not comprise treating the first composition with an organic solvent.
  • Embodiment 29 is the method of any one of the preceding embodiments, wherein the method comprises treating the platelets with Prostaglandin El.
  • Embodiment 30 is the method of any one of the preceding embodiments, wherein the method does not comprise treating the platelets with Prostaglandin EL
  • Embodiment 31 is the method of any one of the preceding embodiments, wherein the method comprises treating the plates with GR144053.
  • Embodiment 32 is the method of any one of the preceding embodiments, wherein the method does not comprise treating the platelets with GR144053.
  • Embodiment 33 is the method of any one of the preceding embodiments, wherein the method comprises treating the platelets with cptifibatidc.
  • Embodiment 34 is the method of any one of the preceding embodiments, wherein the method does not comprise treating the platelets with eptifibatide.
  • subjects who are administered any of the imaging agent-loaded, for example MRI agent-loaded platelets or platelet derivatives herein, are afflicted with one or more of having an indication selected from the group consisting of Acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), Breast cancer, Gastric cancer, Hodgkin lymphoma, Neuroblastoma, Non - Hodgkin lymphoma, Ovarian cancer, Cervical cancer, Small cell lung cancer, Non-small cell lung cancer (NSCLC), Soft tissue and bone sarcomas, Thyroid cancer, Transitional cell bladder cancer, Wilms tumor Neuroendocrine tumors, Pancreatic cancer, Multiple myeloma, Renal cancer, Glioblastoma Prostate cancer, Sarcoma, Colon cancer, Melanoma, Colitis, Chronic inflammatory demyelinating polyneuropathy, Guillain - Barre syndrome, Immune Thrombocytopenia, Kawas
  • subjects who are administered any of the imaging agent-loaded, for example MRI agent-loaded platelets or platelet derivatives herein, are afflicted with one or more of the following: Aneurysm(s), artherosclerosis, cancer, cardiovascular diseases (post - myocardial infarction remodeling, cardiac regeneration, cardiac fibrosis, viral myocarditis, cardiac hypertrophy, pathological cardiac remodeling), genetic disorder(s), metabolic disease(s), neoangiogenesis, ophthalmic conditions (retinal angiogenesis), and pulmonary hypertension.
  • the CPP can comprise, consist essentially of, or consist of any one of SEQ ID NOs: 1 to 87.
  • Protocol 1 Loading platelets with an MRI agent
  • Platelet pool was acidified to pH 6.6-6.8 using 4 pL IM Acid Citrate Dextrose solution per 1 mL pooled platelet rich plasma.
  • Platelet count in solution was obtained using Coulter AcT Diff hematology analyzer.
  • Platelets were isolated via centrifugation at 845 x g for 10 minutes at room temperature, with gentle acceleration and braking.
  • FITC-CPP FITC-TAT
  • FITC-labeled magnetic nanoparticles were prepared.
  • Platelets were resuspended in loading buffer (Table 1) or desired incubation solution at a concentration of 500,000 plate lets/ pL and incubated at 37°C with low frequency agitation on a rocker for up to 3 hours.
  • Platelets were washed with loading buffer (Table 1) 3x to remove unloaded agents (e.g., FITC-TAT, FITC-labeled magnetic nanoparticles) and use the remaining sample for plate reader, flow cytometer, and/or microscope analysis.
  • loading buffer Table 1
  • Platelets were isolated and pooled by centrifugation to adjust concentration that the isolation technology can achieve.
  • the typical concentration is 5 x 10 6 platelets/pl.
  • the platelet medium can be altered to change the proportion of excipients, or to exchange excipients for similar products.
  • the platelet medium is then replaced with a buffer composed of:
  • the platelets were incubated with either 5-100 pM CPP (L-TAT 49-57, See Mishra., R., (2009)) conjugated to FITC and Gd-DOTA or FeCh nanoparticles at about between 1 x IO’ 19 and about 1 x 10' 15 nanoparticles/mL buffer (average particle diameter of about 20-30 nm, labeled with FITC) in the loading buffer for up to 4 hours at 37°C.
  • the loaded platelets are then used for applications which include, but are not limited to, cryopreservation, ly opreservation, and immediate use in therapeutic functional or diagnostic assays.
  • the results for the above formulation are provided herein.
  • ATecan Infinite M200 Pro plate reader was used for quantification of MRI agent loading.
  • ANovocyte flow cytometer was used to determine both MRI agent loading and percent of platelets effectively loaded.
  • An Olympus microscope was used to visualize the loading into platelets.
  • FIG. 1 shows pooled apheresis platelets incubated with FITC-labeled TAT peptide in loading buffer. The platelets were incubated for 15 to 60 minutes at 37°C with low frequency agitation on a rocker. After incubation, the platelet counts were analyzed on an Ac T-Diff hemacytometer. The data show that platelet counts are stable over time up to a concentration of 50 iiM FITC-TAT.
  • FIG. 2A shows the mean fluorescence values for each sample.
  • FIGs. 2B and 2C are histograms are for 50 pM (FIG. 2B) or 25 pM (FIG. 2C) FITC-TAT concentrations during loading, respectively.
  • Negative controls included 0 pM FITC-TAT in both loading buffer (Table 1) and HMTA and 50 pM fluorescein in both loading buffer and HMTA.
  • FIG. 3 shows a flow cytometry histograms of samples incubated with 100 pM FITC-CPP for 60 minutes in the presence of platelet anti-aggregation compounds, including PGE1, GR144053, and eptifibatide.
  • Negative controls included 0 pM FITC-TAT in loading buffer and 50 pM fluorescein in loading buffer. All samples were incubated in loading buffer at 37°C with low frequency agitation on a rocker.
  • FIG. 4 shows the effect of different buffers on FITC-TAT loading into platelets as measured by fluorescence intensity. Minimal fluorescence was detected with HMTA buffer and PBS with 3% dextrose.
  • FIGs. 5A-5C shows brightfield, FITC, and overlaid images of 0 pM FITC-TAT (“Vehicle”) (FIG. 5 A), 100 pM fluorescein (FIG. 5B), and 100 pM FITC-TAT (“FITC-CPP”) (FIG. 5C) incubated for 30 minutes at 37°C with low frequency agitation on a rocker in loading buffer containing the additive indicated herein. Each image is representative. The scale bars are 10 pm. The overlay image in the bottom right corner shows FITC-TAT co-localizing with platelets.
  • FIGs. 7A-7D shows Texas Red (FIG. 7A), brightfield (FIG. 7C), FITC conjugated nanoparticles (FIG. 7B), and overlaid (FIG. 7D) images of samples incubated with nanoparticles (size range between 20-30 nm) labeled with FITC-TAT for 3 hours at 37°C with low frequency agitation on a rocker in loading buffer. Each image is representative. The scale bars are 10 pm.
  • FIG. 8 shows flow cytometry histograms of platelet samples incubated with either loading buffer (left peak) or a 50 pM FITC-CPP-Gd-DOTA solution (right peak) for 30 minutes. The data show that 76% of flow events showed fluorescent signal above background. All platelet samples were incubated in loading buffer at 37°C with low frequency agitation on a rocker.
  • FIGs. 9A-9B show a schematic (FIG. 9A) and magnetic resonance imaging (FIG. 9B).
  • samples 1A, IB, and 2B are negative controls
  • sample 2A includes Gd-DOTA- FITC-CPP with platelets (400K/pL)
  • samples indicated with either 100 mM or 100 pM GdCE are positive controls.
  • Sample 1 A included loading buffer and platelets (400K/pL)
  • sample IB included loading buffer alone
  • sample 2A included loading buffer, Gd-DOTA-CPP-FITC, and platelets (400K/pL)
  • sample 2B included loading buffer and Gd-DOTA-FITC-CPP only (washed) as another negative control.
  • the magnetic resonance imaging data shown in FIG. 9B shows detection in the two positive control samples and also sample 2A which included loading buffer, Gd-DOTA-CPP- FITC, and platelets (400K/pL), thus showing loading of Gd-DOTA-CPP-FITC into platelets.
  • FIG. 10 is a graph showing post-cry opreservation occlusion time of platelets loaded with Gd- DOTA-FITC-CPP with plasma only (negative control) (showm as bottom line), pooled, unloaded platelets (positive control), and Gd-DOTA-FITC-CPP loaded platelets.
  • Gd-DOTA-FITC-CPP loaded platelets had a similar occlusion time to unloaded platelets indicating that such loaded platelets retain hemostatic function.
  • the platelet donor units were initially pooled into a common vessel.
  • the platelets may or may not be initially diluted with an acidified washing buffer (e.g., a control buffer) to reduce platelet activation during processing.
  • the platelets can undergo two processing pathways; either washed with control buffer until a desired residual component is reached (e.g., donor plasma) before being concentrated to a final product concentration or the platelets arc concentrated to a final product concentration before being washed with control buffer until a desired residual component is reached (e.g., donor plasma).
  • the platelet donor units were initially diluted or further diluted 1 : 1 in Buffer A before being loaded onto a TFF machine for further processing. TFF processed platelets are then filled into vials, l ophilized and thermally treated.
  • Buffer A was used for all steps of the TFF process in this Example. The process was carried out at a temperature of 18-24°C.
  • Platelets were initially diluted in Buffer A (1 : 1) and loaded onto the TFF (PendoTECH controller sy stem), which was prepared with a Repligen TFF Cassette (XPM45L01E). The TFF process was performed using a membrane with a pore size of 0.45 pm. The platelets were diluted with an equal weight ( ⁇ 10%) of Buffer A. The platelets were concentrated to about 2250 x 10 3 cells/pL ( ⁇ 250 x 10 3 ) and then washed with approximately 2 diavolumes (DV) of Buffer A. The target plasma percentage was typically less than 15% relative plasma (as determined by plasma protein content). Removal of plasma proteins was monitored through 280 nm UV absorbance against known correlations.
  • TFF Repligen TFF Cassette
  • the cells were either diluted with Buffer A or were concentrated to fall within this range. Under all circumstances whenever the cells are contacted with the buffer A, it was done at a temperature in the range of 18-24°C. For a better clarity, the cells were loaded with the reagents of the buffer A at a temperature in the range of 18-24°C. The cells were typically then frcczc-dricd (lyophilized) and subsequently heated (thermally treated) at 80 °C for 24 hours, thereby forming thrombosomes, but sometimes the cells were used before lyophilization (sometimes called thrombosomes ‘pre-lyo’).
  • the thrombosomes were typically rehydrated with water over 10 minutes at room temperature. In general, the rehydration volume is equal to the volume used to fill each vial of thrombosomes prior to drying.
  • the thrombosomes which were heated (thermally treated) after lyophilization are also referred to as baked thrombosomes. Whereas the thrombosomes which were not heated (thermally treated) after lyophilization are referred to as unbaked thrombosomes.
  • Platelet derivatives are also referred to herein as thrombosomes. It would be clear to a skilled artisan that the thrombosomes which are obtained after lyophilization in the form of a powder can be used for commercial application, like providing the platelet derivative composition (thrombosomes) in dried form in vials to, for example, a medical practitioner who can rehydrate the vials with an appropriate amount of a liquid.
  • samples were drawn at UV readings correlating to about 51% relative plasma volume, about 8.1% relative plasma volume, about 6.0% relative plasma volume, and about 1.3% relative plasma volume. Low volume aliquots were sampled throughout each processing step with the about 6.0% and under samples.
  • FDPDs batch were produced by the TFF method described in Example 2 and assayed for cell surface marker expression or presence or absence using flow cytometry.
  • FDPD samples were rehydrated and diluted 1 :2 in deionized water.
  • a stock of anti-CD41 was diluted by adding 47.6 pL of antibody to 52.4 pL of HMTA.
  • Samples stained with anti-CD41 were made by adding 10 pL of diluted FDPDs to 10 pL HMTA and 10 pL of diluted CD41 antibody.
  • An anti-CD62 master mix was prepared by combining 12 pL anti-CD62 with 23.8 pL anti-CD41 and 64.2 pL of HMTA. An isoty pe control mix was made in the same manner.
  • Samples stained with anti-CD62 were made by adding 10 pL of diluted FDPDs to 20 pL of the anti-CD62 master.
  • the isotypc master mix was used to make isotype control samples in the same manner.
  • An annexin V (AV) master mix was prepared by combining 11.7 pL of AV with 83.3 pL of anti-CD41 and 80 pL of HMTA.
  • Sample stained with AV were made by adding 20 pL of diluted FDPDs containing 50 mM GPRP to 20 pL of HMTA containing 15 mM CaCT and 20 pL of the AV master mix.
  • Negative gating control samples were made in the same manner using HMTA without calcium to prevent AV binding to PS. All samples were incubated at room temperature for 20 minutes. After incubation 1 mL HBS was added to all samples.
  • HBS used to dilute AV test samples contained 5 mM CaCb
  • Anti-CD41 binding was used to identify the population of interest. CD62 and PS expression or presence was assessed by anti- CD62 and AV binding within the CD41 positive population.
  • Glycoprotein lib also known as antigen CD41 expression or presence was assayed using an anti-CD41 antibody (4.8 pL, Beckman Coulter part #IM1416U). The assayed FDPDs demonstrated CD41 positivity (Table 2; Fig. 11)
  • PS Phosphatidylserine
  • AV annexin V
  • P-selectin also called CD62P expression or presence was assayed using an anti-CD62P antibody (2.4 pL, BD Biosciences Cat. No. 550888). The assayed FDPDs demonstrated CD62 positivity (Table 4, Fig. 13)
  • Thrombin generation was measured at 4.8xl0 3 FDPDs/pl in the presence of PRP Reagent containing tissue factor and phospholipids using the below protocol. On average, the Thrombin Peak Height (TPH) for a FDPDs sample was 60.3 nM. Cephalin was used as a positive control. (Table 5;
  • a rehydrated sample of FDPDs was diluted to 7,200 particles per pL based on the flow cytometry particle count using 30% solution of Octaplas in control buffer.
  • sample wells were generated by adding 20 pL of PRP reagent (Diagnostica Stago Catalog No. 86196) and 80 pL of diluted FDPDs.
  • Calibrator wells were generated by adding 20 pL of Thrombin Calibrator reagent (Diagnostica Stago Catalog No. 86197) to 80 pL of diluted FDPDs. The plate was loaded into the plate reader and incubated in the dark at 40°C for 10 minutes.
  • FluCa solution was prepared by adding 40 pL of FluCa substrate (Diagnostica Stago Catalog No. 86197) to 1.6 mL of Fluo -Buffer (Diagnostica Stago Catalog No. 86197) warmed to 37°C and vortexed to mix.
  • the FluCa solution was aspirated in to the dispensing syringe and 20 pL was mechanically dispensed in to each reaction well, bringing the final FDPDs concentration in each well to 4,800 particles per pL and starting the thrombin generation reaction. Thrombin generation was measured via fluorescence in each well over the course of 75 minutes.
  • LTA Light transmission aggregometry
  • FDPDs also referred as “TFF FDPDs”, were produced by the TFF method described in Example 2.
  • Fresh platelets in Platelet Rich Plasma (PRP) were prepared from whole blood collected in acid-citrate-dextrose (ACD) collection tubes (BD Vacutainer ACD Solution A Blood Collection Tubes ref# 364606).
  • Platelet rich plasma (PRP) was prepared by centrifugation of ACD-whole-blood at 180g for 15 minutes at 22°C using a Beckman Coulter Avanti J-15R centrifuge.
  • Platelet poor plasma (PPP) was prepared by centrifugation of ACD-whole-blood at 2000g for 20 minutes at 22°C.
  • TFF FDPDs lyophilized and thermally treated, were prepared using tangential flow filtration as described in Example 2.
  • A30mL vial of FDPDs was rehydrated using 30 mL of cell culture grade water (Coming Cat# 25-055-CI). The vial was incubated at room temperature for a total of 10 minutes. During the 10-minute rehydration period, the vial was gently swirled at 0, 5, and 10 minutes to promote dissolution of the lyophilizate.
  • Fig. 15A PRP samples from a final concentration of 20 pM ADP, 10 pg/mL collagen, 200 pM epinephrine (ADP, collagen, and epinephrine reagents from Helena Laboratories Platelet Aggregation Kit cat. # 5369), 0.5 mg/mL arachidonic acid (Helena Arachidonic Acid Reagent cat.), Img/mL ristocetin (Helena Ristocetin for Aggregation Assays cat.), and 10 pM thrombin receptor activator peptide 6 (TRAP-6) (Sigma Aldrich Cat# T1573-5MG).
  • ADP ADP
  • collagen collagen
  • epinephrine reagents from Helena Laboratories Platelet Aggregation Kit cat. # 5369
  • 0.5 mg/mL arachidonic acid Helena Arachidonic Acid Reagent cat.
  • Img/mL ristocetin Helena Ristocetin
  • PPP, buffer, or buffer with 20% citrated plasma were used as blanks for the PRP, FDPDs, and FDPDs with 20% citrated plasma samples, respectively.
  • 225 pL of FDPDs or PRP sample was reverse pipetted in a test tube containing a stir bar. The test tube was then placed into the aggregometer’s non-stirred incubation well for 1 minute. The sample was then placed into a stirred incubation well for 1 minute. The sample was then placed into the stirred test well and the aggregation test was initiated. After 1-minute of baseline observation the sample was treated with agonist and the aggregation response was recorded. Using the same procedure as the test runs, a negative control of 25 pL buffer was included simultaneously with all runs to determine spontaneous baseline-aggregation responses of all sample groups.
  • ADP Fig. 15C
  • collagen Fig. 15D
  • epinephrine Fig. 15E
  • ristocetin Fig. 15F
  • TRAP-6 Fig.
  • TFF FDPDs did not cause an aggregation response in TFF FDPDs when measured by LTA.
  • TFF FDPDs ‘ response from the aforementioned agonists was equivalent to baseline aggregation values that would be obtained from no agonist or a negative control of buffer.
  • AA arachidonic acid
  • Fig. 15H there was an apparent aggregation response, however after visual inspection of the aggregometry cuvette it was observed that the solution had become visibly clear and aggregates were not observed, indicating that the apparent aggregation response was from lysis of FDPDs and not AA induced aggregation.
  • Example 5 FDPDs are maximally activated - Binding of Annexin V to FDPDs in the presence of TRAP
  • FDPDs prepared using the TFF process and treated with TRAP-6, were tested for the presence of phosphatidylserine (PS), indicative of an activated platelet, on the surface of the FDPDs.
  • PS phosphatidylserine
  • the presence of PS was assessed by analy sis of Annexin V (AV) binding to the FDPDs.
  • AV Annexin V
  • HMTA Albumin buffer
  • T1573-5MG Thrombin Receptor Activating Peptide 6
  • HMTA buffer or TRAP-6 After incubation with HMTA buffer or TRAP-6, the samples were further diluted 1:20 by adding 10 pL of the FDPD sample to 190 pL HMTA.
  • These diluted samples of FDPDs incubated with HMTA and FDPDs incubated with TRAP-6 were both stained in 1.7 mL microcentrifuge tubes as follows: unstained control samples were generated by combining 10 pL of FDPDs and 20 pL HMTA; calcium free control samples were generated by combining 10 pL of FDPDs, 5 pL of Annexin V - ACP (BD Pharmingen Cat# 550475), and 15 pL HMTA; Annexin V (AV) stained test samples were generated by combining 10 pL of FDPDs, 5 pL of AV - ACP ,and 15 pL HMTA supplemented with 9 mM CaC12 (Cellphire RGT-012 Lot#
  • the final concentration of CaC12 in the Abstained test samples was 3 mM. All stained samples for both FDPDs incubated with HMTA and FDPDs incubated with TRAP-6 were generated in triplicate. The samples were incubated at room temperature, protected from light, for 20 minutes.
  • HBS HEPES buffered saline
  • TRAP-6 activity was confirmed by measuring CD62P expression in human apheresis platelets with and without exposure to TRAP-6. Two 475 pL aliquots of apheresis platelets were transferred to two separate 1.7 mL microcentrifuge tubes. Twenty -five microliters of HMTA buffer was added to the sample in the first tube to generate apheresis platelets without TRAP-6. Twenty-five microliters of 400 pM TRAP-6 was added to the second tube to generate FDPDs with TRAP-6. The final concentration of TRAP-6 during incubation was 20 pM. Both tubes were inverted 5 times to mix and incubated at room temperature for 10 minutes.
  • HMTA buffer or TRAP-6 After incubation with HMTA buffer or TRAP-6, the samples were further diluted 1 :20 by adding 10 pL of apheresis platelets to 190 pL HMTA. These diluted samples of apheresis platelets incubated with HMTA and apheresis platelets incubated with TRAP-6 were both stained in 1.7 mL microcentrifuge tubes as follows: unstained control samples were generated by combining 10 pL of apheresis platelets and 20 pL HMTA; Anti-CD62P stained test samples were generated by combining 10 pL of apheresis platelets, 5 pL of anti-CD62P-PE antibody (BD Pharmingen Cat# 550561 Lot# 6322976), and 15 pL HMTA. All stained samples for both apheresis platelets incubated with HMTA and apheresis platelets incubated with TRA
  • phosphate buffered saline (Corning Cat# 21-040-CV1) was added to all samples. One hundred microliters from each sample was transferred to an individual well in a 96 well plate, and the samples were analyzed using an Agilent Quanteon flow cytometer.
  • FDPDs manufactured using the TFF process were incubated with either TRAP-6 or buffer and stained with Annexin V (AV) to determine the relative presence of phosphatidylserine (PS). Apheresis platelets were used to confirm TRAP-6 activity (Fig.
  • FDPDs manufactured using the TFF process, were shown to contain phosphatidylserine (PS) on the membrane as evident by the binding of Annexin V (AV) to the FDPDs.
  • PS phosphatidylserine
  • AV Annexin V
  • TRAP-6 was shown to activate apheresis platelets, as evident by increased CD62P expression, and increased the binding of AV to the activated platelet, it was not the case for the FDPDs.
  • the FDPDs with or without a TRAP-6 incubation exhibited same high level of AV binding, and indicate that TRAP-6 does not promote further surface expression of PS for FDPDs, likely because the FDPDs are maximally activated during the lyophilization and/or rehydration process, and further stimulation/activation is not possible.
  • Example 6 Presence of Thrombospondin (TSP1) on the surface of the FDPDs
  • TSP1 Thrombospondin-1
  • Fresh platelet rich plasma was isolated by centrifuging whole blood collected in acid citrate dextrose (ACD) at 180g for 10 minutes. Isolated PRP was centrifuged again at 823g for an additional 10 minutes. The plasma was then removed and discarded, and the platelet pellet was resuspended in HEPES Modified Tyrode’s Albumin (HMTA) buffer. An aliquot of the resulting washed platelet sample was activated by incubated the platelets at room temperature for 10 minutes in the presence of 2 mM GPRP peptide (BaChem Cat# H-1998.0025), 2 mM CaCT.
  • ACD acid citrate dextrose
  • the diluted samples were analyzed on the Quanteon flow cytometer and the concentrations of the platelets and FDPDs were determined. Based on these concentrations, an aliquot of each FDPDs or fresh platelet sample was diluted to a concentration of 100,000 FDPDs per microliter.
  • Stained samples from each vial of FDPDs and the resting and activated fresh platelets were generated by adding 10 pL of diluted FDPDs or platelets to 20 pL of HMTA containing 4 pg/mL of anti-Thrombospondin-1 (TSP-1) antibody (Santa Cruz Biotech Cat# sc-59887 AF594).
  • Unstained control samples were generated by adding 10 pL of diluted FDPDs or platelets to 20 pL of HMTA. All The samples were incubated at room temperature, protected from light, for 20 minutes. After incubation, 500 pL of PBS was added to all samples. One hundred microliters from each sample were transferred to an individual well in a 96 well plate, and the samples were analyzed using an Agilent Quanteon flow cytometer.
  • Example 7 Presence of von Willebrand factor (vWF) on the surface of the FDPDs
  • Fresh platelet rich plasma (PRP) was isolated by centrifuging whole blood collected in acid citrate dextrose (ACD) at 180g for 10 minutes. Isolated PRP was centrifuged again at 823g for an additional 10 minutes. The plasma was then removed and discarded, and the platelet pellet was resuspended in HEPES Modified Tyrode’s Albumin (HMTA) buffer.
  • ACD acid citrate dextrose
  • the anti-Von Willebrand Factor antibody (Novus Biologicals Cat# NBP2- 54379PE) was diluted by a factor of 10. Stained samples from each vial of FDPDs and the resting and activated fresh platelets were generated by adding 10 pL of diluted FDPDs or platelets to 10 pL of diluted antibody and 10 pL of HMTA. Unstained control samples were generated by adding 10 pL of diluted FDPDs or platelets to 20 pL of HMTA. All The samples were incubated at room temperature, protected from light, for 20 minutes. After incubation, 500 pL of PBS was added to all samples. One hundred microliters from each sample was transferred to an individual well in a 96 well plate, and the samples were analyzed using an Agilent Quanteon flow cytometer.
  • vWF is present on the surface of rehydrated FDPDs, and that the amount of vWF present is greater than that seen on resting platelets.
  • the data suggests that even in the absence of any activation, the FDPDs exhibit properties that is superior to resting platelets and similar to the activated platelets.
  • Example 8 Lyophilized Fixed Platelet and FDPDs Flow Cytometry
  • thrombosomes were prepared using the TFF process as described in Example 2. All vials of thrombosomes were rehydrated using the appropriate amount of cell culture grade water. After water was added, the vials were incubated for 10 minutes at room temperature. Gentle swirling of the vials was performed every 2 minutes during the 10-minute period to promote full dissolution of the cake. Fixed lyophilized platelets (Chrono-Log Corp Cat#299-9-) were rehydrated using Tris buffered saline (TBS) (Chrono-Log Corp Cat# 299-5) according to the manufacturer’s instruction.
  • TBS Tris buffered saline
  • PBS phosphate buffered saline
  • forward scatter also can indicate the membrane integrity of the sample via optical density (i.e., light transmission); a cell with less cytosolic material and a porous membrane would transmit more light (have a lower FSC) than the same cell if intact, despite being the same size.
  • optical density i.e., light transmission
  • Example 2 The FDPDs of Example 2 were studied to determine if FDPDs were permeable to IgGs (150 kDa) by the use of an antibody against a stable intracellular antigen, P-tubulin. Fresh platelets, unbaked FDPDs, and baked FDPDs were fixed and stained with anti- tubulin IgG with and without cell penneabilization. Fresh platelets showed a dramatic increase in IgG binding with permeabilization, whereas both baked and unbaked FDPDs showed no change in response to penneabilization (Table 11).
  • Unstained samples of FDPDs and human apheresis platelets containing 10 6 total cells in HMTA were diluted with 500 pL of PB. One hundred microliters from each sample were transferred to an individual well in a 96 well plate, and the samples were analyzed using an Agilent Quanteon flow cytometer.
  • the plasma membrane of FDPDs is likely damaged by the drying (sublimation) or rehydration processes as freezing in cryopreserved platelets appears to be insufficient to induce severe membrane dysfunction.
  • Example 10 Gadolinium loaded freeze dried platelet derivatives and gadolinium loaded cryopreserved platelets
  • Gadolinium (MRI agent) was loaded onto platelets to create gadolinium loaded freeze dried platelet derivatives (FDPDs) and gadolinium loaded cryopreserved platelets.
  • Two loading mechanism were performed: (1) Using a cell-penetrating peptide - TAT; and (2) covalently binding an MRI agent complex via surface protein conjugation via NSH ester (linker).
  • the TAT peptide that was used herein had a sequence as represented by [K]LRKKRRQRRR (SEQ ID NO: 2).
  • Gadolinium (III) chloride hexahydrate obtained from Sigma Aldrich
  • Ethylenediaminetetraacetic acid obtained from Sigma Aldrich
  • 0.1g of EDTA was rehydrated with 2.69ml of water to create 0. IM stock of EDTA in water.
  • the 0.1M stock of EDTA in water was then diluted in 1:10 in water.
  • Next lOmg of gadolinium chloride was added to achieve a 9mM concentration. The excess was stored at -20 Celsius.
  • the platelet pool plasma (PPP) was aspirated and discarded. The remaining solution was resuspended in a control buffer.
  • the target count was 500 X 10'3/TCI.
  • the solution was divided into 6 groups (target 40-50mLper group). Five groups were loaded with different combinations of MRI agent and/or fluorescent marker and one group was kept unloaded as a control.
  • Pre-Freezing The condenser was turned on and the temperature was set to -40°C before loading the lyophilizer. The lyophilizer was equilibrated in approximately 1-2 hours.
  • Step 1 - Shelf was ramped to -40°C for 0 minutes.
  • Step 2 - Shelf was held at -40°C for 180 minutes.
  • Step 1 - Shelf was ramped to -10°C for 360 minutes (pressure at 0 m Torr)
  • Step 2 - Shelf was held at -10°C for 360 minutes (pressure at 0 m Torr)
  • Step 3 Shelf was ramped to +5 °C for 180 minutes (pressure at 0 m Torr)
  • Step 4 - Shelf was held at +5°C for 360 minutes (pressure at 0 m Torr)
  • Step 5 - Shelf was ramped to +30°C for 300 minutes (pressure at 0 m Torr)
  • Step 1 - Shelf was held at +30°C for 720 minutes (pressure at 0 m Torr)
  • Step 2 - Shelf was held at +30°C for 720 minutes (pressure at 200 m Torr)
  • Step 3 - Shelf was held at +30°C for 9999 minutes (pressure at 0 m Torr)
  • the step 3 of secondary drying was held for a minim um of 1 hour.
  • the FDPD samples were rehydrated with sterile water equivalent to fill volume prior lyophilization, for analysis. For instance, vials were filled with 1ml liquid product were rehydrated with 1ml sterile water. The cryopreserved samples thawed in a 37°C water bath for 8-10 minutes.
  • FIG. 21 is a comparison of the pre-lyo, CPP, and FDPD platelet count for the six sample group using Beckman Coulter Ac - T Diffi2 Hematology Analyzer.
  • FIG. 21 shows both cr opreserved and FDPDs gave a good recovery from the formulated liquid material (pre-lyo) to the reconstituted final product. There were only minor differences in the counts between the different treatment groups, suggesting that none of the treatment groups introduced significant cytotoxicity.
  • FIG. 22 are the flow cytometry measurements of mean fluorescence intensity (MFI) in the FITC channel for all six samples of FDPDs and CPP. Average values are presented with the unloaded product value subtracted out as background. Error bars are the standard deviation of measurements. NovoCyte Quanteon flow cytometer was used to take measurements. Loading of fluorescent markers (NHS-fluorescein, fluorescein, and TAT-FITC-DOTA-GD) was confirmed by flow cytometry (FIG. 22). As expected, the fluorescein without any loading agent gave very low fluorescence.
  • MFI mean fluorescence intensity
  • the NHS-fluorescein that covalently bonds amine groups on proteins gave strong fluorescence; interestingly, the fluorescence was lower in cryopreserved platelets than in FDPDs.
  • the fluorescence intensity from the TAT-FITC-DOTA-GD was low and decreased from cry opreservation to FDPDs.
  • FIG. 23 are the flow cytometry measurements of forward scatter for all six samples of FDPDs and CPP. Average values are presented. Error bars are the standard deviation of measurements. NovoCyte Quanteon flow cytometer was used to take measurements. Forward scatter results did not indicate any significant defects in the cryopreserved platelet or FDPDs (FIG. 23)
  • FIG. 24 is a graph of the Thrombin generation results for all six samples of FDPDs and CPP using the CLARIOstar Plus microplate reader.
  • Thrombin generation potency (TGPU) are equivalent to NIH Units of Thrombin per 1 million particles. Thrombin generation results confirmed that all products maintained hemostatic activity.
  • FIGs. 25 A and 25B are the Total Thrombus System (T-TAS®) results for the six samples of cryopreserved platelet (FIG. 25 A) and six samples of FDPDs (FIG. 25B). The measurements were taken at a platelet concentration of 80k/pl in Octoplas® using a collagen and tissue factor coated AR chip. T-TAS® results confirmed that all products maintained hemostatic activity.

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Abstract

L'invention concerne des plaquettes cryoconservées chargées d'agent d'imagerie et des dérivés de plaquettes, tels que des plaquettes chargées d'agent d'IRM et des dérivés de plaquettes, et des procédés de préparation et d'utilisation de ceux-ci. Dans certains modes de réalisation, des procédés de chargement d'agents d'IRM dans des plaquettes comprennent la mise en contact de plaquettes avec un agent d'IRM et un peptide pénétrant les cellules. Dans certains modes de réalisation, l'invention concerne des procédés de préparation de plaquettes cryoconservées chargées d'agent d'IRM ou de dérivés de plaquettes dans une poudre séchée, les plaquettes cryoconservées chargées d'agent d'IRM ou les dérivés de plaquettes chargés d'agent d'IRM comprenant un complexe d'agent d'IRM lié de manière covalente à la surface des plaquettes cryoconservées ou des dérivés de plaquettes.
PCT/US2023/066904 2022-05-12 2023-05-11 Compositions de plaquettes chargées d'agent d'irm et leurs procédés de préparation et d'utilisation WO2023220694A1 (fr)

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