WO2015176025A9 - Nanoconstructions polymères discoïdes et méthodes d'utilisation dans la théranostique du cancer - Google Patents

Nanoconstructions polymères discoïdes et méthodes d'utilisation dans la théranostique du cancer Download PDF

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WO2015176025A9
WO2015176025A9 PCT/US2015/031226 US2015031226W WO2015176025A9 WO 2015176025 A9 WO2015176025 A9 WO 2015176025A9 US 2015031226 W US2015031226 W US 2015031226W WO 2015176025 A9 WO2015176025 A9 WO 2015176025A9
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dpns
imaging
composition
cancer
particles
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WO2015176025A1 (fr
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Paolo Decuzzi
Jaehong KEY
Santosh AYRAL
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The Methodist Hospital
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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
    • A61K49/001—Preparation for luminescence or biological staining
    • A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
    • A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
    • A61K49/0089—Particulate, powder, adsorbate, bead, sphere
    • A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
    • A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/337—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having four-membered rings, e.g. taxol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • 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
    • A61K49/1818—Nuclear magnetic resonance [NMR] contrast preparations; Magnetic resonance imaging [MRI] contrast preparations characterised by a special physical form, e.g. emulsions, microcapsules, liposomes particles, e.g. uncoated or non-functionalised microparticles or nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/12—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules
    • A61K51/1241—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins
    • A61K51/1244—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by a special physical form, e.g. emulsion, microcapsules, liposomes, characterized by a special physical form, e.g. emulsions, dispersions, microcapsules particles, powders, lyophilizates, adsorbates, e.g. polymers or resins for adsorption or ion-exchange resins microparticles or nanoparticles, e.g. polymeric nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides
    • A61K9/5153—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5146—Organic macromolecular compounds; Dendrimers obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, polyamines, polyanhydrides

Definitions

  • the present invention relates generally to the fields of molecular biology and medicine, and in particular, to the development of polymeric-based theranostic compositions.
  • the invention provides discoidal, polymeric nanoconstructs for delivery of therapeutic and/or diagnostic molecules to cells of interest within the body of a mammal.
  • the present invention overcomes various limitations inherent in the prior art by providing discoidal polymeric nanoconstruct compositions that are useful in a variety of biological and medical applications, including, without limitation, in the delivery of one or more imaging agents, one or more therapeutic agents, or one or more combinations thereof, to selected mammalian cells and, to selected human cancer cells in particular.
  • the disclosed methods of manufacture provide superior advantages over conventional protocols for preparing such delivery agents, particularly with respect to improved control of particle parameters such as size, shape, surface properties, and mechanical stiffness.
  • the invention provides a method for fabrication of these non-spherical, particles, in which multiple particle parameters (e.g., size, shape, surface properties, and mechanical stiffness - i.e., the "4S parameters") over a wide size range.
  • the invention further provides methods for using such particles in a variety of medical modalities, including, without limitation, as systemic (i.e., intravascular) delivery systems to deliver on eor more imaging and/or and therapeutic agents to one or more particular cell types within or about the body of a mammal, and in particular, in a human.
  • systemic i.e., intravascular
  • the inventors have demonstrated unprecedented tumor accumulation (over 10% ID/g tumor, without application of any molecular or magnetic targeting components), and have also demonstrated their use as in vivo contrast enhancement agents in imaging modalities such as PET and magnetic resonance (MR) imaging.
  • MR magnetic resonance
  • the newly-described particles are discoidal, or substantially discoidal, and preferably have either a substantially circular or a substantially rectangular base.
  • the particles have a characteristic size of -1,000 nm, and are composed of poly(lactic acid co-glycolic acid) (PLGA) and polyethylene glycol (PEG).
  • PLGA poly(lactic acid co-glycolic acid)
  • PEG polyethylene glycol
  • the resulting particles may be loaded with a variety of payloads, including, for examples, ultrasmall superparamagnetic iron oxides (USPIOs), superparamagnetic iron oxides (SPIOs), or the like, for use as imaging agents in magnetic resonance imaging (MRI) modalities.
  • USPIOs ultrasmall superparamagnetic iron oxides
  • SPIOs superparamagnetic iron oxides
  • MRI magnetic resonance imaging
  • the disclosed particles may be tagged with one or more suitable isotopes, including, for example, 64 Cu, for use in nuclear imaging modalities.
  • the disclosed DPNs may further include one or more fluorophores, or other imaging agents, to facilitate their use in optical imaging, microscopy, and the like.
  • the particles of the The same particles can be readily loaded with anticancer drugs, such as doxorubicin, vinblastine, carboplatin, cz ' s-platin, carmustine, paclitaxel, docetaxel, curcumin, temozolomide, and so on, for developing theranostic agents.
  • anticancer drugs such as doxorubicin, vinblastine, carboplatin, cz ' s-platin, carmustine, paclitaxel, docetaxel, curcumin, temozolomide, and so on, for developing theranostic agents.
  • these particles can be used for thermal ablation therapy and magnetic dragging/steering.
  • the invention also provides methods for treating, reducing, alleviating, and/or ameliorating one or more symptom of a disease in an animal, particularly one or more cancers in a mammal, such as a human.
  • the invention provides a method that involves, in an overall and general sense, providing to the mammal an amount of a composition that comprises, consists essentially of, or alternatively, consists of, a therapeutically-effective amount of a population of non-spherical, substantially discoidal, nano/microparticles each comprised of a polymer of poly(lactic acid co-glycolic acid) (PLGA) and polyethylene glycol (PEG); one or more of which nano/microparticles comprises a diagnostic agent, an imaging agent, a therapeutic agent, or any combination thereof; for a time effective to treat, reduce, alleviate, and/or ameliorate one or more symptom of the cancer in the mammal.
  • PLGA poly(lactic acid co-glycolic acid)
  • PEG polyethylene glycol
  • the invention also provides a therapeutic kit that includes one or more of the disclosed DPN compositions, one or more pharmaceutical excipients, diluents, vehicles, or buffers, and a set of instructions for using the DPN compositions, either alone, or in combination with one or more additional chemotherapeutic agents for treating one or more mammalian cancers.
  • the invention provides methods for treating, reducing, alleviating, and/or ameliorating one or more symptomsof a disease, disorder, dysfnction, deficit, injury, or trauma, in a mammal, and in a human, in particular.
  • This method involves, in an overall and general sense, providing to the mammal an amount of a composition that comprises, consists essentially of, or alternatively, consists of, a therapeutically-effective amount of a population of non-spherical, substantially discoidal, nano/microparticles each comprised of a polymer of poly(lactic acid co-glycolic acid) (PLGA) and polyethylene glycol (PEG); one or more of which nano/microparticles comprises a diagnostic agent, an imaging agent, a therapeutic agent, or any combination thereof; for a time effective to treat, reduce, alleviate, and/or ameliorate one or more symptoms of the disease, disorder, dysfnction, deficit, injury, or trauma, in the mammal.
  • PLGA poly(lactic acid co-glycolic
  • the invention also provides methods of treating, reducing, alleviating, or ameliorating one or more symptoms of cancer in a mammal, and in a human in particular.
  • Such method generally involves, in an overall and general sense, providing to such a human in need thereof, a chemotherapeutically-effective amount of one or more of the disclosed DPN drug-delivery agents disclosed herein formulated to contain one or more chemotherapeutic agents.
  • the use of such DPN-based theranostics to diagnose and/or treat cancer, or to ameliorate at least one symptom thereof in a human, and the use of such compositions in the manufacture of medicaments for diagnosing and/or treating such cancers are also important aspects of the invention.
  • Another important aspect of the present invention concerns methods for using the disclosed DPN compositions or formulations including them in the preparation of medicaments for treating or ameliorating the symptoms of one or more diseases, dysfunctions, or deficiencies in an animal, such as a vertebrate mammal.
  • Use of the disclosed DPN-based theranostic compositions is also contemplated in therapy and/or treatment of one or more diseases, disorders, dysfunctions, conditions, disabilities, deformities, or deficiencies, and any symptoms thereof.
  • Such use generally involves administration to an animal in need thereof one or more of the disclosed DPN-based theranostic compositions, either alone, or further in combination with one or more additional therapeutic or diagnostic agents, in an amount and for a time sufficient to treat, lessen, or ameliorate one or more of a disease, disorder, dysfunction, condition, disability, deformity, or deficiency in the affected animal, or one or more symptoms thereof, including, without limitation one or more cancers, and particularly, human tumors.
  • compositions including one or more of the disclosed pharmaceutical formulations also form part of the present invention, and particularly those compositions that further include at least a first pharmaceutically acceptable excipient for use in the therapy and/or imaging of one or more diseases, dysfunctions, disorders, or such like, including, without limitation, one or more cancers or tumors of the human body.
  • compositions are also contemplated, particularly in the manufacture of medicaments and methods involving one or more therapeutic (including chemotherapy, phototherapy, laser therapy, etc.) prophylactic (including e.g., vaccines), or diagnostic regimens, (including, without limitation, in diagnostic imaging, such as CT, MRI, PET, ultrasonography, or the like).
  • therapeutic including chemotherapy, phototherapy, laser therapy, etc.
  • prophylactic including e.g., vaccines
  • diagnostic regimens including, without limitation, in diagnostic imaging, such as CT, MRI, PET, ultrasonography, or the like.
  • the pharmaceutical formulations of the present invention may optionally further include one or more additional distinct active ingredients, detection reagents, vehicles, additives, adjuvants, therapeutic agents, radionuclides, gases, or fluorescent labels as may be suitable for administration to an animal.
  • routes of administration are known to and may be selected by those of ordinary skill in the art, and include, without limitation, delivery devices including intramuscular, intravenous, intra-arterial, intrathecal, intracavitary, intraventricular, subcutaneous, or direct injection into an organ, tissue site, or population of cells in the recipient animal.
  • compositions for use in administration to an animal host cell, and to a mammalian host cell in particular are also provided by the invention.
  • the invention provides for formulation of such compositions for use in administration to a human, or to one or more selected human host cells, tissues, organs in situ, or to an in vitro or ex situ culture thereof, for the purpose of diagnosis, imaging, and/or treatment of one or more aberrant cells or tissues within or about the body of the animal, with uses for the treatment of cells being particularly preferred.
  • the present invention also provides for the use of one or more of the disclosed DPN-based theranostic compositions in the manufacture of a medicament for the treatment of one or more mammalian cancers, including, without limitation, in the preparation of one or more therapeutic regimens for the treatment or amelioration of one or more symptoms of solid tumors in humans.
  • the invention also provides methods for providing a therapeutic amount of a DPN-based therapeutic or diagnostic agents to a population of cells or to one or more tissues within the body of a mammal, and particularly, within the body of a human.
  • a method generally includes providing to a mammal in need thereof an effective amount of one or more DPN-based theranostic compositions as disclosed herein for a time effective to provide the desired therapy in the selected cells or tissue within the mammal.
  • the DPN nanoconstructs of the present invention will generally be formulated for systemic and/or localized administration to an animal, or to one or more cells or tissues thereof, and in particular, will be formulated for systemic and/or localized administration to a mammal, or to one or more cells or tissues thereof.
  • the compounds and methods disclosed herein will find particular use in the therapy of cancerous cells or tissues, such as a tumor, within or about the body of a mammal.
  • the present invention also provides for the use of one or more of the disclosed pharmaceutical compositions in the manufacture of a medicament for therapy, and particularly for use in the manufacture of a medicament for treating, and/or ameliorating one or more symptoms of a disease, dysfunction, or disorder in a mammal, and particularly for the treatment and/or amelioration of one or more symptoms of cancer in a human in particular.
  • FIG 1 shows an exemplary fabrication process for DPNs in accordance with one aspect of the present invention.
  • the polymeric paste including the imaging and therapeutic agents, is deposited directly over the silicon template to fill the holes. After drying and cleaning the silicon surface, a PVA back-layer is deposited on the wafer and it is used to peel off the particles. The particles loosely attached to the PVA template are collected via mechanical steering in warm water and centrifugation;
  • FIG. 2A and FIG. 2B show a comparison of the novel fabrication scheme (FIG. 2A) compared to that of prior art methods (see, e.g., Park et ah, U.S. Pat. Appl. Publ. No. 2009/0136583; specifically incorporated herein in its entirety by express reference thereto) (FIG. 2B).
  • the fabrication protocols disclosed herein do not require intermediate PDMS and/or PVA templates, thus leading to i) less material being used; ii) shorter times and lower costs for synthesis of the nanoconstructs; iii) precise control of the nanoconstructs' shape and size (the silicon template does not deform under thermal and mechanical forces needed to spread the polymeric paste within the wells, whereas intermediate PDMS and PVA templates are susceptible to large deformation over time and during storage thus affecting the quality of the nanoconstructs); iv) faster release of the nanoconstructs (only a thin back-layer of PVA must be dissolved as compared to a thick PVA sacrificial template) and higher yielding; v) less payload lost during particle collection (because of the faster release less drug molecules or contrast agents are released from the nanoconstructs during the dissolution of the thin back-layer); In the new fabrication method, a mixture of PVA and Ethanol is utilized to generate a thin back-layer and allow for the effective peeling of the deposited polymeric
  • FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D show the geometrical characterization for DPNs.
  • FIG. 3A shows SEM images of the 1,000 x 500 nm DPNs made out of PLGA/PEG adhering to the PVA back-layer. These particles are loaded with a red fluorescent dye, Pvhodamine B.
  • FIG. 3B shows fluorescent microscopy image of the 1,000 x 500 nm DPNs sitting on the PVA back-layer.
  • FIG. 3C shows bright- field images of 1,000 x 500 nm DPNs with a rectangular cross section (1,000 x 500 nm) sitting on the PVA back-layer.
  • FIG. 3D shows fluorescence microscopy image ofs the 1,000 x 500 nm DPNs with, a rectangular cross section, sitting on the PVA back-layer;
  • FIG. 4A, FIG. 4B, and FIG. 4C illustrate the geometrical characterization for DPNs.
  • FIG. 4 A shows a bright- field image of the detached PVA back-layer for the 1,000 x 500 nm DPNs with a rectangular cross section.
  • FIG. 4B shows fluorescent microscopy image of the 1,000 x 500 nm DPNs sitting on the PVA back-layer.
  • FIG. 4C shows fluorescent microscopy image of the 1,000 x 500 nm DPNs released from the PVA back-layer;
  • FIG. 5 shows the facility of DPNs as enhanced T2 contrast agents.
  • the table shows the enhanced r2 relaxivity.
  • DPNs show 33.4-fold of r2 value and 27.8 fold of r2/rl .
  • Different number of DPNs was suspended in water and filled in each well, respectively (bottom).
  • the 96-well plates were imaged in a 3-Tesla (3T) magnetic resonance (MR) imaging (MRI) device with T2-weighted MR pulse sequences: TR 2500 msec, TE 50-300 msec, and DFOV 80 mm. Darker MR intensities were observed in higher number of particles, showing the T2 contrast effect by the particles;
  • FIG. 6A and FIG. 6B show 3T MRI phantom image of DPNs.
  • FIG. 6A is a Tl -weighted image (Tl W) was utilized by 5000 msec repetition time, 7.6 msec echo time, and 900 msec inversion time. In T1W, DPNs showed brighter signal than water.
  • FIG. 6B shows a T2-weighted image (T2W), in which 3000 msec repetition time and 120 msec echo time were utilized. DPNs showed a darker signal than water. This result demonstrated the capability of DPNs as a MR contrast agent, shortening relaxation time of water in both Tl and T2 mode;
  • FIG. 7 A and FIG. 7B show the magnetic performance of exemplary DPNs prepared in accordance with one aspect of the present invention. These exemplary DPNs provided the best performance and significantly enhanced the MRI response.
  • FIG. 7A and FIG. 7B show T2- weighted MR images of a mouse bearing a breast tumor on the right flank before, and after, intratumoral injection of USPIO-loaded DPNs. The injection point is delimited by white dotted squares, and the site view is magnified in the lateral inset demonstrating a significant darkening of the region of interest upon DPNs injection;
  • FIG. 8A and FIG. 8B show TEM images of DPNs that were -1,000 nm in average diameter (FIG. 8A) and -500 nm in average height (FIG. 8B). Also clearly visible are clusters of USPIOs inside the DPNs. Scale bars are 200 nm;
  • FIG. 9A, FIG. 9B, and FIG. 9C show the in vitro magnetic guidance experiments.
  • a micro fluidic system was used comprising a commercially-available, parallel-plate, flow chamber (Glycotech, Rockville, MD, USA), mounted on a 35-mm cover slip; a syringe pump (Harvard Apparatus, Boston, MA, USA) and an epi- fluorescence-inverted microscope (Nikon Ti-Eclipse).
  • the solution of USPIO-loaded nanoconstructs (10 7 /mL) was infused in the system with a shear rate of 25/sec (SiMPs) and 10/sec (DPNs).
  • the magnetic guidance was performed by placing a discoidal magnet (D401-N52, K&J Magnetics, Inc., Pipersville, PA, USA) under the cover slip, before the flow was started. Movies were taken during the experiments focusing on different regions of interest up to -1,000 ⁇ away from the magnet. Inflow and drifting velocity have been calculated via offline analysis of the x- and y-displacement of particles within the selected time interval;
  • FIG. 10 shows the stiffness measurement of DPNs using atomic force microscopy (AFM).
  • AFM atomic force microscopy
  • the stiffness of DPNs was measured under the following conditions: contact mode and force parameters (sens. Delfsens: 45.0 nm/V, spring constant: 0.0124 N/m, tip radius: 10.0 nm, tip half-angle: 0.314, sample Poisson's ratio: 0.5).
  • contact mode and force parameters as compared using the Young's modulus, DPNs demonstrated a very flexible and spongy-like stiffness, which was similar to that of mammalian cell membranes;
  • FIG. 11A and FIG. 11B show the different internalization effects of DPN and polystyrene.
  • FIG. 12A, FIG. 12B, FIG. 12C, FIG. 12D, FIG. 12E, and FIG. 12F show that there was less internalization of DPNs in J774.
  • DPNs were incubated with J774 macrophages for four hrs. Fluorescent live imaging was recorded during which DPNs were observed to move around macrophages, and even collect on the cell membranes, but the DPNs did not internalize into the macrophages.
  • the white arrows indicate the direction of movement of the DPN;
  • FIG. 13A, FIG. 13B, FIG. 13C, FIG. 13D, FIG. 13E and FIG. 13F show live imaging of liver blood vessels using intravital microscopy (IVM).
  • IVM intravital microscopy
  • 500 million DPNs were injected via the eyes of a mouse under anesthesia.
  • White arrows indicate the location of each DPN at a given time -point.
  • IVM imaging was taken continuously for about two hrs. During the period, only a few DPNs were observed in liver blood vessels, and these did not stay at the same location over time, indicating that the DPNs might move slowly and they were not uptaken by Kupffer cells;
  • FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G, FIG. 14H, and FIG. 141 show PET-CT imaging in tumor-bearing mice.
  • 500 million 64 Cu-labeled DPNs were injected via the tail vein, and then imaged over time using a microPET/CT system.
  • most DPNs were observed in the lungs as expected for all systemically injected and circulating agents. As the lungs cleared, however, more and more DPNs accumulated in the tumor;
  • FIG. 15A and FIG. 15B show the biodistribution of exemplary DPNs. 20-hrs' post-injection of 64 Cu-labeled DPNs, mice were sacrificed, the organs explanted, and the residual radioactivity was measured using a gamma camera to precisely assess the organ biodistribution of the DPNS;
  • FIG. 16 A, FIG. 16B, FIG. 16C, and FIG. 16D show the release curves and cytotoxicity of the discoidal polymeric nanoconstructs (DPNs).
  • RhB hydrophilic Pvhodamine B
  • the SEM micrographs in the insets document the progressive degradation of the DPNs under acidic conditions.
  • FIG. 16B show the cytotoxicity on HeLa cells exposed at different concentrations of DPNs for 24 hrs. No significant reduction in cell viability was measured via an XTT assay.
  • FIG. 16C shows HeLa cells incubated with RhB-loaded DPNs for four hrs.
  • RhB was released from the DP s and diffused into the HeLa cells coloring their cytoplasm around the blue stained nucleus. Most of the DPNs were washed away after four hrs, and very few were observed inside the HeLa cells.
  • FIG. 16D shows a control experiment with HeLa cells not incubated with DPNs;
  • FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D show the release curves and cytotoxicity of the doxorubicin loaded discoidal polymeric nanoconstructs (DPNs).
  • FIG. 17B shows fluorescent imaging that demonstrated DPNs around the cells, and showed fragments of doxorubicin released out of the DPNs.
  • FIG. 17C and FIG. 17D Cytotoxicity on SKBR3 cells exposed at different concentrations of DPNs for 24 and 48 hrs.
  • FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18F show DPNs are loaded with two different drug molecules, Doxorubicin (DOX) and Curcumin (CURC), and incubated with breast cancer cell (MDA-MD-231).
  • FIG. 18A, FIG. 18B, and FIG. 18C, respectively, show: (PI) the cell nuclei (blue); (CURC) the curcumin released in the cell cytosol (small green dots) and the curcumin still loaded into the DPNs (large green dots); (DOX) doxorubicin released in the cell (small red dots) and the doxorubicin still loaded into the DPNs (large red dots);
  • FIG. 18A, FIG. 18B, FIG. 18C, FIG. 18D, FIG. 18E, and FIG. 18F show DPNs are loaded with two different drug molecules, Doxorubicin (DOX) and Curcumin (CURC), and incubated with breast
  • FIG. 18D, FIG. 18E, and FIG. 18F respectively, show: (PI+CURC+DOX) the overlap of the previous three images; (BF+PI+CURC+DOX) the overlap of the three previous four images; (DPN) DPNs appear red (loaded with DOX), green (loaded with CURC); and yellow (co-loaded with DOX and CURC);
  • FIG. 19A, FIG. 19B, FIG. 19C, FIG. 19D, and FIG. 19E show the loading characterization and quantification for curcumin and doxorubicin in DPNs.
  • FIG. 19A and FTG. 19B show the calibration curves for assessing the different loaded amounts of DOX and CURC as a function of the fluorescence intensity (FIG. 19C and FIG. 19D).
  • FIG. 19E UV absorbance spectra showing that the two maxima, for CURC and DOX, are well separated. HPLC was used to confirm the UV absorbance data;
  • FIG. 20 shows the in vitro efficacy of Doxorubicin/Curcumin at 1 : 1 ratio on MCF7 cells.
  • the IC50 for DOX/CURC combined in the same particle (purple dotted line - DOX CURC (1:1)) is 50% lower than the IC 50 for the free DOX and free CURC mixed together in the same solution (green, dashed line - free DOX/CURC (1 :1)).
  • Higher efficacy i.e., lower IC 5 0
  • DOX/CURC i.e., 1 :5, 1 :10 and 1 :20
  • FIG. 21A, FIG. 21B, FIG. 21C, FIG. 21D, and FIG. 2 IE show the preparation steps and constituents of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • the facile preparation of DPNs follows four sequential steps: FIG. 21A, FIG. 2 IB: Filling the cylindrical wells (1000 x 400 nm) in a silicon template with a polymer paste, obtained by intimately mixing together poly(lactic-co- glycolic acid) (PLGA); polyethylene glycol-diacr late (PEG-diacrylate); Lipid-DOTA; Lipid- Rhodamine B; and 20-nm iron oxide nanocubes (NCs).
  • PLGA poly(lactic-co- glycolic acid)
  • PEG-diacrylate polyethylene glycol-diacr late
  • Lipid-DOTA Lipid- Rhodamine B
  • NCs 20-nm iron oxide nanocubes
  • FIG. 21C Forming a hydrophilic, thin PVA film on top of the silicon template and extracting DPNs from the silicon template via peeling off the PVA film.
  • FIG. 21D Dissolving the PVA film in water and efficiently collecting DPNs by centrifugation, magnetic dragging, and filtration.
  • FIG. 21E For PET/CT imaging, the purified DPNs are reacted with Cu salts to form 64 Cu-DOTA molecules stably entangled with the polymer matrix;
  • FIG. 22A, FIG. 22B, FIG. 22C, FIG. 22D, FIG. 22E, FIG. 22F, FIG. 22G, and FIG. 22H show the physico-chemical properties of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • FIG. 22A a SEM image of DPNs on a thin PVA film is shown, before separation, revealing a characteristic average diameter of ⁇ 1,000 nm. A single DPN is shown in the top-right inset.
  • FIG. 22B shows an atomic force microscopy (AFM) image of DPNs confirming their discoidal shape and size.
  • FIG. 22C shows the fluorescent microscopy image of an aqueous solution of DPNs.
  • AFM atomic force microscopy
  • FIG. 22D shows the distribution of the DPN hydrodynamic diameter as measured by a multi-sizer instrument presenting a peak around 866 nm.
  • FIG. 22E is an AFM image of a DPN showing multiple locations (white crosses) for the indentation experiments.
  • FIG. 22F shows force-displacement curves for DPNs interpreted within the Hertz theory for extracting the mechanical stiffness (Young's Modulus of ⁇ 1.3 kPa).
  • FIG. 22G shows TEM image showing 20-nm iron oxide nanocubes loaded in the DPN polymer matrix.
  • FIG. 22H shows a magnified view of the DPN inner core clearly documenting the size and shape of the iron oxide nanocubes;
  • FIG. 23A, FIG. 23B, and FIG. 23C show PET/CT imaging and circulation half-life of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • FIG. 23A shows the transversal (top) and coronal (bottom) PET/CT images of a U87-MG tumor-bearing mouse acquired at 1, 6, 24, and 48 hrs p.i. of radiolabeled DPNs.
  • the PET images are converted to percentage injected dose per gram (%ID/g) and decay-corrected to the time of scanning.
  • %ID/g percentage injected dose per gram
  • FIG.23B shows the quantification of DPN accumulation in U87-MG and B16-10 tumor bearing mice, expressed in terms of percentage injected dose per gram tissue (%ID/g).
  • FIG.23C shows the DPN concentration in blood measured via scintillation counter, at different time points (0, 2, 7, 24, 48 hrs' .
  • FIG. 24A, FIG. 24B, FIG. 24C, FIG. 24D, FIG. 24E, FIG. 24F, FIG. 24G, and FIG. 24H show intravital microscopy (IVM) analysis of liver and tumor accumulation of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • IVM intravital microscopy
  • FIG. 24A is a representative image showing, immediately p.i., several moving DPNs within the complex liver microvasculature, and a few not moving DPNs (dashed white ovals around red spots).
  • FIG. 24B is a representative image demonstrating that most not-moving DPNs are associated with macrophages in the liver (Kupffer cells).
  • FIG. 24C is a representative image showing, at 2-hrs' p.i., several moving DPNs and a few not-moving DPNs (dashed white ovals around red spots), similar to the immediately p.i. image.
  • FIG. 24D is a representative image of the liver microvasculature, immediately post systemic injection of green fluorescent 750-nm spherical polystyrene beads (PS) showing massive colocalization with Kupffer cells. The PS almost immediately accumulates in the liver without any further movement.
  • FIG. 24E shows the quantification of DPN and PS accumulation in the liver and spleen tissues respectively at -2 hr and ⁇ 2 min p. i.
  • FIG. 24F is a representative image showing, 2-his' p.L, several DPNs firmly accumulating within the tumor microvasculature, supporting the PKT/CT data.
  • FIG. 24G is a representative image demonstrating that only very few not-moving DPNs are associated with macrophages (dashed white ovals around red spots), in the tumor microvasculature. (Moving RBCs appear dashed white ovals around blue spots).
  • FIG. 25A, FIG. 25B, FIG. 25C, and FIG. 25D show the analysis of macrophages interaction with DPNs.
  • DPNs were incubated with J-774 murine macrophages.
  • FIG. 25A shows the trajectories of the centers of J-774 macrophages (dashed line) and DPNs (solid line), as derived from imaging post-processing of a live-cell microscopy movie, over a 2-hr incubation period.
  • Four representative snapshots of the full-length movie are presented on the right. DPNs are observed to move on the surface and near the macrophages without triggering any internalization stimulus, (the macrophage nuclei were stained with Hoechst 33342 dye).
  • FIG. 25B is a representative fluorescent microscopy image of J-774 macrophages co-incubated with DPNs (red spots) and 750 nra spherical polystyrene beads (PS green spots).
  • FIG. 25C the bar chart quantifies the number of DPNs and PS per macrophage, as from the analysis of the fluorescent microscopy images. PS beads are much more associated to macrophages than DPNs.
  • FIG. 25D shows the flow-cytometry analysis of J-774 uptake of DPNs (red) and PS (green curve), as compared to control (gray curve). This analysis confirms again the much higher macrophage uptake of PS over DPNs;
  • FIG. 26A, FIG. 26B, FIG. 26C, FIG. 26D, and FIG. 26E show the toxicity analysis of DPNs.
  • FIG. 26A shows the cell proliferation assays performed on human umbilical vascular endothelial cells (HUVECs) showing moderate cytotoxic effects only at the highest doses (50 DPNs: l HUVEC) and longer incubation time points (48 hrs).
  • FIG. 26B shows the blood levels of serum cytokines, namely IL-6, lL-10, and TNF-a, showing no significant difference between control and DPN injected animals, at 2 and 24 hrs' p.i.
  • FIG. 26C, FIG. 26D, and FIG. 26E show serum levels of ALT (alanine transaminase), AST (aspartate aminotransferase) and creatinine showing no significant difference between control and DPN injected animals, at 24-hrs' p.i;
  • FIG. 27A and FIG. 27B show an exemplary fabrication process for the preparation of particular DPNs in accordance with one aspect of the present invention.
  • FIG. 27A shows the sequential steps in the fabrication of DPNs involving the preparation of silicon, PDMS and sacrificial PVA templates. The resulting nanoconstructs were harvested upon dissolution of the sacrificial PVA templates via centrifugation. FTG.
  • 27B shows the sacrificial PVA template is filled with a mixture including the basic forming polymers (carboxyl-PLGA and PEG dimethacrylate): the payload (Rhodamine B dye- RhB-and ultra-small super-paTamagnetic iron oxide nanoparticles - USPIOs); and the photo-initiator.
  • the basic forming polymers carboxyl-PLGA and PEG dimethacrylate
  • the payload Rhodamine B dye- RhB-and ultra-small super-paTamagnetic iron oxide nanoparticles - USPIOs
  • the photo-initiator Upon exposure to UV light, the DPNs were polymerized taking up the size and the shape of the wells within the sacrificial PVA template;
  • FIG. 28A, FIG. 28B, IG. 28C, and FIG. 28D illustrate the geometrical analysis of the templates and DPNs.
  • FIG. 28A shows the SEM image of the Si template showing the wells with a diameter of 1 ,280 run and a depth of 518.9 nm.
  • FIG. 28B shows the AFM image of the PDMS template presenting a regular array of pillars.
  • FIG. 28C shows the fluorescent optical image of the PVA template filled with the polymeric mixture and Rhodamine B dye.
  • FTG. 28D illustrates exemplary DPNs loaded with Rhodamine B dye harvested upon dissolution of the PVA template;
  • FIG. 29A, FIG. 29B, FIG. 29C, and FIG. 29D illustrate physico-chemical characterization of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • FIG. 2 A is an AFM image of an individual DPN indicating the diameter (968 in nm), height (509 in nm), and profile.
  • FIG. 29B is an SEM image of individual DPNs confirming their geometrical properties.
  • FIG. 29C shows DLS and zeta potential of DPNs measured in an aqueous solution.
  • FIG. 29D shows Rhodamine B and USPIO loaded- DPNs dragged on the left of the vial towards an external magnet.
  • FIG. 30A, FIG. 30B, FIG. 30C, and FIG. 30D illustrate release curves and cytotoxicity of the discoidal polymeric nanoconstructs (DPNs).
  • FIG. 30B shows the cytotoxicity on HeLa cells exposed at different concentrations of DPNs for 24 his. No significant reduction in cell viability was measured via a XTT assay.
  • FIG. 30C illustrates HeLa cells incubated with RhB-loaded DPNs for four hrs.
  • RhB was released from the DPNs and diffused into the HeLa cells coloring their cytoplasm around the blue stained nucleus. Most of the DPNs were washed away after four hours, and very few were observed inside the HeLa cells.
  • FIG. 30D is a control experiment with HeLa cells that were not incubated with DPNs;
  • FIG.31 A and FIG. 3 IB show the magnetic performance of exemplary DPNs prepared in accordance with one aspect of the present invention.
  • the DPNs provided the best performance and significantly enhanced the MRI response.
  • FIG. 31A and FIG. 31B are T 2 -weighted MR images of a mouse bearing a breast tumor on the right flank before and after intratumoral injection of USPIO-loaded DPNs. The injection point is delimited by white dotted squares and the site view is magnified in the lateral inset demonstrating a significant darkening of the region of interest upon DPNs injection;
  • FIG.32A, FIG. 32B, FIG. 32C, and FIG. 32D show fluorescent optical images of PVA templates filled with Rhodamine B-dye-loaded DPNs.
  • a thin scum layer is covering the whole area as demonstrated by the low contrast between the red wells, where the nanoconstructs are housed, and the surroundings.
  • a stripe of a thicker scum layer is also visible.
  • FIG. 32B gives a magnified view from the same region. Multiple and accurate washing cycles with DCM solvent are performed to remove almost completely the scum layers (FIG. 32C), and in the magnified view (FIG. 32D);
  • FIG. 33A and FIG. 33B show SEM images of the scum layer. Thick scum layers prevent the nanoconstructs from being freely dispersed within the solution, and dramatically reduces the fabrication yield to ⁇ l-5%;
  • FIG. 34A and FIG. 34B show the dissolution of the sacrificial PVA template and DPNs trapping.
  • FIG. 34A shows dissolved PVA templates in water showed high viscosity and complex networks in SEM. The PVA solution made it hard to separate the nanoconstructs, resulting in a lower yield.
  • FIG. 34B shows some PVA templates were not completely dissolved in water, and many " nanoconstructs were entangled by the PVA templates;
  • FIG. 35A, FIG. 35B, and FIG. 35C show the fluorescent microscopy images of individual DPNs.
  • FIG. 35A DPNs separated from PVA templates showed homogeneous shape in optical images. DPNs in water clearly appeared discoidal in shape, a fact that was validated by recording their movements where DHPs were continuously rotating and moving in water, showing a unique morphology at different angles of the DPN. A DHP showed a round shape at 9.99 sec of acquisition time (FIG. 35B) and the DPN showed a long and flat side at 13.41 sec by rotating itself (FIG. 35C);
  • FIG. 36A and FIG. 36B show 3T MR phantom image of DPNs.
  • FIG. 36A Ti-weighted image (TiW) was utilized by 5000 ms of repetition time and 7.6 ms of echo time, and 900 ms of inversion time. In TjW, DPNs showed brighter signal than water.
  • FIG. 36B In T 2 -weighted image (T 2 W), 3000 msec repetition time and 120 msec echo time were utilized. DPNs gave a darker signal than water. This result demonstrated the capability of DPNs as a MR contrast agent, by shortening the relaxation time of water in both Ti and T 2 mode;
  • FIG. 37 A, FIG. 37B, and FIG. 37C show deliver ⁇ ' of nanoparticles to cancers developing in the lungs (FIG. 3 A).
  • Spherical nanoparticles typically smaller than 200 nm in size, accumulate within tumor tissue by crossing endothelial fenestrations, whereas sub-micron discoidal nanoconstructs are designed to lodge within the tortuous tumor vasculature without relying on the enhanced permeation and retention (EPR) effect (FIG. 37B).
  • EPR enhanced permeation and retention
  • 37C can be loaded with a variety of chemotherapeutic molecules (such as doxorubicin, docetaxel, paclitaxel), and with multiple imaging agents, including infra-red and near infra-red dyes (for optical imaging), Gd 3+ -ions and iron oxide nanocrystals (for magnetic resonance imaging), radioisotopes (for nuclear imaging), and iodine molecules and gold nanoparticles (for CT imaging);
  • chemotherapeutic molecules such as doxorubicin, docetaxel, paclitaxel
  • imaging agents including infra-red and near infra-red dyes (for optical imaging), Gd 3+ -ions and iron oxide nanocrystals (for magnetic resonance imaging), radioisotopes (for nuclear imaging), and iodine molecules and gold nanoparticles (for CT imaging);
  • FIG. 38A, FIG. 38B, FIG. 38C, FIG. 38D, FIG. 38E, and FIG. 38F show cancer imaging with discoidal polymeric nanoconstructs.
  • FIG. 38A and FIG. 38B Scanning electron micrographic (SEM) and optical fluorescence images of discoidal polymeric nanoconstructs, lying in an orderly fashion over a polymeric sacrificial template.
  • FIG. 38C Transmission electron micrograph of discoidal polymeric nanoconstructs loaded with 5-nm iron oxide nanocrystals (black dots); and
  • FIG. 38D, FIG. 38E, and FIG. 38F PET/CT imaging of breast tumor-bearing mice following systemic injection of 50 ⁇ of discoidal polymeric nanoconstructs labeled with 64 Cu(DOTA), at 1, 6, and 20-hrs p. i. , respectively;
  • FIG. 39 A, FIG. 39B, FIG. 39C, FIG. 39D, FIG. 39E, and FIG. 39F show PET/CT imaging of pulmonary metastasis with discoidal nanoconstructs.
  • PET/CT imaging of mice, bearing breast cancer metastasis in the lungs (FIG. 39A, FIG. 39B, FIG. 39C), following systemic injection of 50 ⁇ of discoidal polymeric nanoconstructs labeled with 64 Cu(DOTA). Images are taken at 1, 6 and 24 hrs' p.i. (FIG. 39D, FIG. 39E, and FIG. 39F, respectively) and show the presence of hot spots in the lungs at the later time point (24 hr), possibly associated with metastasis. .
  • the liver activity reduced progressively over time. Note that PET images were corrected for the decay time of the radioisotope so that they could be directly compared; and
  • FIG. 40A, FIG. 40B, FIG. 40C, and FIG. 40D show ex vivo imaging of pulmonary metastasis with discoidal nanoconstructs.
  • FIG. 40A Bioluminescence imaging of MDA-MB-231/Luc cells forming pulmonary metastasis in the right and left lungs of a mouse (right). The control mouse (left), which was not injected with tumor cells, shows no bioluminescence signal. The MDA-MB-231/Luc cells are injected via the tail vein ( ⁇ 10 6 cells) and require three to five weeks to develop stably- growing pulmonary metastasis.
  • FIG. 40B Bioluminescence imaging of the harvested lungs showing clusters of tumor cells in the upper portion of the right lung and lower portion of the left lung.
  • FIG. 40C PET/CT imaging of the harvested lungs showing hot spots in the upper and central portions of the right lung and central and lower portions of the left lung.
  • FIG. 40D Photograph of the harvested lungs showing regions (yellow arrows) with multiple nodules on the right and left lungs.
  • Nanoparticles have emerged as intravenous delivery systems of therapeutic and imaging agents for the early detection and cure of cancer, cardiovascular, metabolic and other diseases (Ferrari, 2005; Peer et al, 2007; Gil and Parak, 2008; Faroklizad and Langer, 2009).
  • Nanoparticles with different compositions have been proposed made out of polymers, lipids, metals, carbon, silica, silicon, and their combinations. The largest majority of nanoparticles exhibit a spherical shape and a diameter ranging from tens to a few hundreds of nanometers (10-300 nm).
  • vascular density and size of the fenestrations vary and depend on the type, site, and stage of development of the malignancy. Moreover, growing evidence in large animal studies, such as dogs and humans, are suggesting that the EPR may have less relevance in larger species (Petersen et al, 2012; Moghimi et al, 2012). Importantly, fenestrations appeared solely in the tumor vasculature, and were not observed to the same extent in other diseases, such as cardiovascular disease.
  • Tn vascular targeting the authors and others have extensively documented the advantage of utilizing non-spherical particles - oblate and discoidal - to enhance vascular deposition (Decuzzi et al, 2009; Mitragotri and Lahann, 2009; Lee et al, 2009; Tao et al, 2011 ; Geng et al, 2007; Muro et al, 2008).
  • the nanoparticle size and shape affect the balance between hemodynamic forces, tending to dislodge nanoparticles away from the vessel wall along the flow direction; and molecular interactions, regulating the firm adhesion of the nanoparticles to the diseased vessel walls.
  • a discoidal shape offers a much larger area of interaction with blood vessel walls, thus supporting the formation of a larger number of ligand-receptor bonds and enhancing the adhesive strength (Decuzzi and Ferrari, 2006; Adriani et al.,. 2012; van de Ven et al, 2012); and favors the lateral drifting of the nanoparticles - margination - enhancing their periodic interaction with the vessel walls in search of vascular abnormalities - endothelial receptors as well as fenestrations (Gentile et al, 2008; Lee et al, 2009).
  • Spherical nanoparticles are generally synthesized by favoring the self-assembly of molecules, polymeric chains or grains under vigorous steering (Graf et al, 2012; Nehilla et al, 2008; Park et al, 2009).
  • non-spherical particles are fabricated via a top-down approach, involving techniques widely utilized in the microelectronic industry.
  • Very few research groups have documented the fabrication and in vivo application of non-spherical particles for biomedical applications (Tanaka et al, 2009; Rolland et al, 2005; Champion and Mitragotri, 2006; Acharya et al, 2010).
  • Park and coworkers have developed a sacrificial hydrogel template approach for synthesizing variously-shaped particles, mostly in the micron-sized regime, for controlling the long term release of therapeutic agents (Acharya et al, 2010a; Acharya et al, 2010b).
  • the present method possesses several advantages over the process described in US 8,263,129 B2 and US 2009/0136583 Al, and in particular: the silicon wafer does not adsorb any solvent, differently from PLGA or PVA sacrificial templates; the silicon wafer is rigid and does not deform under mechanical or thermal stresses occurring during the fabrication process, thus preserving accurately the size and shape of the particles; and the PVA back-layer can be more easily and efficiently removed as compare to a full sacrificial template. All of these enhance both the yield and the quality of the particles as compared to various prior-art methods.
  • Fabrication Methods Using e-beam lithography, multiple wells of desired sizes are generated within a silicon wafer. The wells are fabricated to have the same size and shape as that of the final particles.
  • the polymeric paste including any imaging and/or therapeutic agent(s), is deposited over the silicon template to fill all the wells. The filled wafer is then moved into the oven and kept at 40-60°C for 10-30 min to dry the polymer paste deposited into the wells. The precise temperature and time selected is dependant on the selected polymers and/or incorporated agent(s). Then, DCM, chloroform, or another suitable solvent is carefully dispersed over the surface to remove the excess polymeric material.
  • the cleaned wafer is moved into an oven and kept at 40-60°C for 10- 30 min for a second drying step.
  • PVA solution is deposited on the wafer surface, and the wafer is again dried in an oven at 40-60°C for 10-30 min.
  • molecular bonds are formed between the PVA back-layer and the polymer paste within the wells.
  • This PVA back-layer is then peeled off to permit extraction of the polymeric particles from the silicon wafer (PVA and the polymeric particles do not adhere to the silicon wafer).
  • the PVA back-layer containing the particles is dissolved by continuous stirring in warm water. The liberated particles can then be collected via centrifugation or magnetic dragging from the solution.
  • FIG. 2A and FIG. 2B compare one aspect of the instant fabrication method with the prior-art "Park" method.
  • SEM and fluorescent images of exemplary particles prepapred by the methods of the present invention are shown in FIG. 3A, FIG. 3B, FIG. 3C, and FIG. 3D.
  • DPNs have shown an unprecedented tumor accumulation in tumor-bearing mice well above 10% ID/g tumor, because of the precise control of their size, shape, surface and stiffness (FIG. 14A, FIG. 14B, FIG. 14C, FIG. 14D, FIG. 14E, FIG. 14F, FIG. 14G, FIG. 14H, FIG. 141, FIG. 15A, and FIG. 15B).
  • the low macrophage uptake limits drastically the sequestration of the DPNs in the liver, which shows a very low particle concentration compared to the tumor accumulation (tumor-to-liver ration > 0.5).
  • the DPNs are spread within the tumor mass, and generate a clear contrast at 20 hrs, as compared with the surrounding tissue (FIG.
  • the spleen activity is still elevated at 20 hrs, but it represents only 4% of the absolute ID (FIG. 15 A and FIG. 15B).
  • FIG. 15 A and FIG. 15B These data demonstrate the precise control of the size and shape for the DPNs; the magnetic properties of the particles; the control of the mechanical stiffness and cell uptake in vitro and in vivo, and the in vivo PET imaging and biodistribution.
  • FIG. 16B, FIG. 16C, and FIG. 16D the release of a drug (in this case, doxorubicin) from DPNs, and a tumor cell viability analysis has also been demonstrated (FIG. 17A, FIG. 17B, FIG. 17C, and FIG. 17D).
  • a drug in this case, doxorubicin
  • spherical polymeric nanoparticles with a diameter of -150 nm
  • discoidal mesoporous silicon nanoconstructs and discoidal polymeric nanoconstructs with a diameter of -1,000 nm and a height of 400 and 500 nm, respectively.
  • the spherical nanoparticles accumulate in tumors by means of the well-known enhanced permeation and retention effect, whereas sub-micrometer discoidal nanoconstructs are rationally designed to adhere firmly to the tortuous tumor vasculature.
  • CXR chest radiographs
  • CT computed tomography
  • PET/CT fused positron emission tomography
  • Chest radiography is the most commonly used imaging modality and often the first modality used in assessing the lungs for pulmonary metastasis from many non-thoracic primary malignancies.
  • chest radiographs have low sensitivity, especially for primary lung cancer.
  • Low-dose CT is now the recommended modality for early detection of primary lung cancer (National Lung Screening Trial Research Team et al, 2011); diagnostic CT is the primary method used for staging many non-thoracic malignancies.
  • long-term use of CT for surveillance of metastatic lung disease is not common because of the expense and potential radiation exposure (Pretreatment evaluation of non- small-cell lung cancer, 1997).
  • CT guidance is often required for radiation therapy and histological sampling.
  • FDG F-deoxyglucose
  • buffer includes one or more compositions, or aqueous solutions thereof, that resist fluctuation in the pH when an acid or an alkali is added to the solution or composition that includes the buffer.
  • buffers or buffer solutions This resistance to pH change is due to the buffering properties of such solutions, and may be a function of one or more specific compounds included in the composition. Thus, solutions or other compositions exhibiting buffering activity are referred to as buffers or buffer solutions. Buffers generally do not have an unlimited ability to maintain the pH of a solution or composition; rather, they are typically able to maintain the pH within certain ranges, for example from a pH of about 5 to 7.
  • carrier is intended to include any solvent(s), dispersion medium, coating(s), diluent(s), buffer(s), isotonic agent(s), solution(s), suspension(s), colloid(s), inert(s), or such like, or a combination thereof that is pharmaceutically acceptable for administration to the relevant animal or acceptable for a therapeutic or diagnostic purpose, as applicable.
  • DNA segment refers to a DNA molecule that has been isolated free of total genomic DNA of a particular species. Therefore, a DNA segment obtained from a biological sample using one of the compositions disclosed herein refers to one or more DNA segments that have been isolated away from, or purified free from, total genomic DNA of the particular species from which they are obtained. Included within the term “DNA segment,” are DNA segments and smaller fragments of such segments, as well as recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • the term "effective amount,” as used herein, refers to an amount that is capable of treating or ameliorating a disease or condition or otherwise capable of producing an intended therapeutic effect.
  • engineered and recombinant cells are intended to refer to a cell into which an exogenous polynucleotide segment (such as DNA segment that leads to the transcription of a biologically active molecule) has been introduced. Therefore, engineered cells are distinguishable from naturally occurring cells, which do not contain a recombinantly introduced exogenous DNA segment. Engineered cells are, therefore, cells that comprise at least one or more heterologous polynucleotide segments introduced through the hand of man.
  • epitope refers to that portion of a given immunogenic substance that is the target of, i.e., is bound by, an antibody or cell-surface receptor of a host immune system that has mounted an immune response to the given immunogenic substance as determined by any method known in the art.
  • an epitope may be defined as a portion of an immunogenic substance that elicits an antibody response or induces a T-cell response in an animal, as determined by any method available in the art (see, for example, Geysen et ah, 1984).
  • An epitope can be a portion of any immunogenic substance, such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • immunogenic substance such as a protein, polynucleotide, polysaccharide, an organic or inorganic chemical, or any combination thereof.
  • epitope may also be used interchangeably with “antigenic determinant” or “antigenic determinant site.”
  • heterologous is defined in relation to a predetermined referenced DNA or amino acid sequence.
  • a heterologous promoter is defined as a promoter that does not naturally occur adjacent to the referenced structural gene, but which is positioned by laboratory manipulation.
  • a heterologous gene or nucleic acid segment is defined as a gene or segment that does not naturally occur adjacent to the referenced promoter and/or enhancer elements.
  • the term “homology” refers to a degree of complementarity between two polynucleotide or polypeptide sequences.
  • the word “identity” may substitute for the word “homology” when a first nucleic acid or amino acid sequence has the exact same primary sequence as a second nucleic acid or amino acid sequence.
  • Sequence homology and sequence identity can be determined by analyzing two or more sequences using algorithms and computer programs known in the art. Such methods may be used to assess whether a given sequence is identical or homologous to another selected sequence.
  • homologous means, when referring to polypeptides or polynucleotides, sequences that have the same essential structure, despite arising from different origins.
  • homologous proteins are derived from closely related genetic sequences, or genes.
  • an “analogous” polypeptide is one that shares the same function with a polypeptide from a different species or organism, but has a significantly different form to accomplish that function.
  • Analogous proteins typically derive from genes that are not closely related.
  • identity in the context of two or more peptide sequences, refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues that are the same, when compared and aligned for maximum correspondence over a comparison window, as measured using a sequence comparison algorithm or by manual alignment and visual inspection.
  • isolated or “biologically pure” refer to material that is substantially, or essentially, free from components that normally accompany the material as it is found in its native state.
  • an isolated peptide in accordance with the invention preferably does not contain materials normally associated with that peptide in its in situ environment.
  • kit may be used to describe variations of the portable, self-contained enclosure that includes at least one set of reagents, components, or pharmaceutically-formulated compositions to conduct one or more of the diagnostic methods of the invention.
  • mammal refers to the class of warm-blooded vertebrate animals that have, in the female, milk-secreting organs for feeding the young. Mammals include without limitation humans, apes, many four-legged animals, whales, dolphins, and bats. A human is a preferred mammal for purposes of the invention.
  • Naturally occurring refers to the fact that an object can be found in nature.
  • a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by the hand of man in a laboratory is naturally-occurring.
  • laboratory strains of rodents that may have been selectively bred according to classical genetics are considered naturally occurring animals.
  • nucleic acid includes one or more types of: polydeoxyribonucleotides (containing 2-deoxy-D-ribose), polyribonucleotides (containing D-ribose), and any other type of polynucleotide that is an N-glycoside of a purine or pyrimidine base, or modified purine or pyrimidine bases (including abasic sites).
  • nucleic acid also includes polymers of ribonucleosides or deoxyribonucleosides that are covalently bonded, typically by phosphodiester linkages between subunits, but in some cases by phosphorothioates, methylphosphonates, and the like.
  • Nucleic acids include single- and double-stranded DNA, as well as single- and double-stranded RNA.
  • nucleic acids include, without limitation, gDNA; hnRNA; mRNA; rRNA, tRNA, micro RNA (miRNA), small interfering RNA (siRNA), small nucleolar RNA (snORNA), small nuclear RNA (snRNA), and small temporal RNA (stRNA), and the like, and any combination thereof.
  • the term "patient” refers to any host that can serve as a recipient of one or more of the therapeutic or diagnostic formulations as discussed herein.
  • the patient is a vertebrate animal, which is intended to denote any animal species (and preferably, a mammalian species such as a human being).
  • a "patient” refers to any animal host, including but not limited to, human and non-human primates, avians, reptiles, amphibians, bovines, canines, caprines, cavines, corvines, epines, equines, felines, hircines, lapines, leporines, lupines, murines, ovines, porcines, racines, vulpines, and the like, including, without limitation, domesticated livestock, herding or migratory animals or birds, exotics or zoological specimens, as well as companion animals, pets, and any animal under the care of a veterinary practitioner.
  • polypeptide is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and includes any chain or chains of two or more amino acids.
  • terms including, but not limited to “peptide,” “dipeptide,” “tripeptide,” “protein,” “enzyme,” “amino acid chain,” and “contiguous amino acid sequence” are all encompassed within the definition of a “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with, any of these terms.
  • polypeptides that have undergone one or more post-translational modification(s), including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • post-translational modification(s) including for example, but not limited to, glycosylation, acetylation, phosphorylation, amidation, derivatization, proteolytic cleavage, post-translation processing, or modification by inclusion of one or more non-naturally occurring amino acids.
  • Conventional nomenclature exists in the art for polynucleotide and polypeptide structures.
  • amino acids Alanine (A; Ala), Arginine (R; Arg), Asparagine (N; Asn), Aspartic Acid (D; Asp), Cysteine (C; Cys), Glutamine (Q; Gin), Glutamic Acid (E; Glu), Glycine (G; Gly), Histidine (H; His), Isoleucine (I; He), Leucine (L; Leu), Methionine (M; Met), Phenylalanine (F; Phe), Proline (P; Pro), Serine (S; Ser), Threonine (T; Thr), Tryptophan (W; Trp), Tyrosine (Y; Tyr), Valine (V; Val), and Lysine (K; Lys).
  • Amino acid residues described herein are preferred to be in the "L” isomeric form. However, residues in the "D" isomeric form may be substituted for any L-amino acid residue provided the desired
  • Protein is used herein interchangeably with “peptide” and “polypeptide,” and includes both peptides and polypeptides produced synthetically, recombinantly, or in vitro and peptides and polypeptides expressed in vivo after nucleic acid sequences are administered into a host animal or human subject.
  • polypeptide is preferably intended to refer to all amino acid chain lengths, including those of short peptides of from about 2 to about 20 amino acid residues in length, oligopeptides of from about 10 to about 100 amino acid residues in length, and polypeptides including about 100 amino acid residues or more in length.
  • sequence when referring to amino acids, relates to all or a portion of the linear N-terminal to C-terminal order of amino acids within a given amino acid chain, e.g., polypeptide or protein; "subsequence” means any consecutive stretch of amino acids within a sequence, e.g., at least 3 consecutive amino acids within a given protein or polypeptide sequence.
  • sequence and “subsequence” have similar meanings relating to the 5' to 3' order of nucleotides.
  • a compound or entity may be partially purified, substantially purified, or pure.
  • a compound or entity is considered pure when it is removed from substantially all other compounds or entities, i.e., is preferably at least about 90%, more preferably at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than 99% pure.
  • a partially or substantially purified compound or entity may be removed from at least 50%, at least 60%, at least 70%, or at least 80% of the material with which it is naturally found, e.g., cellular material such as cellular proteins and/or nucleic acids.
  • subject describes an organism, including mammals such as primates, to which treatment with the compositions according to the present invention can be provided.
  • Mammalian species that can benefit from the disclosed methods of treatment include, but are not limited to, apes; chimpanzees; orangutans; humans; monkeys; domesticated animals such as dogs and cats; livestock such as horses, cattle, pigs, sheep, goats, and chickens; and other animals such as mice, rats, guinea pigs, and hamsters.
  • treatment includes but is not limited to, alleviating a symptom of a disease or condition; and/or reducing, suppressing, inhibiting, lessening, ameliorating or affecting the progression, severity, and/or scope of a disease or condition.
  • Nanoparticles have been shown to modulate the biodistribution, organ specific accumulation, and circulation half- life of the original encapsulated agents providing enhanced therapeutic and imaging performance while limiting off-target effects (Mitragotri and Lahann, 2009; Chauhan and Jain, 2013; van de Ven et al, 2012; and Wang et al, 2011).
  • nanoparticles can carry and deliver multiple agents attaining spatiotemporal control on their release enhancing the efficacy of combinatorial drug therapies, multimodal imaging, and enabling the integration of diagnosis and therapy - theranostic (Torchilin, 2006; McCarthy and Weissleder, 2008; Khemtong et al, 2009; and Massoud and Gambhir, 2003).
  • diagnosis and therapy - theranostic Torchilin, 2006; McCarthy and Weissleder, 2008; Khemtong et al, 2009; and Massoud and Gambhir, 2003.
  • a myriad of nanoparticles have been developed exhibiting different geometric features - size and shape; surface properties - such as electrostatic charge, and type and density of ligand moieties; and composition - including, for example, lipid, polymer, metal, and/or hybrid.
  • nanoparticles are synthesized following a bottom-up approach where the basic constituents are mixed together and self-assemble to form larger structures relying on weak, non-covalent molecular interactions.
  • This strategy has been successfully used for the synthesis of micelles (Cabral et al., 2011), liposomes (Moghimiand Szebeni, 2003), and polymeric nanoparticles (Zhang et al., 2008) typically with a spherical shape and a size ranging from tens to a few hundreds of nanometers.
  • nanoconstructs are formed by mixing two conventional polymers - poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) diacrylate - with lipid chains and other agents.
  • the resulting polymer matrix incorporates three different agents, namely iron oxide nanocubes (NCs) with an edge size of - 20 nm; 64 Cu(DOTA) radioactive molecules and red fluorescent Pvhodamine B dyes directly chelated to the lipid chains.
  • NCs iron oxide nanocubes
  • DPNs red fluorescent Pvhodamine B dyes directly chelated to the lipid chains.
  • Polymer Paste Polyvinyl alcohol) (PVA), Poly(DL-lactide-co- glycolide) acid terminated (PLGA, lactide:glycolide 50:50), poly(ethylene glycol) diacrylate, 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone (Photo-initiator), and oleic acid (Sigma, St. Louis, MO, USA).
  • Sylgard 184 kit as polydimethylsiloxane (PDMS) and elastomer were purchased from Dow Coming Corp (Midland, MI, USA).
  • 1,2- dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) was ordered from Avanti Polar Lipids Inc. (Alabaster, AL, USA).
  • PLGA and PEG diacrylate (5: 1 ratio) were dissolved in co-solvent of DCM and CHCI 3 (1 :1 ratio) and 10 ⁇ g of 2-hydroxy-40-(2-hydroxyethoxy)-2-methylpropiophenone (Photo-initiator) was added.
  • 30 ⁇ g of Rhodamine-lipid, 30 ⁇ g of iron oxide nanocube, or 3 mg of lipid-DOTA were mixed together.
  • This polymer paste was then used to fill the wells of a silicon template and generate DPNs with different geometrical configurations.
  • two DPN configurations were considered: 1,000x400 nm discs with a circular base and 1,000x500x500 nm discs with a rectangular base.
  • Lipid-DOTA chains were synthesized by following our previous method. 100 mg of DSPE was dissolved in chloroform and methanol that showed a cloudy solution. With this solution, 100.3 mg of DOTA-NHS was added drop by drop in water, producing in the final ratio of co-solvent of 1.0: 0.5: 0.07 (chloroform : ethanol : water). The reaction was performed for 6h in the presence of a catalytic amount of triethylamine (TEA). The reaction mixture was precipitated in diethyl ether for the further purification.
  • TEA triethylamine
  • NCs Iron Oxide Nanocubes
  • Iron acetylacetonate Fe(acac) 3 , 99%
  • oleic acid OA, technical grade 90%
  • 1- benzyl ether 98%>,
  • 4-biphenylcarboxylic acid 99%, Acros Organics
  • acetone 99.5%
  • hexanes 98.5%) were purchased from Fisher Scientific.
  • the synthesis of clustered iron oxide NCs was modified from previous protocols.
  • iron (III) acetylacetonate (0.71 g, 2 mmol) was mixed with oleic acid (1.27 g, 2 mmol), 4-biphenylcarboxylic acid (0.4 g, 2 mmol), and benzyl ether (10 g, 75 mmol).
  • the mixture was heated to 60°C for 1 hr and further heated up to 200°C for two hrs. After increasing temperature to 280°C, the reaction mixture was maintained at this temperature for one hour with refluxing system under N 2 flow.
  • the resulting black colloidal solution was purified by adding 35 ml acetone and centrifuged at 4150 rpm for 30 min. The black precipitated pellets were re-dispersed using toluene. The purification was repeated for three to five times.
  • iron oxide NCs (20-nm core) was prepared and stored in toluene.
  • oleic acid bilayer coating was used by following established procedures. Briefly, oleic acid (10 ⁇ ) was mixed with 1 mL of NCs solution dispersed in hexanes (1,500 mg/L of iron concentration) and 10 mL sodium bicarbonate buffer (0.1 M, pH 8.5). Two organic/aqueous layers of sample were probe sonicated (UP 50H) at 80% amplitude for 1-2 min. The resulting solution was further stirred and uncovered for overnight to evaporate the residual organic solvents completely.
  • the oleic acid bilayer coated iron oxide NCs were further purified using ultracentrifugation (optima L-90K ultracentrifuge, Beckman coulter) at 35,000 rpm for 2 hrs followed by syringe filtration (pore size 0.45 ⁇ , Whatman, Inc., New York, NY, USA).
  • the silicon master template was fabricated using Electron Beam Lithography (EBL) techniques.
  • the master template is a silicon substrate with textures comprising an array of cylindrical holes with a fixed diameter (1.2 ⁇ ) and and height (400 nm).
  • (100) silicon wafers (from Jocam, Milan, Italy) were cleaned with acetone and isopropanol to remove possible contaminant and then etched with a 4% wet HF solution. The wafers were then rinsed with DI water and dried with N 2 .
  • a high-resolution positive electronic resist (PMMA-A2) was spin-coated for 60 sec at 4000 rpm to obtain a 70-nm thick layer of resist on the substrate.
  • the sample was pre- baked at 170°C for 2 min to remove any residual solvent from the resist. Patterns of ⁇ 10 5 discs with a diameter of 1.2 ⁇ , packed in 1200 x 1200 ⁇ fields, were written on the sample using an Electron Beam Lithography EBL system (CRESTEC), operating at 50 keV. For the exposure, a current of 10 nA, a dose of 220 ⁇ / ⁇ , and a resolution of 20 nm/pixel were adjusted.
  • CRESTEC Electron Beam Lithography EBL system
  • the silicon wafer was developed in 3: 1 isopropanol:methyl isobutyl ketone (MIB ) solution for 60 sec, and in 1 : 1 isopropanol: MIBK solution for 10 sec, to remove exposed regions of the photoresist.
  • MIB methyl isobutyl ketone
  • the pattern was transferred to the underlying silicon substrate by deep reactive ion etching with SF6/O2 plasma.
  • PLGA poly(lactic-co-glycolic acid)
  • DCM dichloromethane
  • CHCI3 chloroform
  • PEG polyethylene glycol
  • Photo-initiator 2-hydroxy-40-(2- hydroxyethoxy)-2-methylpropiophenone
  • 30 ⁇ g of Pvhodamine-lipid, 30 ⁇ g of iron oxide nanocube, or 3 mg of lipid-DOTA were mixed together.
  • the final polymeric mixture was loaded into the wells engraved on the Si master templates. After loading, the surface of the templates was quickly cleaned by DCM solution and then exposed under the UV-light for further polymerization.
  • the polymer- loaded templates were completely dried at 60°C. 6% (wt./vol.) of poly(vinylalcohol) (PVA) solution was prepared in deionized water. The PVA solution was cooled down and stored at room temperature. The PVA solution was loaded on the surface of the polymer loaded Si master templates and the solution was dried at 60°C. The PVA solution became a thin film and the film was easily peeled off from the Si master template. The film also holds numerous identical discoidal polymeric nanoconstructs (DPNs) formed from the loaded polymer in the Si master template. Under magnetic stirring, the PVA templates were swollen and dissolved in DI water at room temperature releasing many DPNs. The released DPNs were separated from the PVA solution by centrifugation at 3,700 rpm for 20 min. The separation processes were repeated until pure pellet is collected. Finally, using 2- ⁇ filter membranes, DPNs were collected.
  • PVA poly(vinylalcohol)
  • PLGA poly(lactic-co-glycolic acid)
  • DCM dichloromethane
  • CHCI3 chloroform
  • the solution was mixed with 6 mg of polyethylene glycol (PEG) diacrylate and 10 ⁇ g of 2-hydroxy-40-(2- hydroxyethoxy)-2-methylpropiophenone (Photo-initiator).
  • PEG polyethylene glycol
  • Photo-initiator 2-hydroxy-40-(2- hydroxyethoxy)-2-methylpropiophenone
  • 30 ⁇ g of Rhodamine-lipid, 30 ⁇ of iron oxide nanocube, or 3 mg of lipid-DOTA were mixed together.
  • the final polymeric mixture was loaded into the wells engraved on the Si master templates. After loading, the surface of the templates was quickly cleaned by DCM solution and then exposed under the UV-light for further polymerization.
  • the polymer- loaded templates were completely dried in oven. 6% (wt./vol.) of poly(vinylalcohol) (PVA) solution was prepared in deionized water. The PVA solution was cooled down and stored at room temperature. The PVA solution was loaded on the surface of the polymer loaded Si master templates and the solution was dried at 60°C. The PVA solution became a thin film and the film was easily peeled off from the Si master template. The film also holds numerous identical discoidal polymeric nanoconstructs (DPNs) formed from the loaded polymer in the Si master template. Under magnetic stirring, the PVA templates were swollen and dissolved in DI water at room temperature releasing many DPNs.
  • PPNs discoidal polymeric nanoconstructs
  • the released DPNs were separated from the PVA solution either by centrifugation at 3,700 rpm for 20 min or by magnetic dragging, given the presence of the NCs in the DPN matrix. The separation processes were repeated until pure pellet is collected. Finally, purified DPNs were collected after 2- ⁇ filter membranes.
  • DPNs Discoidal Polymeric Nanoconstructs
  • a petri dish (diameter: 6 cm) was coated with poly-l-lysine (Sigma Aldrich) for 20 min at room temperature and washed twice with purified water.
  • a ⁇ - ⁇ , drop of DPNs was spotted onto the poly-l-lysine-coated petri dish and then incubated for 30 min to allow the DPNs to absorb onto the substrate. Then, the substrate was washed twice by purified water.
  • the silicon nitride cantilevers modified from 5 ⁇ -diameter silica microparticles (Novascan Technologies, Inc., Ames, IA, USA) were used for this measurement.
  • the spring constant value (0.06 N/m) was measured by the thermal tuning method and applied for this experiment. Force curves were acquired at a sampling rate of 1 Hz.
  • the Young's modulus, E was calculated from the force curve on the Hertz model (Eq. 1) using Nanoscope analysis program of Bruker corporation.
  • TEM Transmission Electron Microscopy
  • FE-TEM field emission TEM
  • TEM sample was prepared by evaporating a drop of DPNs solution on 300-mesh copper grid (Ted Pella, Inc., Redding, CA, USA) (see e.g., FIG. 22G and FIG. 22H).
  • SEM Scanning Electron Microscopy
  • the size and shape characterization of DPNs were performed using FEI Nova Nano-SEM 230 (Hillsboro, OR, USA).
  • Ultra- high-resolution SEM images were acquired at high vacuum conditions after 5 to 7-nm platinum coating using a Cressington 208HR sputter-coater (Ted Pella, Inc.). 5 to 15 keV of beam energy, and corresponding electron current of 0.98 pA to 0.14 nA were used. In some cases, the Mode 2-configuration was used whereby SEM images were magnified over 2500k (see FIG. 22A).
  • PET/CT Imaging PET/CT scanning was performed with a dedicated PET (dPET, resolution: ⁇ 1.4 mm) and multi-modality system (MM), together function of PET and CT with integration capabilities for in-line fusion imaging (Inveon® Research Workplace Systems, Siemens, Malvern, PA, USA).
  • dPET dedicated PET
  • MM multi-modality system
  • mice were anesthetized in a plastic chamber using isoflurane gas in 100% oxygen.
  • the mice were positioned on the imaging bed equipped with a heating pad and the imaging system was connected with the isoflurane gas system.
  • mice images were acquired for 4 min by high resolution CT imaging, and CT image reconstruction was achieved using the scanner's default common cone-bean reconstruction (COBRA) algorithm (Siemens).
  • COBRA common cone-bean reconstruction
  • PET acquisition completed for 10 min at 1- and 6-hrs' p.i. and 20 min at 24- and 48-hrs' p.i.
  • PET images were reconstructed by 2D-ordered subset expectation maximization (OSEM2D) algorithm (Siemens). PET and CT images were coregistered and viewed using the Inveon Research Workplace software (Siemens).
  • COBRA common cone-bean reconstruction
  • mice had tumor sizes of about 1.0 cm in diameter.
  • the mice were injected intravenously with DPNs (10 ⁇ ).
  • PET/CT scans were performed as described above at 1, 6, 24, and 48 hrs post injection of DPNs.
  • the tumor accumulation of DPNs was estimated by using the Inveon Research Workplace software (Siemens) where PET images were corrected based on the initial injection dose and the decay rate of 64 Cu.
  • CT images co-localized with PET images were utilized to delineate the area of tumors in each slide.
  • the unit of the accumulation was expressed as the percentage of injected dose/gram tissue (% ID/g).
  • HAVECs Human umbilical vein endothelial cells
  • PromoCell Human umbilical vein endothelial cells
  • J774 A.l macrophage and B16-F10 mouse melanoma cell lines were obtained from the American Type Culture Collection (ATCC; Mannassas, VA, USA), and cultured in DMEM medium supplemented with 10% FBS and antibiotics (lOO U/mL penicillin G, lOO mg/mL streptomycin), and grown at 37°C in a humidified 5% C0 2 atmosphere.
  • U87-MG human glioblastoma cells were obtained from ATCC and cultured in EMEM medium supplemented with 10% FBS and antibiotics (lOO U/mL penicillin G, lOO mg/mL streptomycin), and grown at 37°C in a humidified 5% C0 2 atmosphere.
  • Live Cell Microscopy Live cell imaging with DPNs was acquired by 1X81 inverted microscope (Olympus Co. Ltd., Tokyo, Japan). 5 x 10 4 J774 A. l macrophages were seeded into glass bottom dishes and incubated for 2 hrs with lipid-Pvhodamine-DPNs at a concentration of 5 DPNs per cell. Cells were imaged at Live Cell Confocal Image System using a 40x objective for all the incubation period to follow DPNs dynamics and interaction with macrophages. Images were recorded every 5 min until the end of the study. Cell nuclei were stained with Hoechst 33342 dye. [0133] Flow Cytometry.
  • J774A.1 cells were plated in 6-well dishes at a density of 4 x 10 5 and incubated with Rhodamine-Lipid-DPNs and fluorescent polystyrene beads at a concentration of five particles/cell for 3 hrs. After that, cells were detached from the dishes using PBS-EDTA 0.5 mM and processed following standard procedures. Briefly, cells were washed twice in PBS buffer to remove free particles, suspended in a final volume of 300 ⁇ , of PBS + 3% of FBS, and then analyzed by LSRII FACS (BD -Bio sciences, San Jose, CA).
  • MTT Proliferation assay
  • ALT alanine transaminase
  • AST aspartate aminotransferase
  • creatinine levels have been measured by standardized kits (Cayman Chemical, Ann Arbor, MI, USA) according to the manufacturer's instructions.
  • Discoidal polymeric nanoconstructs were synthesized following four main steps, as schematically depicted in FIG. 21A-FIG. 21D.
  • Step 1 deals with the definition of a pattern of wells in a silicon template. These wells represent the geometry of the desired nanoconstructs which in this case were circular discs with a ⁇ l,000 nm diameter and a ⁇ 400 nm height (FIG. 21 A).
  • the silicon template was obtained using a standard electron beam lithographic approach, as described above.
  • Step 2 the paste constituting the DPN polymer matrix was directly transferred over the template to fill in carefully the wells (red spots in FIG. 21B).
  • the polymer paste comprises two conventional biodegradable and biocompatible polymers - poly(D,L- lactide-co-glycolide) (PLGA, MW 38,000-54,000) and polyethylene glycol) diacrylate (PEG diacrylate); a photo -initiator (2-hydroxy-40-(2-hydroxyethoxy)-2- methylpropiophenone) for curing the paste; and multiple agents providing different functionalities, namely 20-nm super-paramagnetic iron oxide nanocubes (NCs), for separation and purification purposes; lipid chains coupled with 1,4,7,10- tetraazacyclododecane-l,4,7,10-tetraacetic acid (Lipid-DOTA) for the stable encapsulation
  • PLGA poly(D,L- lactide-co-glycolide)
  • PEG diacrylate polyethylene glycol) diacrylate
  • a photo -initiator (2-hydroxy-40-(2-hydroxyethoxy)-2- methylpropi
  • Step 3 a hydrophilic PVA solution was deposited over the template and dried in oven to form a film (FIG. 21C). Weak, non-covalent bonds were originated at the interface between the polymerized DPNs and the PVA film, which was peeled away to extract the DPNs from the wells of the template.
  • Step 4 the PVA film was immerged in an aqueous solution and dissolved after gentle stirring for 30 min (FIG.
  • DPNs with different geometrical configurations can be readily obtained by using silicon templates with different wells, in the case of rectangular cylinders or rods.
  • the DPNs were characterized for their physico-chemical properties using multiple techniques to confirm their geometrical and mechanical features. Scanning electron microscopy (SEM) images are presented in FIG. 22A for DPNs on the PVA film and released (top-right inset). The DPNs are orderly arranged in arrays with a pitch of -3,000 nm and present a diameter slightly larger than 1,000 nm. These geometrical features are also confirmed by the atomic force microscopy image of FIG. 22B showing two DPNs, released from the PVA film, under hydrated conditions. The presence of Lipid-RhB chains is appreciated in FIG. 22C where images of DPNs in aqueous solution were taken via a fluorescent microscope.
  • the DPNs facing the objective presented a circular base with ⁇ l,000 nm diameter, whereas inclined DPNs showed a lateral thickness of ⁇ 400 nm, precisely matching the geometry imparted in the silicon template.
  • the DPN size distribution appeared as a single peak around 900 nm, in a multisizer instrument (FIG. 22D).
  • the surface electrostatic charge of the DPNs appeared to be slightly negative with a zeta-potential of - 14.0 mV.
  • the mechanical properties of DPNs were tested using an atomic force microscope and the Young's modulus was calculated by analyzing the indention curves, invoking the Hertz theory for contact mechanics (FIG. 22E and FIG. 22F).
  • FIG. 22G Based on measurements performed on multiple DPNs and at different locations within the same DPN (crosses in FIG. 22E), an average elastic modulus of -1.3 kPA was estimated. Finally, the transmission electron microscopy (TEM) image of FIG. 22G reveals additional information on the inner properties of DPNs and shows the presence of iron oxide nanocubes (NCs) with an edge length of 20 nm (FIG. 22H).
  • TEM transmission electron microscopy
  • radiolabeled DPNs were used for in vivo biodistribution and tumor accumulation studies via PET imaging.
  • Two different tumor models were considered, namely human primary glioblastoma cells (U87-MG) injected in the flank of the animal to develop a brain tumor model, and melanoma cells (B16-F10) injected subcutaneously to induce an orthotopic model for skin cancer.
  • the radioactive DPNs were injected through the tail vein in both experimental groups and mice were monitored longitudinally acquiring images at 1, 6, 24, and 48 firs' post-injection (p.i.).
  • FIG. 23A, FIG. 23B, and FIG. 23C Representative PET/CT images for the transverse (top) and coronal (bottom) sections are presented in FIG. 23A, FIG. 23B, and FIG. 23C for the considered mice.
  • the PET images were converted to percentage injected dose per gram (%ID/g) and decay-corrected to the time of scanning.
  • %ID/g percentage injected dose per gram
  • %ID/g percentage injected dose per gram
  • a significant activity is also observed in the abdominal cavity, specifically in the liver, where most of the blood volume is processed.
  • the liver activity reduces over time and it should most likely be associated with the longevity of DPNs in the blood pool.
  • the tumor uptake of DPNs grows steadily over time (red arrow in FIG. 33A).
  • FIG. 33B shows the percentage of DPNs accumulating within the tumor in terms of percentage injected dose per gram tissue (%ID/g).
  • %ID/g percentage injected dose per gram tissue
  • DPNs were systemically injected via tail vein in Tie-2 mice bearing a skin cancer model. These genetically-engineered mice express green-fluorescent proteins in the endothelial cells, so that the vasculature lights up in green under the microscope. Representative images taken from full-length movies are presented in FIG. 24A, FIG. 24B, FIG. 24C, and FIG. 24D for the liver and FIG. 24F, FIG. 24G, and FIG. 24H for the tumor.
  • DiD-labeled RBCs were injected prior imaging for quantifying blood flow; differentiating between functional and not functional vessels; and identifying phagocytic cells (Kupffer cells and tumor macrophages) which would turn blue upon the internalization of RBCs.
  • FIG. 24E shows that, in the liver, the number of DPNs accumulating per unit area within the first 2 hrs p.i. is 3 x 10 - " 4 #/ ⁇ 2 ; whereas the same quantity for the PS within the first 2 min' p.i. is 9 x 10 - " 4 #/ ⁇ 2.
  • the difference between DPNs and PS becomes much larger, being 2.5 x 10 "6 and 4.5 x 10 "4 #/ ⁇ m 2 -min), respectively.
  • a similar behavior was also observed for the spleen (FIG.
  • DPNs were incubated with murine macrophages (J-774) and observed over time using a fluorescent confocal microscope. The location of the DPN and macrophage centers was recorded over a time up to 2 hrs and the corresponding trajectories were extracted by imaging post processing (FIG. 25A; DPN: solid line; cell: dashed line). DPNs and macrophages were observed to move over time along independent trajectories, with DPNs bouncing against and moving around the J-774 cells without inducing any internalization stimulus. Representative snapshots of the full-length movies are shown in the smaller, lateral insets of FIG. 25A.
  • FIG. 25B A side-by-side comparison between DPNs and PS, co- incubated with J-774 cells for two hrs under quiescent conditions, is provided in FIG. 25B.
  • the representative fluorescent microscopy images demonstrate that PS were uptaken more avidly than DPNs.
  • the number of particles uptaken per macrophage is presented in the adjacent bar chart (FIG. 25B).
  • the few DPNs remaining after washing were continuously moving pushed by Brownian forces, whereas the PS were firm inside the cells.
  • flow cytometry analysis was employed to further assess cell uptake properties and confirmed the same behavior observed in vivo and in vitro with a significantly higher uptake for the 750-nm rigid PS as compared to DPNs (FIG. 25C).
  • the radioactivity of 64 Cu-DOTA-DPNs was measured by dose calibrator (: CAPINTEC, model CRC-25PET) and a solution of ⁇ 7 ⁇ iC ⁇ of 64 Cu-DOTA-DPNs was systemically injected into each mouse via tail vein. PET/CT images were taken at 1 , 6, 24, and 48 hrs' p.i.
  • the 64 Cu-DOTA-DPNs showed continuous accumulation in B16 melanoma and U87 glioblastoma tumors.
  • the tumor volumes were measured by CT imaging and drawing three-dimensional ellipsoidal regions of interest around the malignant mass. In addition, the actual weight of the tumor was measured at 48 hrs p.i. upon animal sacrifice.
  • DPNs Deformability of DPNs.
  • the mechanical stiffness of DPNs was tested using Atomic Force Microscopy, as described in the main text.
  • the actual ability of DPNs to squeeze and pass through small bores remaining intact was demonstrated by using syringe membrane filters with different pore sizes, namely 400, 800, and 1,000 nm.
  • the DPNs were force to pass through the filter by gently pushing on the syringe.
  • DPNs showed an excellent filtering rate (%), defined as the percentage difference between the initial amount of DPNs and the amount of DPNs recovered in the lower chamber, upon filtering.
  • the filtering rates decreases as the pore size reduces going from 57% for the 1,000-nm pores, to 36% for the 800-nm pores, and 9% for the 400-nm pores. It should here be noted that the surface density of pores on the membrane surface was very low with an open area of ⁇ 15%, as documented by the manufacturer. Many DPNs could have not a chance to pass the pores. Therefore, it is expected that a large amount of DPNs could be just deposited on the non-open portion of the membrane, especially for the 400 nm pore membrane. For the 400-nm filters, before and after filtration, the size and shape of DPNs was observed under a fluorescent microscope. No significant difference was observed between the two conditions, implying that DPNs can pass through 400-nm pores and recover their original size and shape configuration.
  • Red blood cell (RBC) labeling with DiD Mice were anesthetized using isoflurane. Blood (200 ⁇ ) was obtained from the retro-orbital sinus and collected in EDTA tubes. Blood was stored at 4°C for RBC staining on the same day. The blood collected by retro-orbital injection was centrifuged at 2000 rpm for 10 min to separate plasma from cellular components. Plasma was then removed, and the pelleted red blood cells were aliquoted in 2-mL tubes, and then incubated two times with Vybrant ® DiD Cell- Labeling Solution (Invitrogen) in PBS-Glucose (1 mg/mL) at 37°C for 15 min to allow the labeling of the RBC with the fluorescent tracer.
  • Vybrant ® DiD Cell- Labeling Solution Invitrogen
  • the stained RBC pellets are collected and stored at 4°C until they were re -injected in the mice by retro-orbital sinus technique. The final injected volume for each mouse was 50 ⁇ L. Two days prior, the stained RBCs were systemically injected to the mice to allow enough time for phagocytosis in the body. [0148] IVM Studies. The liver sequestration and tumor accumulation of DPNs was tested in Tie2-GFP + mice following the published protocols described above.
  • DPNs red, size: 1.0 x 0.4 ⁇ discoidal shape
  • PS nanoparticles green, 750 nm spherical shape
  • 1X81 automated inverted microscope was used. For ⁇ 4 hrs, images were acquired. For tracking both particles, FITC and TRITC filters were utilized. Panel A of the figure shows PS nanoparticles were easily internalized into the J774 macrophages upon contact with the cell membrane; whereas DPNs did not stimulate any internalization.
  • Panels B and C show that after 4 hrs incubation, the petri dishes were gently washed by PBS to remove unbounded nanoparticles. The cells were observed by confocal microscopy. PS nanoparticles did not move, while DPNs were observed to continuously move suggesting that the PS nanoparticles were either firmly adhering the cell surface or internalized, whilst the DPNs were either weakly adhering on the cell surface, or not adhering at all.
  • the proposed top-down fabrication process allows for the facile preparation of polymeric nanoconstructs with a precise control on size, shape, surface properties, and mechanical stiffness - the 4S parameters.
  • a rigid silicon template is directly filled with the polymer paste of interest.
  • ⁇ 1,000 x 500 x 500 nm rectangular discs or short rods - discoidal nanoconstructs with any arbitrary contour can be prepared via electron beam lithography and deep reactive ion etching in any silicon foundry or academic clean room facility.
  • DPNs Another advantage of DPNs is the direct loading of any molecular agents and small nanoparticles that can be uniformly dissolved with the original polymer paste.
  • the mass ratio of each individual agent is dictated by their original concentrations in the paste and is preserved during the synthesis process. Indeed, payload losses could occur during the final DPN release and collection in aqueous solution, depending on the hydrophilicity of the loaded agent, but such losses are limited by efficient centrifugation and magnetic dragging.
  • the DPN matrix here is composed of PLGA, PEG diacrylate, and lipid chains intimately entangled one with the other, which is different from more conventional particle architectures where a uniform polymer core is stabilized by a superficial layer of lipid and polymer chains. Moreover, this intimate entanglement of hydrophobic and hydrophilic polymer chains would facilitate the incorporation of molecules and nanoparticles with different properties and functionalities.
  • iron oxide nanocubes dispersed in DPNs may also be also used for Magnetic Resonance Imaging and localized hyperthermia, as shown by the authors in previous works with different nanoplatforms (Key et al, 2013; Aryal et al, 2014; and Cervadoro et al, 2013).
  • the lipid-DOTA chains which are here reacted with copper salts to provide radioactive 64 Cu-DOTA molecules, could be also used as chelators of gadolinium, in Ti-weighted MR imaging; and other radioisotopes, such as 68 Ga, 90 Y, and 177 Lu, for nuclear imaging and radiotherapy.
  • the nuclear imaging and intravital microscopy data together with the cell- uptake analysis shed light on two other unique features of DPNs: the in vivo longevity in the blood stream with a circulation half-life of ⁇ 20 hrs; the capability of lodging within the tumor vasculature with accumulation doses as high as 20% ID/g, in two different tumor models with malignant masses ranging from 0.03 to 0.9 g.
  • most nanomedicines perform significantly better than the corresponding free agents, the values of circulation half-life and tumor accumulation reported for DPNs are uncommon even among the most successful intravascular nanodelivery systems.
  • DPNs The specific architecture of DPNs, with the entanglement of hydrophobic and hydrophibic polymer chains, imparts a moderately negative surface electrostatic charge (-14 mV) and low mechanical stiffness ( ⁇ 1.3 kPa) which are very similar to those of most cells. In part, this could explain the limited DPN uptake by phagocytic cells, in vitro (murine J-774 cells) and in vivo (Kupffer cells in the liver), resulting in a longer circulation half-life in blood.
  • sub-micron discoidal nanoconstructs are forced to navigate in proximity of the vessel walls by the fast-moving red blood cells (see, e.g., Lee et ah, 2013a; Lee et ah, 2013b), and preferentially lodge within the tumor capillaries, characterized by high tortuosity and low blood- flow (Decuzzi et ah, 2009; Decuzzi et ah, 2010; and Godin et ah, 2012). This could contribute to the observed high accumulation of DPNs in the tumor vasculature.
  • this exmple describes a novel top-down approach for the precise synthesis of polymer nanoconstructs that can be readily implemented in any basic polymer chemistry laboratory.
  • the direct loading of agents into the polymer paste constituting the nanoconstructs allows for a precise control of the mass ratios for each therapeutic and imaging agent.
  • these data suggest that the proper combination of size, shape, surface properties, and mechanical stiffness (the 4S design parameters described herein) support both a prolonged circulation and a high accumulation of DPNs within tumors.
  • the facile preparation, multi-functionality, and unprecedented in vivo performance of DPNs present new and useful applications for non-spherical, sub-micron sized nanoconstructs for both the imaging and the treatment of such tumors.
  • DPNs discoidal polymeric nanoconstructs
  • the resulting DPNs were obtained by a one-step mixing of the constituent polymers - poly(lactic acid-co- glycolic acid) (PLGA) and polyethylene glycol (PEG) dimethacrylate -and a selected payload having particular magneto-optical properties - in this case, 5 nm ultra-small super-paramagnetic iron oxide nanoparticles (SPIOs) and Pvhodamine B dye (RhB).
  • SPIOs ultra-small super-paramagnetic iron oxide nanoparticles
  • RhB Pvhodamine B dye
  • Rhodamine B dye was pH-dependent, and increased under acidic conditions by the enhanced hydrolysis of the polymeric matrix.
  • Each DPN was loaded with ⁇ 100 fg of iron, and that concentration was sufficient for the particles to be efficiently dragged by static and/or external magnetic field(s).
  • the USPIO confinement within the DPN porous structure was responsible for a significant enhancement in MRI relaxivity [r 2 ⁇ 500 (mMs) "1 ], which was up to ⁇ 5 times greater than that of commercially available systems.
  • the resulting nanoconstructs described herein provide a new platform for engineering theranostic systems that are particularly useful as anti-angiogenesis agents, and as tools for improved vascular imaging.
  • DPNs discoidal polymeric nanoconstructs
  • PLGA carboxyl- terminated poly(lactic acid-co-glycolic acid)
  • PEG polyethylene glycol dimethacrylate
  • RhB red fluorescent Rhodamine B dye
  • USPIOs 5-nm ultra-small super-paramagnetic iron oxide nanoparticles
  • Sylgard 184 kit as polydimethylsiloxane (PDMS) and elastomer was purchased from Dow Corning, Corp. (Midland, MI, USA).
  • Methacryloxyethyl thiocarbamoyl rhodamine B (RhB) was purchased from Polysciences, Inc. (Warrington, PA, USA).
  • 1 ,2-dioleoyl-sn- glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) was ordered from Avanti Polar Lipids, Inc. (Alabaster, AL, USA). All reagents and other solvents were used without further purification.
  • the silicon master template was fabricated using advanced Electron Beam Lithography (EBL) techniques.
  • EBL Electron Beam Lithography
  • This master was a silicon substrate with textures comprising a periodic, rectangular lattice of cylindrical holes having a known diameter (1 ⁇ ), pitch (5 ⁇ ) and height (400 nm).
  • 100 silicon wafers (from Jocam, Milan, ITALY) were cleaned with acetone and isopropanol to remove possible contaminants, and then etched with a 4% wet HF solution. The wafers were then rinsed with deionized water and dried under N 2 .
  • a high-resolution positive electronic resist (PMMA-A2) was spin- coated for 60 sec at 4000 rpm to obtain a 70-nm thick layer of resist on the substrate. The sample was then pre-baked at 170°C for 2 min to remove any residual solvent from the resist. Patterns of 1051 ⁇ discs, packed in 1200 x 1200- ⁇ fields, were written on the sample using an Electron Beam Lithography EBL system (CRESTEC), operating at 50 keV. For the exposure, a current of 10 nA, a dose of 220 ⁇ , and resolutions as low as 20 nm/pixels were employed.
  • CRESTEC Electron Beam Lithography EBL system
  • the silicon wafer was developed in 3: 1 isopropanol :methyl isobutyl ketone (MIBK) solution for 60 sec, and in 1 : 1 isopropanol:MIBK solution for 10 sec to remove exposed regions of the photoresist.
  • MIBK isopropanol :methyl isobutyl ketone
  • the pattern was transferred to the underlying silicon oxide by deep reactive ion etching with SF 6 /0 2 plasma.
  • the generated silicon master template was used in the fabrication of hydrogel templates.
  • SEM images of the samples were captured to assess uniformity and reproducibility.
  • a dual-beam SEM-FIB FEI Nova 600 NanoLab system was used for the measurements.
  • beam energies of 5 and 15 keV (corresponding to electron currents of 0.98 pA and 0.14 nA, respectively) were employed.
  • the Mode 2 configuration was used, whereby images could be magnified over 250,000x and ultra-high resolution could be achieved.
  • PVA solution (6% wt./vol.) was prepared in deionized water. PVA powder was dissolved at 200°C for 2 hrs. The PVA solution was cooled down and stored at room temperature. The templates provided by the PVA solution maintained the same patterns as the silicon master template.
  • AFM Atomic Force Microscopy
  • DPN Dynamic Light Scattering
  • NMRD and Relaxivity Measurements T 2 relaxation times of DPNs were recorded by using a time domain NMR relaxometer, Bruker Minispecs mq60 (Karlsruhe, Germany). T 2 relaxation times of hemispherical particles were measured at 60 MHz and 37°C using a 5-mm glass probe. The volume of samples was fixed by 225 ⁇ , and HPLC- grade water was utilized as a control. Three samples of particles for this measurement were prepared and run in the same condition: recycle delay (20 sec), gain (57), tau (1), number of data points (500), and number of not-fitted echoes (19).
  • the Fe amount of particles was measured by using inductively coupled plasma optical emission spectroscopy, Varian ICP-OES 720-ES (Palo Alto, CA, USA).
  • DPNs were digested in 70% HNO 3 and the digested solution was completely dried at 200°C overnight.
  • the digested samples were resuspended in 2% HNO 3 and filtered through a 0.22- ⁇ pore diameter membrane.
  • the actual amounts of Fe in the samples were measured in the ICP spectroscopy against a reference standard solution of Fe ions (purchased from Fisher Scientific, Hampton, NH, USA).
  • Particle Degradation and Releasing Profile of Dye from DPNs About 635,000 DPNs were suspended in 100 ⁇ , of PBS at pH 7.4 and pH 5.4, respectively. The releasing dyes from the DPNs were collected by using 100 KDa centrifugal filters (Millipore, Billerica, MA, USA), and separated at 14,000 rpm for 5 min. 100 of PBS (including the released dyes) was transferred to a 96-well plate, and the DPNs trapped by the filter in the tube were resuspended in 100 ⁇ , of PBS at pH 7.4 and pH 5.4, respectively. Samples were then stored at 36.5°C on a shaker at 100 rpm.
  • the PBS solutions passing through the filter were repeatedly transferred to the 96-well plate at time points between 30 min and 24 hrs. After 24 hrs, the 96-well plate was recorded at excitation (510 nm) and emission (560 nm) spectra in a micro-plate reader (BioTek, Winooski, VT, USA). The accumulative amount of the released dyes was finally calculated at pH 7.4 and pH 5.4, respectively.
  • HeLa cell lines were purchased for this study from American Type Culture Collection, and were cultured in MEM Alpha, GlutaMAX® (Life Technologies, Carlsbad, CA, USA) with 10% fetal bovine serum and 1% penicillin-streptomycin at 37°C. All other reagents were purchased from Life Technologies, and used for cell culture without further purification. HeLa cells were seeded in 8-well EZ-Chamber® slides (Millipore) at a density of 40,000 cells/well, and then incubated in RPMI 1640 medium with 10% fetal bovine serum overnight at 37°C in a humidified atmosphere containing 5% C0 2 .
  • the medium was replaced with fresh medium containing 2% fetal bovine serum, in which DPNs were treated as a 1 :20 ratio of cells to DPNs.
  • the chamber slide was placed back in the incubator at 37°C for ⁇ 3 to 4 hrs. Thereafter, cells were washed three times using PBS to remove free DPNs from the cells, and then fixed with 4% paraformaldehyde. Finally, the cells were mounted with 4',6-damidino-2-phenylindole dihydrochloride, DAPI for nuclear staining.
  • XTT Cytotoxicity Assay To evaluate the cytotoxicity of DPNs, XTT assays (American Type Culture Collection) were performed using the manufacturer's protocol. HeLa cells were seeded onto a 96-well plate (5,000 cells/well) one day before treating with DPNs. The following day, HeLa cells were exposed to DPNs at different concentrations with fresh medium for 24 hrs. Data were expressed as the percentage of surviving cells, with all samples being run in quadruplicate.
  • Ti-weighted MR images (TiW) of the phantom
  • SE spin echo
  • DPNs fabrication of DPNs.
  • the biodegradable polymers namely poly(lactic acid- co-glycolic acid) (PLGA) and polyethylene glycol (PEG) dimethacrylate, were combined to generate discoidal nanoconstructs using a modified hydrogel template strategy (Acharya et al., 2010a).
  • the hydrophilic PEG dimethacrylate is entangled and cross-linked with the hydrophobic PLGA resulting in a highly-porous, spongy, polymeric matrix.
  • the inclusion of PLGA facilitates the entrapment and controls the release of hydrophobic molecules.
  • RhB red fluorescent Rhodamine B dye
  • USPIO 5-nm ultra-small super-paramagnetic iron oxide nanoparticles
  • Control of the nanoconstruct geometry was achieved employing the sacrificial hydrogel template strategy, here modified to improve both yield and particle quality at the sub-micron and nano-scales.
  • the DPN fabrication process employed is schematically described in FIG. 27A and FIG. 27B.
  • electron beam lithography is utilized to generate a silicon template exhibiting millions of discoidal wells with a diameter and depth of 1,280 and 518.9 nm, respectively (FIG. 27A).
  • a polydimethylsiloxane (PDMS) layer was accurately deposited over the Si wafer to produce a second template presenting cylindrical pillars, resembling the same size and shape of the wells in the original Si template.
  • PDMS polydimethylsiloxane
  • the geometrical properties of the templates and DPNs were characterized using different techniques, including scanning electron microscopy (SEM), atomic force microscopy (AFM) and fluorescent optical microscopy (FOM) (FIG. 28A, FIG. 28B, FIG. 28C, and FIG. 28D).
  • SEM scanning electron microscopy
  • AFM atomic force microscopy
  • FOM fluorescent optical microscopy
  • the Si templates manufactured by electron beam lithography presented a regular matrix of wells with a diameter of 1,280 nm and a depth of 518.9 nm, as from the SEM micrographs shown (FIG. 28A).
  • the corresponding pillars on the completely dried PDMS template were visualized using AFM and showed a quasi- discoidal shape (FIG. 28B).
  • FIG. 28C A FOM image of the PVA template filled with the discoidal PLGA/PEG nano constructs is shown in FIG. 28C.
  • the fluorescent signal was derived from the RhB dye loaded into the DPN polymeric mixture.
  • the contrast between the PVA template (dark) and the DPNs (red) demonstrated the loading efficiency as well as the absence of any residual, scum layer on the PVA template.
  • DPNs were separated from the mixture by centrifugation and appeared as bright and red spots (FIG. 28D). Note that the image shows DPNs on different focal planes, thus exhibiting different sizes.
  • FIG. 29A The physico-chemical properties of the DPNs, loaded with RhB and USPIOs, were further characterized, as shown in FIG. 29A, FIG. 29B, FIG. 29C, and FIG. 29D.
  • An AFM and SEM analysis conducted on an individual particle confirmed the size and shape of the DPNs.
  • the AFM image of the DPN is provided in FIG. 29A, together with its geometrical profile demonstrating 968 nm diameter and 509 nm height.
  • a total of 40 points was recorded while moving the AFM cantilever along the surface of the DPN at every 25 to 30 nm distance using high-speed Bruker MultiMode AFM.
  • the SEM images are shown in FIG. 29B.
  • the hydrodynamic diameter and zeta potential of the DPNs were measured in an aqueous solution resulting in 529 ⁇ 76 nm and -19 ⁇ 8 mV, respectively (FIG. 29C).
  • the non-spherical shape of the nanoconstructs returned an average hydrodynamic diameter varying between the actual diameter (-1,000 nm) and thickness (-500 nm) of the DPNs.
  • the negative surface charge has to be ascribed to the carboxyl-terminated PLGA polymeric chains exposed on the DPN surface.
  • FIG. 30C show HeLa cells of 4 hrs' post-incubation with the RhB and USPIO-loaded DPNs.
  • FIG. 30D shows a control experiment without DPNs. Interestingly, most DPNs were easily removed upon cell washing implying that only few nanoconstructs could enter the cell over the 4-hr period. However, the RhB released from DPNs passively diffused through the cell membrane, and accumulated within the cytoplasm of the HeLa cells, as shown by the red areas surrounding the blue stained nucleus in FIG. 30C.
  • Rhodamine B dye was used as a reference molecule, although it can be readily substituted with one or more anti-cancer drugs (e.g., doxorubicin, paclitaxel, etc.) in commercial application of the present invention.
  • anti-cancer drugs e.g., doxorubicin, paclitaxel, etc.
  • the PLGA/PEG polymeric matrix allows for the encapsulation and controlled release of both hydrophilic and hydrophobic drug molecules.
  • loading and release can be tuned by tailoring the PLGA/PEG weight ratio during the synthesis process.
  • the longitudinal and transversal relaxivities, ri and r 2 were calculated.
  • the transversal relaxivity and the r 2 /ri ratio are the most important parameters.
  • Such an enhancement could be ascribed to the agglomeration of multiple USPIOs to form larger clusters (Paquet et al, 2011; Roca et al, 2009), or to a change in the mobility of the water molecules surrounding the USPIOs confined within the polymeric matrix (Ananta et al, 2010; Sethi et al, 2012).
  • the r 2 /ri ratio was >10, confirming the high quality of the nanoconstructs, and their suitability as T 2 MR imaging agents.
  • Fe loading was also remarkably high reaching the value of approximately 100 fg per DPN.
  • the mass of iron has been loaded in each DPN corresponding to a bulk iron oxide sphere with a diameter larger than 300 nm. Note that such large beads cannot be used in biomedical applications for their poor bioavailability and low biodegradability.
  • the present nanoconstructs encapsulate thousands of small, 5-nm USPIOs that would be readily released in the circulation upon DPN degradation and eventually excreted or metabolized.
  • the magnetic performance of the USPIO-loaded discoidal polymeric particles was also characterized using a commercial MRI device.
  • T 2 -weighted MR images of DPNs were compared to pure water, demonstrating a huge contrast for both imaging modalities.
  • the same DPNs were also injected in a breast tumor mass developed in a mouse flank, and then imaged using the same 3T MRI instrument.
  • T 2 -weighted MR images were taken before (FIG. 31 A) and immediately following intratumoral injection of the DPNs (FIG. 3 IB).
  • a comparison between the two MRI images showed a significant darkening of the tumor tissue at the site of injection.
  • the nanoconstructs could be guided in vivo via external, static magnetic fields to facilitate their localization at specific sites and vascular areas. This improved biodistribution, combined with their high relaxivity, facilitate a significant shortening of the relaxation times and stronger MRI contrast in vivo.
  • DPNs Discoidal Polymeric Nanoconstructs
  • the scum layer is generated by the same polymeric mixture utilized for the final nanoconstructs. When this mixture is deposited over the sacrificial PVA template, as the wells in the template are progressively filled, a thin film (scum layer) covers the PVA template and connects multiple nanoconstructs together.
  • FIG. 32A, FIG. 32B, FIG. 32C, and FIG. 32D show the cleansing process on the top of the PVA templates.
  • FIG. 32A and FIG. 32B show mixtures of polymer loaded in PVA template, which showed many thin or thick scum layer that interconnected the particles each other, resulting in the low yielding rate.
  • FIG. 32C and FIG. 32D show PVA templates after the cleansing process using DCM, which could decrease the amount of the scum layer.
  • SEM micrographs of the final centrifuged solution with nanoconstructs demonstrate even more clearly the presence of scum layers and the deleterious effect that these have on the fabrication yielding. This is illustrated in the insets (FIG. 33A and FIG. 33B).
  • FIG. 33A shows a thick scum layer, wrapped on itself, still holding several nanoconstructs that have not been released in the solution. Note also the several filaments of PVA surrounding the complex.
  • FIG. 33B shows multiple films of scum layer with a characteristic size of several tens of microns, still encapsulating several tens of thousands of nanoconstructs.
  • FIG. 34A illustrates with a SEM micrograph the complex fiber structures forming upon PVA dissolution.
  • the inset FIG. 34B provides a fluorescent microscopy image of individual DPNs, loaded with Pvhodamine B dye, entrapped in the PVA fibrous network.
  • Rhodamine B dye-loaded DPNs are shown via fluorescent microscopy in the images of FIG. 35A, FIG. 35B, and FIG. 35C.
  • the nanoconstructs are suspended in an aqueous solution and move chaotically as driven by thermal agitation.
  • Some DPNs present their circular base at the objective, whiles other are inclined and present their side view, thus confirming their anisotropic, discoidal shape.
  • Table 1 provides the experimental values for the transversal relaxation time T 2 , iron concentration C and corresponding relaxivity r 2 for three different samples of USPIO- loaded DPNs.
  • Table 2 provides information of the DPN loading with the UPIOs, as derived from the ICP-OES analysis.
  • Multifunctional nanoconstructs with precisely controlled geometrical and surface properties have been prepared using a modified hydrogel-template strategy.
  • the resulting nanoconstructs were fabricated from a PLGA/PEG mixture, and exhibited a discoidal shape with a diameter of -1,000 nm and a height of -500 nm.
  • 5-nm USPIOs and Pvhodamine B dye were dispersed within the DPN polymeric matrix to provide enhanced magneto-optical properties. Stability of the resulting DPNs was tested in under various biological conditions, and it was shown that acidic pH triggered dissolution of the polymeric matrix, and thus accelerated the release of Pvhodamine B dye.
  • NPs nanoparticles
  • NPs are fabricated objects small enough to be injected intravascularly and navigate safely within the circulatory system, and they are designed to perform several, useful functions (Ferrari, 2005; Peer et al., 2007). They can efficiently carry multiple, and different, imaging and/or therapeutic molecules from the site of injection to one or more biological targets ⁇ e.g., malignant tissue), where they can provide contrast enhancement and exert a curative action.
  • biological targets e.g., malignant tissue
  • a large variety of NPs having different sizes, shapes, surface properties, and compositions have been developed for diverse oncological applications.
  • NPs in cancer imaging and therapy, the advantage of using NPs over freely administered molecules can be summarized in the following two points: i) the size, shape, surface properties and composition of NPs can be finely tuned during their synthesis to enhance their accumulation at the diseased site while limiting off-site targeting ⁇ engineer ability of nanoparticles); and ii) multiple and different therapeutic and imaging molecules can be simultaneously loaded within the same NP, without interfering with the original organ-specific biodistribution ⁇ multi-functionality of nanoparticles).
  • the main paradigm in the design of NPs for oncological applications is based on the observation that the tumor vasculature is discontinuous and presents openings (fenestrations) measuring several hundreds of nanometers.
  • NPs typically being smaller than 200 nm, progressively accumulate within the tumor tissue by crossing these endothelial fenestrations (FIG. 37A, FIG. 37B, and FIG. 37C) (Jain and Stylianopoulos, 2010). This phenomenon, first documented by Maeda (1989), is generally known as the enhanced permeation and retention (EPR) effect.
  • EPR enhanced permeation and retention
  • organs of the reticulo -endothelial system such as the liver, spleen, and bone marrow, are also characterized by a discontinuous endothelium and tend to avidly sequester foreign circulating objects, including NPs.
  • tumor masses are also characterized by a tortuous vascular network, low mean blood velocity, impaired lymphatic system, and high interstitial fluid pressure (Jain and Stylianopoulos, 2010).
  • the mean blood velocity in most tumors is 1-10 ⁇ /sec, which is about ten times lower than that recorded in healthy vessels (Jain and Stylianopoulos, 2010).
  • our group has proposed sub-micron, discoidal nanoconstructs as vehicles for preferentially targeting the malignant vasculature, without relying on the EPR effect (FIG. 37) (Decuzzi et al, 2009).
  • these discoidal nanoconstructs are designed to recognize the altered tumor vasculature and to adhere firmly to the endothelial walls, without crossing the fenestrations (Decuzzi and Ferrari, 2008; Decuzzi and Ferrari, 2006). They are sub- micron in size and tend to be pushed laterally in the so-called cell free layer by the fast moving red blood cells. Therefore, by design, these nanoconstructs tend to move in close proximity to the vessel walls, continuously sensing for local vascular abnormalities (Lee et al, 2013). Also, the discoidal shape of these nanoconstructs favors firm, stable adhesion to the vessel walls in regions of lower blood velocity (Decuzzi and Ferrari, 2008; Decuzzi and Ferrari, 2006).
  • Both conventional spherical NPs and discoidal nanoconstructs can be loaded with a variety of drug molecules, including potent chemotherapeutic molecules, such as doxorubicin, docetaxel, or paclitaxel (Aryal et al, 2010; Key et al, 2013; and Shen et al, 2013).
  • potent chemotherapeutic molecules such as doxorubicin, docetaxel, or paclitaxel
  • constructs can also be labeled with multiple contrast agents, including infrared and near-infrared dyes for optical imaging (Key et al, 2013); Gd -ions and iron oxide nanocrystals for magnetic resonance imaging (Shen et al, 2013; Ananta et al, 2010; Sethi et al, 2012; Aryal et al, 2013; and Gizzatov et al, 2014); radioisotopes for nuclear imaging (Aryal et al, 2014); and iodine molecules and gold nanoparticles for CT imaging.
  • contrast agents including infrared and near-infrared dyes for optical imaging (Key et al, 2013); Gd -ions and iron oxide nanocrystals for magnetic resonance imaging (Shen et al, 2013; Ananta et al, 2010; Sethi et al, 2012; Aryal et al, 2013; and Gizzatov et al, 2014); radioisotopes for nuclear imaging (Ary
  • Discoidal Polymeric Nanoconstructs are the third example of NPs described herein for cancer theranosis.
  • DPNs were synthesized using a top-down approach as described above by combining electron beam lithography and polymer chemistry (see Key et al, 2013). The fabrication process starts with the definition of a geometrical pattern of wells in a master silicon template, using lithographic techniques. The well represents the final geometry of the nanoconstruct. From the silicon template, sacrificial templates are originated for the actual DPN synthesis.
  • FIG. 38A and FIG. 38B show SEM and optical fluorescence images, respectively of DPNs still adhering to a sacrificial template.
  • DPNs were observed as arranged in an orderly fashion of rows and columns, which were about 3,000-nm apart, with an average diameter of -1,000 nm.
  • the upper-right insets in both images present details of the DPN geometry.
  • TEM of DPNs demonstrated the presence of 5-nm USPIOs (black spots) within the polymeric matrix (see FIG. 38C).
  • DPNs were obtained by photo-polymerizing a polymeric paste comprising PLGA chains, multi-arm PEG acrylate chains, lipid-DOTA chains, lipid-Pvhodamine B dye (RhB), and USPIOs. While RhB and USPIOs were used for providing the optical and MRI capabilities, the lipid-DOTA chains, uniformly distributed within the polymer matrix, were reacted with copper chloride salts after DPN formation to generate 64 Cu-DPNs for PET imaging.
  • FIG. 39A, FIG. 39B, FIG. 39C, FIG. 39D, FIG. 39E, and FIG. 39F show PET/CT images of murine lungs taken at 1, 6, and 24 hrs' p.L
  • FIG. 40A shows the accumulation of MDA-MB-231/Luc cells within the lungs of a living mouse. These cells required three-to-five weeks to develop into pulmonary nodules.
  • FIG. 40B, FIG. 40C, and FIG. 40D show ex vivo images of harvested lungs acquired via bioluminescence (BL) (FIG. 40B), which identifies the location of the metastasis; PET/CT (FIG.
  • FIG. 40C which served to localize DPNs and quantify their accumulation in the microvasculature; and optical imaging, and which helped in delineating regions with multiple tumor nodules (see yellow arrows in FIG. 40D).
  • the spatial distribution of the hot spots observed via BL, PET/CT, and the surface nodules correlated well.
  • a large metastatic site was identified in the upper portion of the right lung, as confirmed by the images in FIG. 40B, FIG. 40C, and FIG. 40D, and appeared to correlate with the PET hot spot detected in the live animal at 24 hrs (see FIG. 39A, FIG. 39B, FIG. 39C, FIG. 39D, FIG. 39E, and FIG 39F).
  • nanoconstructs can improve the bioavailability and biodistribution of systemically injected agents by exploiting the altered architecture and biology of the tumor vasculature.
  • the size, shape, surface properties, and composition of nanoconstructs can be tailored during the fabrication process to support their preferential deposition within the tumor vasculature or passive permeation through tumor fenestrations ⁇ engineer ability).
  • nanoconstructs can encapsulate large amounts of different therapeutic and imaging agents (multi-functionality) .
  • nanoconstructs can be loaded with USPIOs and tagged with Gd-based macromolecules (Gd(DOTA), Gd(DTPA) for MRI; labeled with different types of radionuclides ( 18 F, 64 Cu) for PET and ( 99 Tc, m In) SPECT imaging; decorated with a variety of dyes (RhB, Cy5.5) for optical imaging; and loaded with iodine molecules and gold nanoparticles for CT imaging. All these modalities (MRI, PET, SPECT, optical, CT) can coexist within the same nanoconstructs, without interfering with any moieties for molecular targeting, thus nanoconstructs enable both multi-modal and molecular imaging.
  • Gd(DOTA), Gd(DTPA) for MRI
  • radionuclides 18 F, 64 Cu
  • RhB a variety of dyes
  • iodine molecules and gold nanoparticles for CT imaging. All these modalities (MRI, PET, SPECT, optical, CT) can coexist within
  • NPs can deliver multiple agents providing different curative approaches.
  • Potent chemotherapeutic molecules such as doxorubicin, docetaxel, paclitaxel, and so on, can be encapsulated within the same nanoconstructs and released, at the diseased site, following precise schedules.
  • metal nanoparticles, such iron oxides, and gold nanoparticles, loaded into the nanoconstructs can deploy, upon external excitation, significant doses of thermal energy to induce localized hyperthermia and tissue ablation. Indeed, truly combinatorial drug delivery and synergistic therapies can be achieved only by using nanoconstructs.
  • nanoconstruct configurations can be envisioned which could improve on the current clinically available intervention protocols.
  • sub-micrometer discoidal nanoconstructs can be tagged with 64 Cu and loaded with gold nanorods for spotting small malignant masses via PET imaging and for guiding radiation therapy via CT.
  • the presence of gold nanorods could locally enhance the efficacy of radiation therapy, limiting side effects and radiation doses.
  • the same nanoconstructs could also slowly release locally one or more chemotherapeutic drugs to ameliorate the efficacy of the radiation therapy.
  • 64 Cu-tagged discoidal nanoconstructs could be loaded with USPIOs and exposed to focused alternating magnetic fields to induce local hyperthermia and enhance blood perfusion and vessel permeability.
  • conventional spherical NPs loaded with chemotherapeutic molecules, could more efficiently accumulate deeper into the tumor matrix, upon systemic injection. Indeed, the possible combinations are limited only by the available resources and clinical needs.
  • nanoconstructs will always appear more complicated and more difficult to handle than single molecules in the eyes of pharmaceutical companies, approval agencies, and some scientists. But the opportunities to be derived from merging, in a single entity, multiple imaging modalities and different therapeutic strategies are offered only by nanoparticles, and more effort should be dedicated to developing nanoconstructs for theranosis in pulmonary cancers.
  • ADRIANI G et al, "The preferential targeting of the diseased microvasculature by disk-like particles," Biomaterials, 33(22):5504-5513 (Aug. 2012)
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  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically- and/or physiologically-related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

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Abstract

L'invention concerne des procédés pour la fabrication de nano/micro-particules non sphériques présentant des propriétés contrôlées de taille, de forme et de surface, et une rigidité du mécanisme sur une large gamme de tailles. L'invention concerne également des méthodes permettant d'utiliser ces particules en tant que système d'administration systémique (i.e., intravasculaire) pour l'administration d'agents d'imagerie et/ou d'agents thérapeutiques à des cellules et/ou à des tissus choisis du corps du mammifère. Dans certaines applications, les particules décrites sont discoïdes avec une base circulaire ou rectangulaire ; ont une taille caractéristique de ~ 1 000 nm, et sont composées de différentes combinaisons de copolymère d'acide lactique et d'acide glycolique (PLGA) et de polyéthylène glycol (PEG). L'invention concerne également des méthodes destinées à une accumulation sans précédent des particules dans la tumeur (proportion supérieure à 10 % de dose injectée/g, sans ciblage moléculaire ni magnétique), et leur utilisation comme agents d'augmentation de contraste in vivo pour des modalités d'imagerie à base de tomographie par émission de positons ou de résonance magnétique. Certaines des particules décrites peuvent également être utilisées pour une thérapie par ablation thermique, et/ou un entraînement/guidage magnétique.
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IT202200002486A1 (it) 2022-02-11 2023-08-11 Fondazione St Italiano Tecnologia Microparticelle di idrogelo per la veicolazione di agenti terapeutici e diagnostici

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