WO2015168393A1 - Nanostructures de modulation de communication intercellulaire, et leurs utilisations - Google Patents

Nanostructures de modulation de communication intercellulaire, et leurs utilisations Download PDF

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Publication number
WO2015168393A1
WO2015168393A1 PCT/US2015/028494 US2015028494W WO2015168393A1 WO 2015168393 A1 WO2015168393 A1 WO 2015168393A1 US 2015028494 W US2015028494 W US 2015028494W WO 2015168393 A1 WO2015168393 A1 WO 2015168393A1
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nanostructure
cell
synthetic
vesicle
shell
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PCT/US2015/028494
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English (en)
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Michael P. PLEBANEK
C. Shad Thaxton
Raja Kannan Mutharasan
Nicholas L. ANGELONI
Kaylin M. MCMAHON
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Northwestern University
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Publication of WO2015168393A1 publication Critical patent/WO2015168393A1/fr
Priority to US15/339,500 priority Critical patent/US10413565B2/en

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    • 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
    • A61K9/5123—Organic compounds, e.g. fats, sugars
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
    • 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
    • A61K9/5115—Inorganic compounds

Definitions

  • the present invention generally relates to nanostructures and compositions for modulating or monitoring intercellular communication via vesicles and uses thereof.
  • Vesicles are crucial for intercellular communication. Vesicles, such as exosomes, are recognized to be crucial mediators of several biological processes. The ability to specifically modulate intercellular communication processes, such as exosome uptake and release, in a targeted manner may provide significant therapeutic value to several diseases. Moreover, modulating or monitoring intercellular communication may provide significant diagnostic or research value.
  • Exosomes are 30-100 nm nanovesicles responsible for the transport of a myriad of molecular cargo including protein, lipids, mRNA and miRNA [Valadi et al., 2007].
  • Exosome signaling is linked to a number of pathologies including cancer [Martins et al., 2013; Jung et al., 2009], neurological disorders [Rajendran et al., 2006], and cardiovascular disease [Hergenreider et al., 2012].
  • cancer Patents et al., 2013; Jung et al., 2009
  • neurological disorders [Rajendran et al., 2006]
  • cardiovascular disease Hergenreider et al., 2012.
  • local and distal communication between tumor and supporting cells is critical for tumor progression [Peinado et al., 2012; Ghajar et al., 2013].
  • nanostructures that inhibit intercellular communication may be effective for slowing or halting tumor progression or the nanostructures may be effective for monitoring tumor progression.
  • tumor cell derived exosomes are reported to target specific sites and deliver pro-tumor molecules leading to a preparation of the metastatic microenvironment, or niche
  • nanostructures that can associate with vesicles may be able to be specifically delivered to specific sites for therapeutic, diagnostic, or research purposes.
  • the cell membrane has a critical role in intercellular communication because the cell membrane is the interface between individual cells and their external environment.
  • a number of critical cellular events including signal transduction, membrane compartmentalization and endosomal trafficking, are coordinated in lipid rafts.
  • Lipid rafts are complex membrane domain structures that are characterized by an excess of cholesterol, sphingolipids, and proteins
  • Scavenger receptor type B-l (SR-B1) is one of the many receptors that are expressed in lipid rafts [Umemoto et al. 2013]. Because of this, tumor progression is often associated with an increased expression of SR-B1 aiding in the procurement of cholesterol needed for maintaining cell membrane integrity and other cellular processes
  • lipid raft cholesterol content inhibits downstream second messenger signaling events such as ERK 1/2 signaling which have been reported as critical for exosome uptake.
  • nanostructures that can change the cell membrane by associating with lipid rafts or binding receptors in the cell membrane, such as SR- Bl may be useful for therapeutic, diagnostic, or research purposes.
  • Synthetic nanostructures have been shown to be useful for therapeutic, diagnostic, and research purposes.
  • nanostructures having a corona of nucleic acids extending radially from the center have been shown to useful for inhibiting gene expression (as described in International Patent Publication No. WO/2006/6138145 entitled “Nucleic acid functionalized nanoparticles for therapeutic applications,” filed 8 June 2006)
  • nanostructures having a detectable marker have be shown to be useful for detecting intracellular targets in living cells (as described in International Patent Publication No.
  • WO/2009/131704 describes the use of nanostructures for treating cancers generally and International Patent Publication No. WO/2013/126776 describes the use of nanostructure for treating cancer cells having an SR-B1 receptor, neither publication describes the use of nanostructures to modulate or monitor intercellular communication.
  • SR-B1 could bind synthetic nanostructures and that the binding of the nanostructures may lead to apoptosis of certain cell types, e.g. lymphoma
  • SR-B1 binding of a nanostructure in a viable cell would exhibit modulated intercellular communication. It was unexpected, therefore, that nanostructures such as those described herein could be used for the treatment, diagnosis, or research of vesicle-mediated diseases, as the role of these particles in modulating vesicle uptake or release was not envisioned.
  • the present invention generally relates to nanostructures and compositions for modulating intercellular communication processes for research, diagnostic and/or therapeutic purposes.
  • One aspect the invention is a method for inhibiting intercellular communication, comprising contacting a cell with an effective amount of a synthetic nanostructure.
  • the synthetic nanostructure comprises a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associated with the shell.
  • the interaction between the cell and the vesicle results is release of the vesicle by the cell.
  • the interaction between the cell and the vesicle results is uptake of the vesicle by the cell.
  • the vesicle may be is an exosome.
  • the cell may cell expresses a receptor and the synthetic nanostructure binds the receptor, and, optionally the receptor is SR-B1.
  • contacting the cell with the effective amount of synthetic nanostructure induces a change in the cell membrane, and, in certain cases, the change in the cell membrane may be clustering of lipid rafts in the cell membrane.
  • the effective amount of synthetic nanostructure is a therapeutically effective amount of synthetic nanostructure to treat a vesicle-mediated disorder.
  • the vesicle-mediated disorder may be an exosome-mediated disorder or may be a cancer, a viral infection, a neurological disorder, or rheumatic disease.
  • One aspect of the invention is a method for loading a vesicle, the method comprising contacting a cell with a synthetic nanostructure, wherein the synthetic nanostructure is capable of being taking up by the cell, and wherein the synthetic nanostructure is capable of being secreted in a vesicle comprising the synthetic nanostructure.
  • synthetic nanostructure comprises a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associated with the shell.
  • the vesicle may be an exosome.
  • the synthetic nanostructure is taken up by the cell and/or the synthetic nanostructure is secreted in the vesicle comprising the synthetic nanostructure.
  • the synthetic nanostructure further comprises an agent.
  • the agent may be a diagnostic agent, a therapeutic agent, or both a diagnostic agent and a therapeutic agent.
  • the diagnostic agent may be a tracer lipid.
  • the tracer lipid may be a chromophore, a biotin subunit, or both a chromophore and a biotin subunit.
  • the therapeutic agent may be a nucleic acid, antiviral agent, antineurological agent,
  • the nucleic acid may be siRNA.
  • the method further includes collecting the vesicle comprising the synthetic nanostructure and/or isolating the vesicle comprising the synthetic nanostructure.
  • the cell may be in culture, the cell may be a cancer cell, the cell may have been removed from a patient, or the cell may be contacted with the synthetic nanostructure ex vivo.
  • the method further includes contacting the vesicle comprising the nanostructure with a second cell. The second cell may be in culture or may be in a patient.
  • One aspect of the invention is a method for the treatment of a vesicle-mediated disorder, the method comprising administering a therapeutically effective amount of synthetic
  • the synthetic nanostructure comprises a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associated with the shell.
  • the synthetic nanostructure further comprises a therapeutic agent.
  • the therapeutic agent may be a nucleic acid, antiviral agent, antineurological agent, antirheumatologic agent.
  • the nucleic acid may be siRNA.
  • the administering step comprises contacting a cell with a vesicle comprising the synthetic nanostructure.
  • the vesicle comprising the synthetic nanostructure is prepared by contacting a second cell with the synthetic nanostructure.
  • One aspect of the invention is a method for cellular analysis, the method comprising contacting a cell with a vesicle comprising a synthetic nanostructure.
  • the synthetic nanostructure comprises a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associated with the shell.
  • the method further includes detecting the nanostructure.
  • the synthetic nanostructure comprises a diagnostic agent.
  • the diagnostic agent may be a tracer lipid.
  • the tracer lipid may be a chromophore, a biotin subunit, or both a chromophore and a biotin subunit.
  • Figures 1A-C show characterization of A375 melanoma exosomes: size, morphology and molecular markers, (a) Transmission electron micrograph (TEM) of A375 exosomes isolated by differential ultracentrifugation. The isolated exosomes display typical cup-shaped
  • Figures 2A-F show free and esterified cholesterol content of hHDL and HDL NP, cholesterol efflux, and specific targeting of SR-B1 in lipid rafts
  • (a) Pie charts show the content of free cholesterol and cholesteryl ester to hHDL and HDL NPs before (left) and after (right) cholesterol efflux assay in A375 melanoma cells
  • Cells were fractionated using FocusTM Global Fractionation (G
  • A375 cells expressing a GFP-SR-B1 fusion protein (green) are stained with an Alexafluor-647 conjugated CTx-B (red) to label and image lipid rafts, (e) A375 melanoma cell lipid rafts were stained with an Alexafluor-488 conjugated CTx-B (green) after treatment with 20 nM DiD-labeled HDL NPs (red) (f) A375 melanoma cells expressing a GFP-SR-Bl fusion protein (green) were treated with DiD labeled HDL NPs (20 nM, red).
  • FIG. 3 shows HDL NPs have no effect on cellular viability.
  • MTS 3-(4,5- dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
  • FIG. 4 shows expression of SR-B1 and GFP-SR-Bl in A375 cells and HMVECs.
  • Western blot shows for SR-B1 in both A375 cells and HMVECs using an anti-SR-Bl monoclonal antibody.
  • GFP-SR-Bl fusion protein characterized by increased molecular weight in A375 cells trans fected with appropriate construct (lane 1).
  • Figures 5A-D show clustering of SR-B1 following HDL NP treatment.
  • Time-lapse images of A375 melanoma cells expressing GFP-SR-Bl were taken in the presence (HDL NP) and absence (i.e. untreated, labeled in the figure as "untx") of HDL NPs (30 nM) 24 hours after treatment
  • Raw images (left) were segmented using a wavelet-based method (see Materials and Methods) to define and measure GFP-SR-Bl -positive domains.
  • Whiskers extend between the 10th and the 90th percentile,
  • Figures 6A-C show HDL NPs lead to reduced mobility and dispersion of SR-B 1 containing domains.
  • (b) Average speeds per puncta for each condition (***P ⁇ 0.00005 via permutation t-test).
  • FIGS 7A-G show HDL NPs block the uptake of exosomes by A375 melanoma cells
  • the exosome uptake by untreated cells serves as a negative control
  • FIGS 8A-B show HDL NP treatment inhibits exosome uptake in A375 melanoma cells expressing GFP-SR-Bl .
  • A375 cells expressing GFP-SR-Bl were treated with exosomes in the presence of 0, 5, and 50 nM HDL NPs.
  • exosomes and HDL NP treatment reduces exosome uptake A375 cells expressing GFP-SR-Bl .
  • *** represents P ⁇ 0.001 as compared to exosome only condition).
  • Figures 9A-B show the inhibition of exosome uptake after treatment with HDL NP is not due to extracellular interaction of exosomes and HDL NP.
  • A375 melanoma cells were pre- treated for 12 hours with HDL NP 5 and 50 nM. Excess HDL NPs were then washed 2 times in PBS (a) Flow cytometry analysis of exosome uptake after A375 cell pre-treatment with HDL NP.
  • Graph (b) shows the average fluorescence intensity of cells analyzed by flow cytometry in b. represents P ⁇ 0.001 as compared to exosome only condition).
  • Figures 10A-B show hHDL has only a modest effect on exosome uptake by A375 cells. Exosomes were labeled using Dil and their uptake by A375 cells in the presence of 0, 5, 50, 500 nM hHDL was measured using flow cytometry. In contrast to HDL NP treatment, there the reduction in exosome uptake does not exceed 15%, even at 500 nM hHDL . (* represents P ⁇ 0.05 as compared to exosome only condition) [0024] Figures llA-O show targeting SR-B1 to induce receptor clustering and inhibit exosome uptake.
  • A375 melanoma cells were analyzed for exosome uptake by flow cytometry and clustering of GFP-SR-B1 containing domains was measured using fluorescent microscopy after 24 hrs treatment with the following agents: (a, b) 50 nM HDL NPs; (c, d) 50 nM PEG-NPs; (e, f) 50 nM hHDL; (g, h) SR-B1 neutralizing antibody; (i, j) siRNA targeting SR-B1 expression (siSR-Bl); and, (k, 1) 1 ⁇ BLT-1.
  • FIGS 12A-B show that Rhodamine lipid is present in exosomes treated with
  • Rhodamine HDL NPs Rhodamine HDL NPs.
  • Figure 13 shows that scheme for identifying biotin presence on membrane of exosomes.
  • Intercellular communication is cell-to-cell transfer of chemicals and signals that lead to some sort of response by the receiving cell.
  • Vesicles are important to the process as intercellular communication and provide a means to transport those chemicals and signals between cells, often in a targeted manor.
  • Exosomes are one example of vesicles that transport molecular cargo to and from cells as a means of intercellular communication [Valadi et al. 2007; Martins et al. 2013].
  • Exosomes are nano-sized, and these vesicles contribute to multiple diseases, including cancers [Valadi et al. 2007; Rajendran et al. 2006; Ramakrishnaiah et al. 2013; Jung et al.
  • cancer cells enhance their production of exosomes as a means of facilitating disease progression [Yu et al. 2006; Filipazzi et al. 2012].
  • exosomes produced by melanoma cells have been shown to target endothelial cells to enhance angiogenesis [Ekstrom et al. 2014], as well as macrophages and dendritic cells causing immune suppression [Marton et al. 2012].
  • considerable data are accumulating showing that enhanced exosome production by cancer cells facilitates metastasis by conditioning the pre -metastatic niche [Peinado et al. 2011] through the mobilization of bone marrow cells and the delivery of pro-tumorigenic cargo to metastatic sites [Peinado et al. 2012].
  • modulation of intercellular communication is the inhibition of intercellular communication and an effective amount of the nanostructure inhibits the interaction between a cell and a vesicle.
  • the interaction may be the uptake of a vesicle by the cell.
  • the interaction may be the release of a vesicle by the cell. Further embodiments and illustrations are provided below.
  • the present disclosure also teaches nanostructures and methods for loading a vesicle with the nanostructure.
  • the loaded vesicle can be used in a number of different applications, including but not limited to therapeutic or diagnostic applications. Because vesicles can specifically target, the loaded vesicles can be used to specifically target certain cells to deliver the nanostructure as a payload.
  • the nanostructure payload may be used for therapeutic or diagnostic purposes. In certain cases the nanostructure may further comprises an agent, such as a therapeutic agent or diagnostic agent. Further embodiments and illustrations are provided below.
  • a range includes each individual member.
  • a group having 1-3 members refers to groups having 1, 2, or 3 members.
  • the modal verb "may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb "may” refers to an affirmative act regarding how to make or use an aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb "may” has the same meaning and connotation as the auxiliary verb "can.”
  • a number of different types of vesicles play a role in intercellular communication.
  • Exosomes are one example of vesicles that play a role in intercellular communication. Exosomes are 30-100 nm nano vesicles responsible for the transport of a myriad of molecular cargo including protein, lipids, mRNA and miR A. Exosomes are linked to a number of difference pathologies including cancers, neurological diseases, rheumatic diseases, and viral infections.
  • Receptors on the cell surface also play a role in intercellular communication, as reception of a target may initiate the intercellular communication.
  • Scavenger receptor type B-l (SR-B1) is one example of receptor that has a role in intercellular communication, SR-B1 may also specific targets of biological as well as synthetic origin.
  • SR-B1 is found in lipid raft in the cell membrane, and SR-B1 is a high-affinity receptor for cholesterol-rich high-density lipoproteins (HDL).
  • HDL cholesterol-rich high-density lipoproteins
  • SR-B1 may also bind synthetic nanostructures, like cholesterol-poor biomimetic HDL-like nanoparticles (HDL NPs) as described below.
  • Changes in the cell membrane may lead to modulation of intercellular communication. Changes in the cell membrane may be the result of receptors binding the synthetic nanostructure. As mentioned above, SR-B1 may be found in lipid rafts in the cell membrane and SR-B1 binding of synthetic nanoparticle may result in clustering of the lipid rafts. These morphological changes in the cell membrane modulate intercellular communication, as the cluster of the lipid rafts interfere with uptake of exosomes. In certain cases, the interference may result in the inhibition of vesicle uptake. In other cases, the interference may result in the inhibition of the release of a vesicle.
  • One aspect of the invention is the inhibition of intercellular communication by contacting a cell with an effective amount of a synthetic nanostructure, wherein contacting the cell with the effective amount of synthetic nanostructure inhibits an interaction between the cell and the vesicle.
  • the synthetic nanoparticle may be any synthetic nanoparticle that may modulate or inhibit intercellular communication when a cell is contacted with an effective amount of synthetic nanostructure.
  • the synthetic nanostructure may comprise a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associate with the shell. Examples of synthetic nanostructures useful for the present purposes are described below.
  • the synthetic nanoparticle is a synthetic cholesterol binding nanostructure.
  • the synthetic cholesterol binding nanostructure may be a biomimic of mature, spherical HDL, e.g., in terms of the size, shape, surface chemistry and/or function of the structures.
  • HDL-NP high-density lipoprotein synthetic nanoparticles
  • Au- NP gold nanoparticle
  • other suitable entity or material e.g., lipids, proteins, etc.
  • the synthetic nanostructures may have a substantially similar size, shape and/or surface chemistry to that of natural HDL, but may differ in at least one
  • the at least one characteristic may be, for example, the presence or absence of one or more components in the nanostructure, the positioning of one or more components in or on the nanostructure, the materials used to form the nanostructure, the makeup of the shell of the nanostructure, the makeup of the core of the nanostructure, and combinations thereof.
  • the synthetic nanostructures can be made substantially free of cholesterol (e.g., in the core, and/or prior to administration of the nanostructures to a subject or sample) as the Au-NP or other suitable entity occupies the real- estate at the core. This configuration differs from that of natural HDLs, which have a core formed of cholesteryl esters and triglycerides.
  • nanostructures described herein may have certain characteristics and/or functions similar to that of natural HDL (e.g., cholesterol binding constant) but may have other characteristics and/or functions that differ from that of natural HDL (e.g., ability to deliver cholesterol to cells). The differences between the two HDL (e.g., cholesterol binding constant) but may have other characteristics and/or functions that differ from that of natural HDL (e.g., ability to deliver cholesterol to cells). The differences between the
  • nanostructures described herein and natural HDLs may contribute to the effectiveness of the nanostructures in treating the cells, diseases and conditions described herein.
  • the interaction between the cell and the vesicle may be any type of interaction associated with intercellular communication.
  • the interaction between the cell and the vesicles results in the release of the vesicle by the cell.
  • the interaction between the cell and the vesicle results in the uptake of the vesicles by the cell.
  • the interaction between the cell and the vesicles may result in a signaling event.
  • inhibition of ER 1/2 signaling was shown to reduce exosome uptake, HDLs induce ERK 1/2 signaling by phosphorylation of ERK 1/2 and AKT.
  • HDL NP treatment drastically reduces both ERK 1/2 and AKT phosphorylation.
  • the vesicle may be any vesicle associated with intercellular communication.
  • the vesicles is an exosome, a virus, an apoptotic body, a synthetic lipid particle (e.g. liposome), a bacteria, or a fungus.
  • the cell may be any cell capable of intercellular communication.
  • the cell is a cell that expresses a receptor and the receptor may bind the synthetic nanostructure.
  • the receptor is a scavenger receptor, a receptor in the tetraspanin family, a receptor known to be a pattern receptor, a receptor known to exist in areas of the cell membrane known to be involved in particle uptake, e.g. caveolin and clathrin.
  • the scavenger receptor may be SR-B1.
  • the cell may be in vivo.
  • the cell may be in an animal, a human, or a patient.
  • the patient may be a patient suffering from a vesicle-mediated or an exosome-mediated disorder.
  • vesicle-mediate disorders include, but are not limited to, cancers, viral infection, neurological disorders, rheumatic diseases, immunological disorders, inflammation, antigen presentation, blood disorders, bacterial infection.
  • the cell may be in vitro or ex vivo.
  • that cell may be in culture or in a biological sample.
  • Another aspect of the invention is a method for preparing vesicles comprising synthetic nanostructures.
  • the vesicles comprising the synthetic nanostructures may be useful for targeted delivery of the synthetic nanostructures for therapeutic, diagnostic, or research purposes.
  • the method for vesicle loading comprises contacting a cell with a synthetic
  • a vesicle comprising the synthetic nanostructure may also be referred to as a loaded vesicle.
  • the loaded vesicle may be prepared by having the synthetic nanostructure taken up by the cell and by having the cell secret a vesicle comprising the synthetic nanostructure.
  • the synthetic nanostructure may be any synthetic nanostructure having the property of being able to be taken up by the cell and having the property of being able to be secreted in a vesicle comprising the synthetic nanostructure.
  • the synthetic nanostructure may comprise a nanostructure core, a shell, the shell comprising a lipid layer surrounding and attached to the nanostructure core, and a protein associate with the shell. Examples of synthetic nanostructures useful for the present purposes are described below.
  • the synthetic nanostructure may be a synthetic cholesterol binding nanostructure, i.e. a biomimic of mature, spherical HDL, e.g., in terms of the size, shape, surface chemistry and/or function of the structures.
  • HDL-NP high-density lipoprotein synthetic nanoparticles
  • Au-NP gold nanoparticle
  • other components e.g., lipids, proteins, etc.
  • synthetic nanostructure may further include an agent.
  • the agent may be a diagnostic agent (which may also be known as an imaging agent), a therapeutic agent, or both a diagnostic agent and a therapeutic agent.
  • the diagnostic agent is a tracer lipid. Tracer lipids may comprise a chromophore, a biotin subunit, or both a chromophore and a biotin subunit.
  • the therapeutic agent may be a nucleic acid, antiviral agent, antineurological agent, antirheumatologic agent.
  • the nucleic acid may be siRNA.
  • the vesicle may be any vesicle associated with intercellular communication such that the vesicle may be secreted by one cell and interact with another.
  • the interaction between the cell and the vesicle comprising the synthetic nanostructure may be any type of interaction associated with intercellular communication.
  • the interaction between the cell and the vesicles results in the release of the vesicle by the cell.
  • the interaction between the cell and the vesicle results in the uptake of the vesicles by the cell.
  • the interaction between the cell and the vesicles may result in a signaling event.
  • the vesicle may be any vesicle associated with intercellular communication.
  • the vesicles is an exosome, a virus, an apoptotic body, a synthetic lipid particle (e.g. liposome), a bacteria, or a fungus.
  • the cell may be any cell capable of secreting a vesicle for intercellular communication.
  • the cell may be in vivo.
  • the cell may be in an animal, a human, or a patient.
  • the patient may be a patient suffering from a vesicle-mediated or a exosome-mediated disorder.
  • vesicle-mediate disorders include, but are not limited to, cancers, viral infection, neurological disorders, rheumatic diseases, immunological disorders, inflammation, antigen presentation, blood disorders, bacterial infection.
  • the cell may be in vitro or ex vivo.
  • that cell may be in culture or in a biological sample. Delivering loaded vesicles for therapeutic, diagnostic, and research applications
  • Another aspect of the invention is delivering loaded vesicles for therapeutic, diagnostic, and research applications.
  • the vesicle comprising the synthetic nanostructure is collected.
  • the vesicle comprising the synthetic nanostructure may be isolated.
  • the vesicle comprising the synthetic nanostructure may be contacted with a second cell.
  • the contacting step may be accomplished by contacting the second cell with a loaded vesicle that has been collected and/or isolate.
  • the contacting step may also be accomplished by having the cell secreting the vesicle comprising the synthetic nanostructure in intercellular
  • the second cell may be in vivo.
  • the cell may be in an animal, a human, or a patient.
  • the patient may be a patient suffering from a vesicle- mediated or a exosome-mediated disorder.
  • vesicle-mediate disorders include, but are not limited to, cancers, viral infection, neurological disorders, rheumatic diseases
  • the second cell may be in vitro or ex vivo.
  • the second cell may be in vitro or ex vivo.
  • cell may be in culture or in a biological sample.
  • Therapeutic applications include methods for the treatment of a vesicle-mediated disorder comprising administering a therapeutically effective amount of synthetic nanostructure to a patient in need thereof.
  • the administering step comprises contacting a cell with a vesicle comprising the synthetic nanostructure.
  • the synthetic nanostructure may comprise a therapeutic agent.
  • HDL NPs can also be functionalized with other types of cargo such as nucleic acids. This cargo would be loaded into exosomes and specifically delivered to appropriate locations using the intrinsic targeting capabilities of the exosomes. Existing tumors can be injected with the HDL NPs containing anti-cancer therapies, which would then package the particles into exosomes and deliver this cargo to appropriate targets.
  • cargo such as nucleic acids.
  • cancer cells can be removed from the patient and treated with HDL NPs under laboratory culture. Exosomes can be harvested and returned to the patient, where they will deliver their cargo for use as therapy or for imaging.
  • Examples of therapeutic applications include, but are not limited to, methods of treating an exosome meditated disorder by administering to a subject having a exosome meditated disorder an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the exosome meditated disorder; methods of treating cancer by administering to a subject having a drug-resistant cancer an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the cancer is also provided; methods of treating a metastatic cancer by administering to a subject having a metastatic cancer an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the metastatic cancer; and methods of treating cancer by administering to a subject having a cancer, wherein the cancer is an ERKl/2 or AKT associated cancer, an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the cancer is provided.
  • the ERKl/2 or AKT associated cancer is lung cancer, colon cancer, breast cancer, or prostate cancer.
  • the lung cancer may be, for instance, NSCLC.
  • the subject has naive or acquired resistance to EGFR-targeted therapy.
  • Methods of treating intracellular viral infection involves administering to a subject infected with an intracellular virus an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of virally infected exosomes in order to treat the infection.
  • Methods for treating a neurological disorder involve administering to a subject having a neurological disorder an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the neurological disorder.
  • the neurological disorder is selected from the group consisting or Alzheimer's, Parkinson's, or prion related disease.
  • Methods for treating a rheumatic disease by administering to a subject having a rheumatic disease an effective amount of a synthetic cholesterol binding nanostructure for inhibiting cellular uptake of exosomes in order to treat the rheumatic disease.
  • Targeted delivery of cancer therapies remains a significant challenge and an area of much research.
  • This technology would exploit the intrinsic ability of cancer cell exosomes to specifically target tissues and cells required for progression, and deliver anti-cancer therapies directly to those populations to interfere with tumor growth and metastasis.
  • Loading exosomes using a cell-based assay is a significant improvement over current strategies such as
  • immunomagnetic-based separation methodologies can be employed to specifically isolate an exosome population of interest for study as biomarkers or other uses.
  • Diagnostic or research applications include method for cellular analysis comprising contacting a cell with a vesicle comprising a synthetic nanostructure.
  • the invention in other aspects relates to methods and products associated with the loading of vesicles with a synthetic nanostructure using a cell-based technology.
  • the vesicles contain the synthetic nanostructure and their components, which allows for tracking of the exosomes, isolation and quantification from mixed populations of exosomes, and packaging of synthetic nanostructures for specific delivery to cells and tissues targeted by the exosomes.
  • the synthetic nanostructure is detected.
  • the synthetic nanostructure further comprises a diagnostic agent, and the diagnostic agent is detected.
  • Synthetic nanostructures can be used for research, diagnostic and/or therapeutic indications, where it is beneficial to label, track and/or isolate exosomes.
  • cells in culture can be treated with HDL NPs containing a tracer lipid (fluorophore, biotin). Exosomes from these cells are now labeled and can be tracked when given to other cells in culture or injected into animal models or humans.
  • HDL NPs with tracer can be injected into cancerous tissue. Exosomes produced by the cancerous tissue can be isolated from blood samples using the tracer for further molecular analysis. Whole-body imaging techniques could also be used to determine what tissues are targeted by the exosomes by looking for signal from the gold particle as well as any tracer molecules functionalized to it.
  • the above properties can also be used to isolate exosomes originating from a single source from complex mixtures. Blood and other body fluids contain many types of exosomes from multiple sources. Flow cytometry and immune - based separation methods could be utilized to separate exosomes tagged with a tracer from these complex mixtures in vitro and in vivo, including human samples. This allows for the study of a particular population of exosomes originating from a certain group of cells. [0073] Quantification of exosomes from cancer cells may also be useful as a biomarker to measure disease progression. Studies suggest increased exosome production is linked with increased tumor activity. The ability to measure levels of tumor-derived exosomes over time may help inform treatment strategies.
  • the synthetic nanostructures described above may be detected or may further include diagnostic agents that may be detected. Imaging agents and diagnostic agents may be used interchangeably.
  • the synthetic nanostructure having a nanostructure core that comprises a material suitable for use as an imaging agent (e.g., gold, iron oxide, a quantum dot, radionuclide, etc.).
  • the synthetic nanostructure having a shell comprises an imaging agent.
  • a nanoparticle or other suitable contrast agent may be embedded within the lipid bilayer of the shell, or associated with an inner or outer surface of the shell.
  • the imaging agents may be used to enhance various imaging methods known to those in the art such as MRI, X-ray, PET, CT, etc.
  • the structure (e.g., a synthetic structure or synthetic nanostructure) has a core and a shell surrounding the core.
  • the core includes a surface to which one or more components can be optionally attached.
  • core is a nanostructure surrounded by shell, which includes an inner surface and an outer surface.
  • the shell may be formed, at least in part, of one or more components, such as a plurality of lipids, which may optionally associate with one another and/or with surface of the core.
  • components may be associated with the core by being covalently attached to the core, physisorbed, chemisorbed, or attached to the core through ionic interactions, hydrophobic and/or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or combinations thereof.
  • the core includes a gold nanostructure and the shell is attached to the core through a gold-thiol bond.
  • components can be crosslinked to one another.
  • Crosslinking of components of a shell can, for example, allow the control of transport of species into the shell, or between an area exterior to the shell and an area interior of the shell.
  • relatively high amounts of crosslinking may allow certain small, but not large, molecules to pass into or through the shell, whereas relatively low or no crosslinking can allow larger molecules to pass into or through the shell.
  • the components forming the shell may be in the form of a monolayer or a multilayer, which can also facilitate or impede the transport or sequestering of molecules.
  • shell includes a lipid bilayer that is arranged to sequester cholesterol and/or control cholesterol efflux out of cells, as described herein.
  • a shell that surrounds a core need not completely surround the core, although such embodiments may be possible.
  • the shell may surround at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 99% of the surface area of a core.
  • the shell substantially surrounds a core.
  • the shell completely surrounds a core.
  • the components of the shell may be distributed evenly across a surface of the core in some cases, and unevenly in other cases.
  • the shell may include portions (e.g., holes) that do not include any material in some cases.
  • the shell may be designed to allow penetration and/or transport of certain molecules and components into or out of the shell, but may prevent penetration and/or transport of other molecules and components into or out of the shell.
  • the ability of certain molecules to penetrate and/or be transported into and/or across a shell may depend on, for example, the packing density of the components forming the shell and the chemical and physical properties of the components forming the shell.
  • the shell may include one layer of material, or multilayers of materials in some embodiments.
  • the structure may also include one or more agents, such as a therapeutic or diagnostic agent.
  • agents may be associated with the core, the shell, or both; e.g., they may be associated with surface of the core, inner surface of the shell, outer surface of the shell, and/or embedded in the shell.
  • agents may be associated with the core, the shell, or both through covalent bonds, physisorption, chemisorption, or attached through ionic interactions, hydrophobic and/or hydrophilic interactions, electrostatic interactions, van der Waals interactions, or combinations thereof.
  • the synthetic nanostructure is a synthetic cholesterol binding
  • Kd is less than or equal to about 100 ⁇ , less than or equal to about 10 ⁇ , less than or equal to about 1 ⁇ , less than or equal to about 0.1 ⁇ , less than or equal to about 10 nM, less than or equal to about 7 nM, less than or equal to about 5 nM, less than or equal to about 2 nM, less than or equal to about 1 nM, less than or equal to about 0.1 nM, less than or equal to about 10 pM, less than or equal to about 1 pM, less than or equal to about 0.1 pM, less than or equal to about 10 fM, or less than or equal to about 1 fM.
  • the core of the nanostructure may have any suitable shape and/or size.
  • the core may be substantially spherical, non- spherical, oval, rod-shaped, pyramidal, cube-like, disk-shaped, wire-like, or irregularly shaped.
  • the core (e.g., a nanostructure core or a hollow core) may have a largest cross-sectional dimension (or, sometimes, a smallest cross-section dimension) of, for example, less than or equal to about 500 nm, less than or equal to about 250 nm, less than or equal to about 100 nm, less than or equal to about 75 nm, less than or equal to about 50 nm, less than or equal to about 40 nm, less than or equal to about 35 nm, less than or equal to about 30 nm, less than or equal to about 25 nm, less than or equal to about 20 nm, less than or equal to about 15 nm, or less than or equal to about 5 nm.
  • a largest cross-sectional dimension or, sometimes, a smallest cross-section dimension of, for example, less than or equal to about 500 nm, less than or equal to about 250 nm, less than or equal to about 100 nm, less than or equal to about 75 nm, less than
  • the core has an aspect ratio of greater than about 1 : 1, greater than 3 : 1 , or greater than 5 : 1.
  • aspect ratio refers to the ratio of a length to a width, where length and width measured perpendicular to one another, and the length refers to the longest linearly measured dimension.
  • the nanostructure core may be formed from any suitable material.
  • the core is formed of a synthetic material (e.g., a material that is not naturally occurring, or naturally present in the body).
  • a nanostructure core comprises or is formed of an inorganic material.
  • the inorganic material may include, for example, a metal (e.g., Ag, Au, Pt, Fe, Cr, Co, Ni, Cu, Zn, and other transition metals), a semiconductor (e.g., silicon, silicon compounds and alloys, cadmium selenide, cadmium sulfide, indium arsenide, and indium phosphide), or an insulator (e.g., ceramics such as silicon oxide).
  • the inorganic material may be present in the core in any suitable amount, e.g., at least 1 wt%, 5 wt%, 10 wt%, 25 wt%, 50 wt%, 75 wt%, 90 wt%, or 99 wt%.
  • the core is formed of 100 wt% inorganic material.
  • the nanostructure core may, in some cases, be in the form of a quantum dot, a carbon nanotube, a carbon nanowire, or a carbon nanorod.
  • the nanostructure core comprises, or is formed of, a material that is not of biological origin.
  • a nanostructure includes or may be formed of one or more organic materials such as a synthetic polymer and/or a natural polymer.
  • synthetic polymers include non-degradable polymers such as polymethacrylate and degradable polymers such as polylactic acid, polyglycolic acid and copolymers thereof.
  • natural polymers include hyaluronic acid, chitosan, and collagen.
  • a shell of a structure can have any suitable thickness.
  • the thickness of a shell may be at least 10 Angstroms, at least 0.1 nm, at least 1 nm, at least 2 nm, at least 5 nm, at least 7 nm, at least 10 nm, at least 15 nm, at least 20 nm, at least 30 nm, at least 50 nm, at least 100 nm, or at least 200 nm (e.g., from the inner surface to the outer surface of the shell).
  • the thickness of a shell is less than 200 nm, less than 100 nm, less than 50 nm, less than 30 nm, less than 20 nm, less than 15 nm, less than 10 nm, less than 7 nm, less than 5 nm, less than 3 nm, less than 2 nm, or less than 1 nm (e.g., from the inner surface to the outer surface of the shell).
  • Such thicknesses may be determined prior to or after sequestration of molecules as described herein.
  • Suitable techniques include dynamic light scattering (DLS) (e.g., using a Malvern Zetasizer instrument), transmission electron microscopy, scanning electron microscopy, electroresistance counting and laser diffraction. Other suitable techniques are known to those or ordinary skill in the art. Although many methods for determining sizes of nanostructures are known, the sizes described herein (e.g., largest or smallest cross-sectional dimensions, thicknesses) refer to ones measured by dynamic light scattering.
  • DLS dynamic light scattering
  • the shell of a structure described herein may comprise any suitable material, such as a hydrophobic material, a hydrophilic material, and/or an amphiphilic material.
  • the shell may include one or more inorganic materials such as those listed above for the nanostructure core, in many embodiments the shell includes an organic material such as a lipid or certain polymers.
  • the components of the shell may be chosen, in some embodiments, to facilitate the sequestering of cholesterol or other molecules. For instance, cholesterol (or other sequestered molecules) may bind or otherwise associate with a surface of the shell, or the shell may include components that allow the cholesterol to be internalized by the structure. Cholesterol (or other sequestered molecules) may also be embedded in a shell, within a layer or between two layers forming the shell.
  • the components of a shell may be charged, e.g., to impart a charge on the surface of the structure, or uncharged.
  • the surface of the shell may have a zeta potential of greater than or equal to about -75 mV, greater than or equal to about -60 mV, greater than or equal to about -50 mV, greater than or equal to about -40 mV, greater than or equal to about -30 mV, greater than or equal to about -20 mV, greater than or equal to about -10 mV, greater than or equal to about 0 mV, greater than or equal to about 10 mV, greater than or equal to about 20 mV, greater than or equal to about 30 mV, greater than or equal to about 40 mV, greater than or equal to about 50 mV, greater than or equal to about 60 mV, or greater than or equal to about 75 mV.
  • the surface of the shell may have a zeta potential of less than or equal to about 75 mV, less than or equal to about 60 mV, less than or equal to about 50 mV, less than or equal to about 40mV, less than or equal to about 30 mV, less than or equal to about 20 mV, less than or equal to about 10 mV, less than or equal to about 0 mV, less than or equal to about -10 mV, less than or equal to about -20 mV, less than or equal to about -30 mV, less than or equal to about -40 mV, less than or equal to about -50 mV, less than or equal to about -60 mV, or less than or equal to about -75 mV.
  • the surface charge of the shell may be tailored by varying the surface chemistry and components of the shell.
  • a structure described herein or a portion thereof, such as a shell of a structure includes one or more natural or synthetic lipids or lipid analogs (i.e., lipophilic molecules).
  • One or more lipids and/or lipid analogues may form a single layer or a multi-layer (e.g., a bilayer) of a structure. In some instances where multi-layers are formed, the natural or synthetic lipids or lipid analogs interdigitate (e.g., between different layers).
  • Non- limiting examples of natural or synthetic lipids or lipid analogs include fatty acyls, glycerolipids, glycerophospholipids, sphingo lipids, saccharolipids and polyketides (derived from condensation of ketoacyl subunits), and sterol lipids and prenol lipids (derived from condensation of isoprene subunits).
  • a structure described herein includes one or more phospholipids.
  • the one or more phospholipids may include, for example, phosphatidylcholine, phosphatidylglycerol, lecithin, ⁇ , ⁇ -dipalmitoyl-a-lecithin, sphingomyelin, phosphatidylserine, phosphatidic acid, N-(2,3-di(9-(Z)-octadecenyloxy))-prop-l-yl-N,N,N-trimethylammonium chloride, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine,
  • dioleoylphosphatidylcholine dipalmitoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dioleoylphosphatidylglycerol, palmitoyl-oleoyl-phosphatidylcholine, di-stearoyl- phosphatidylcholine, stearoyl-palmitoyl-phosphatidylcholine, di-palmitoyl- phosphatidylethanolamine, di-stearoyl-phosphatidylethanolamine, di-myrstoyl- phosphatidylserine, di-oleyl-phosphatidylcholine, 1 ,2-dipalmitoyl-sn-glycero-3- phosphothioethanol, and combinations thereof.
  • a shell (e.g., a bilayer) of a structure includes 50-200 natural or synthetic lipids or lipid analogs (e.g., phospholipids).
  • the shell may include less than about 500, less than about 400, less than about 300, less than about 200, or less than about 100 natural or synthetic lipids or lipid analogs (e.g., phospholipids), e.g., depending on the size of the structure.
  • Non-phosphorus containing lipids may also be used such as stearylamine, docecylamine, acetyl palmitate, and fatty acid amides.
  • lipids such as fats, oils, waxes, cholesterol, sterols, fat-soluble vitamins (e.g., vitamins A, D, E and K), glycerides (e.g., monoglycerides, diglycerides, triglycerides) can be used to form portions of a structure described herein.
  • fat-soluble vitamins e.g., vitamins A, D, E and K
  • glycerides e.g., monoglycerides, diglycerides, triglycerides
  • a portion of a structure described herein such as a shell or a surface of a nanostructure may optionally include one or more alkyl groups, e.g., an alkane-, alkene-, or alkyne-containing species that optionally imparts hydrophobicity to the structure.
  • alkyl groups e.g., an alkane-, alkene-, or alkyne-containing species that optionally imparts hydrophobicity to the structure.
  • An "alkyl” group refers to a saturated aliphatic group, including a straight-chain alkyl group, branched-chain alkyl group, cycloalkyl (alicyclic) group, alkyl substituted cycloalkyl group, and cycloalkyl substituted alkyl group.
  • the alkyl group may have various carbon numbers, e.g., between C 2 and C 4 o, and in some embodiments may be greater than C 5 , Cio, C 15 , C 2 o, C 25 , C30, or C35.
  • a straight chain or branched chain alkyl may have 30 or fewer carbon atoms in its backbone, and, in some cases, 20 or fewer.
  • a straight chain or branched chain alkyl may have 12 or fewer carbon atoms in its backbone (e.g., Ci-Ci 2 for straight chain, C3-Ci 2 for branched chain), 6 or fewer, or 4 or fewer.
  • cycloalkyls may have from 3-10 carbon atoms in their ring structure, or 5, 6 or 7 carbons in the ring structure.
  • alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, tert- butyl, cyclobutyl, hexyl, cyclochexyl, and the like.
  • the alkyl group may include any suitable end group, e.g., a thiol group, an amino group (e.g., an unsubstituted or substituted amine), an amide group, an imine group, a carboxyl group, or a sulfate group, which may, for example, allow attachment of a ligand to a nanostructure core directly or via a linker.
  • the alkyl species may include a thiol group to form a metal-thiol bond.
  • the alkyl species includes at least a second end group.
  • the species may be bound to a hydrophilic moiety such as polyethylene glycol.
  • the second end group may be a reactive group that can covalently attach to another functional group.
  • the second end group can participate in a ligand/receptor interaction (e.g.,
  • the shell includes a polymer.
  • an amphiphilic polymer may be used.
  • the polymer may be a diblock copolymer, a triblock copolymer, etc., e.g., where one block is a hydrophobic polymer and another block is a hydrophilic polymer.
  • the polymer may be a copolymer of an a-hydroxy acid (e.g., lactic acid) and polyethylene glycol.
  • a shell includes a hydrophobic polymer, such as polymers that may include certain acrylics, amides and imides, carbonates, dienes, esters, ethers, fluorocarbons, olefins, sytrenes, vinyl acetals, vinyl and vinylidene chlorides, vinyl esters, vinyl ethers and ketones, and vinylpyridine and vinylpyrrolidones polymers.
  • a shell includes a hydrophilic polymer, such as polymers including certain acrylics, amines, ethers, styrenes, vinyl acids, and vinyl alcohols. The polymer may be charged or uncharged.
  • the particular components of the shell can be chosen so as to impart certain functionality to the structures.
  • a shell includes an amphiphilic material
  • the material can be arranged in any suitable manner with respect to the nanostructure core and/or with each other.
  • the amphiphilic material may include a hydrophilic group that points towards the core and a hydrophobic group that extends away from the core, or, the amphiphilic material may include a hydrophobic group that points towards the core and a hydrophilic group that extends away from the core. Bilayers of each configuration can also be formed.
  • the structures described herein may also include one or more proteins, polypeptides and/or peptides (e.g., synthetic peptides, amphiphilic peptides).
  • the structures include proteins, polypeptides and/or peptides that can increase the rate of cholesterol transfer or the cholesterol-carrying capacity of the structures.
  • the one or more proteins or peptides may be associated with the core (e.g., a surface of the core or embedded in the core), the shell (e.g., an inner and/or outer surface of the shell, and/or embedded in the shell), or both. Associations may include covalent or non-covalent interactions (e.g., hydrophobic and/or hydrophilic interactions, electrostatic interactions, van der Waals interactions).
  • An example of a suitable protein that may associate with a structure described herein is an apolipoprotein, such as apolipoprotein A (e.g., apo A-I, apo A-II, apo A-IV, and apo A-V), apolipoprotein B (e.g., apo B48 and apo B100), apolipoprotein C (e.g., apo C-I, apo C-II, apo C- III, and apo C-IV), and apolipoproteins D, E, and H.
  • apolipoprotein A e.g., apo A-I, apo A-II, apo A-IV, and apo A-V
  • apolipoprotein B e.g., apo B48 and apo B100
  • apolipoprotein C e.g., apo C-I, apo C-II, apo C- III, and apo C-IV
  • apo Ai , apo A 2 , and apo E promote transfer of cholesterol and cholesteryl esters to the liver for metabolism and may be useful to include in structures described herein.
  • a structure described herein may include one or more peptide analogues of an apolipoprotein, such as one described above.
  • a structure may include any suitable number of, e.g., at least 1, 2, 3, 4, 5, 6, or 10, apolipoproteins or analogues thereof.
  • a structure includes 1-6 apolipoproteins, similar to a naturally occurring HDL particle.
  • other proteins e.g., non-apolipoproteins
  • one or more enzymes may also be associated with a structure described herein.
  • lecithin-cholesterol acyltransferase is an enzyme that converts free cholesterol into cholesteryl ester (a more hydrophobic form of cholesterol).
  • cholesteryl ester is sequestered into the core of the lipoprotein, and causes the lipoprotein to change from a disk shape to a spherical shape.
  • structures described herein may include lecithin-cholesterol acyltransferase to mimic HDL and LDL structures.
  • Other enzymes such as cholesteryl ester transfer protein (CETP) which transfers esterified cholesterol from HDL to LDL species may also be included.
  • CETP cholesteryl ester transfer protein
  • the components described herein may be associated with a structure in any suitable manner and with any suitable portion of the structure, e.g., the core, the shell, or both.
  • one or more such components may be associated with a surface of a core, an interior of a core, an inner surface of a shell, an outer surface of a shell, and/or embedded in a shell.
  • such components can be used, in some embodiments, to facilitate the sequestration, exchange and/or transport of materials (e.g., proteins, peptides, polypeptides, nucleic acids, nutrients) from one or more components of a subject (e.g., cells, tissues, organs, particles, fluids (e.g., blood), and portions thereof) to a structure described herein, and/or from the structure to the one or more components of the subject.
  • the components have chemical and/or physical properties that allow favorable interaction (e.g., binding, adsorption, transport) with the one or more materials from the subject.
  • the components described herein may be associated with a structure described herein prior to administration to a subject or biological sample and/or after administration to a subject or biological sample.
  • a structure described herein includes a core and a shell that is administered in vivo or in vitro, and the structure has a greater therapeutic effect after sequestering one or more components (e.g., an apolipoprotein) from a subject or biological sample. That is, the structure may use natural components from the subject or biological sample to increase efficacy of the structure after it has been administered.
  • the synthetic nanostructures may be used in "pharmaceutical compositions" or “pharmaceutically acceptable” compositions, which comprise a therapeutically effective amount of one or more of the structures described herein, formulated together with one or more pharmaceutically acceptable carriers, additives, and/or diluents.
  • the pharmaceutical compositions described herein may be useful for treating cancer or other conditions. It should be understood that any suitable structures described herein can be used in such pharmaceutical compositions, including those described in connection with the figures.
  • the structures in a pharmaceutical composition have a nanostructure core comprising an inorganic material and a shell substantially surrounding and attached to the nanostructure core.
  • compositions may be specially formulated for administration in solid or liquid form, including those adapted for the following: oral administration, for example, drenches (aqueous or non-aqueous solutions or suspensions), tablets, e.g., those targeted for buccal, sublingual, and systemic absorption, boluses, powders, granules, pastes for application to the tongue; parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection as, for example, a sterile solution or suspension, or sustained-release formulation; topical application, for example, as a cream, ointment, or a controlled-release patch or spray applied to the skin, lungs, or oral cavity; intravaginally or intrarectally, for example, as a pessary, cream or foam; sublingually; ocularly; transdermally; or nasally, pulmonary and to other mucosal surfaces.
  • oral administration for example, drenches (aqueous or non-aqueous solutions or suspensions),
  • phrases "pharmaceutically acceptable” is employed herein to refer to those structures, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • phrases "pharmaceutically-acceptable carrier” as used herein means a
  • composition or vehicle such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • a liquid or solid filler such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body.
  • Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient.
  • materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ring
  • wetting agents such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
  • antioxidants examples include: water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium
  • antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like
  • metal chelating agents such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.
  • the structures described herein may be orally administered, parenterally administered, subcutaneously administered, and/or intravenously administered.
  • a structure or pharmaceutical preparation is administered orally.
  • the structure or pharmaceutical preparation is administered intravenously.
  • Alternative routes of administration include sublingual, intramuscular, and transdermal administrations.
  • compositions described herein include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and/or parenteral administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, and the particular mode of administration.
  • the amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, this amount will range from about 1% to about 99% of active ingredient, from about 5% to about 70%, or from about 10% to about 30%.
  • inventive compositions suitable for oral administration may be in the form of capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion, or as an elixir or syrup, or as pastilles (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes and the like, each containing a predetermined amount of a structure described herein as an active ingredient.
  • An inventive structure may also be administered as a bolus, electuary or paste.
  • the active ingredient is mixed with one or more pharmaceutically-acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the following: fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and/or acacia; humectants, such as glycerol; disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium compounds; wetting agents, such as, for example, cetyl alcohol, g
  • absorbents such as kaolin and bentonite clay
  • compositions such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and coloring agents.
  • the pharmaceutical compositions may also comprise buffering agents.
  • Solid compositions of a similar type may also be employed as fillers in soft and hard-shelled gelatin capsules using such excipients as lactose or milk sugars, as well as high molecular weight polyethylene glycols and the like.
  • a tablet may be made by compression or molding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared using binder (for example, gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (for example, sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Molded tablets may be made in a suitable machine in which a mixture of the powdered structure is moistened with an inert liquid diluent.
  • the tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention may optionally be scored or prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical-formulating art. They may also be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and/or microspheres. They may be formulated for rapid release, e.g., freeze-dried.
  • compositions may be sterilized by, for example, filtration through a bacteria-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions that can be dissolved in sterile water, or some other sterile injectable medium immediately before use.
  • These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredient(s) only, or in a certain portion of the gastrointestinal tract, optionally, in a delayed manner.
  • embedding compositions that can be used include polymeric substances and waxes.
  • the active ingredient can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.
  • Liquid dosage forms for oral administration of the structures described herein include pharmaceutically acceptable emulsions, microemulsions, solutions, dispersions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylene glycol, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
  • inert diluents commonly used in the art, such as, for example, water or other solvents,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, perfuming and preservative agents.
  • Suspensions in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.
  • Formulations of the pharmaceutical compositions described herein may be presented as a suppository, which may be prepared by mixing one or more compounds of the invention with one or more suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body and release the structures.
  • suitable nonirritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and which is solid at room temperature, but liquid at body temperature and, therefore, will melt in the body and release the structures.
  • Dosage forms for the topical or transdermal administration of a structure described herein include powders, sprays, ointments, pastes, foams, creams, lotions, gels, solutions, patches and inhalants.
  • the active compound may be mixed under sterile conditions with a pharmaceutically-acceptable carrier
  • the ointments, pastes, creams and gels may contain, in addition to the inventive structures, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.
  • Powders and sprays can contain, in addition to the structures described herein, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane.
  • Transdermal patches have the added advantage of providing controlled delivery of a structure described herein to the body. Dissolving or dispersing the structure in the proper medium can make such dosage forms. Absorption enhancers can also be used to increase the flux of the structure across the skin. Either providing a rate controlling membrane or dispersing the structure in a polymer matrix or gel can control the rate of such flux.
  • Ophthalmic formulations are also contemplated as being within the scope of this invention.
  • compositions described herein suitable for parenteral administration comprise one or more inventive structures in combination with one or more pharmaceutically- acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents.
  • aqueous and nonaqueous carriers examples include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate.
  • polyols such as glycerol, propylene glycol, polyethylene glycol, and the like
  • vegetable oils such as olive oil
  • injectable organic esters such as ethyl oleate.
  • Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.
  • These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents.
  • Prevention of the action of microorganisms upon the inventive structures may be facilitated by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.
  • Delivery systems suitable for use with structures and compositions described herein include time-release, delayed release, sustained release, or controlled release delivery systems, as described herein. Such systems may avoid repeated administrations of the structures in many cases, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art.
  • polymer based systems such as polylactic and/or polyglycolic acid, polyanhydrides, and polycaprolactone
  • nonpolymer systems that are lipid-based including sterols such as cholesterol, cholesterol esters, and fatty acids or neutral fats such as mono-, di- and triglycerides
  • hydrogel release systems silastic systems
  • peptide based systems wax coatings
  • compressed tablets using conventional binders and excipients or partially fused implants.
  • erosional systems in which the composition is contained in a form within a matrix, or diffusional systems in which an active component controls the release rate.
  • compositions may be as, for example, microspheres, hydrogels, polymeric reservoirs, cholesterol matrices, or polymeric systems.
  • the system may allow sustained or controlled release of the active compound to occur, for example, through control of the diffusion or erosion/degradation rate of the formulation.
  • a pump-based hardware delivery system may be used in some embodiments.
  • the structures and compositions described herein can also be combined (e.g., contained) with delivery devices such as syringes, pads, patches, tubes, films, MEMS-based devices, and implantable devices.
  • long-term release implant may be particularly suitable in some cases.
  • Long-term release means that the implant is constructed and arranged to deliver therapeutic levels of the composition for at least about 30 or about 45 days, for at least about 60 or about 90 days, or even longer in some cases.
  • Long-term release implants are well known to those of ordinary skill in the art, and include some of the release systems described above.
  • injectable depot forms can be made by forming microencapsule matrices of the structures described herein in biodegradable polymers such as polylactide-polyglycolide.
  • the rate of release of the structure can be controlled.
  • biodegradable polymers include poly(orthoesters) and poly(anhydrides).
  • the structures described herein are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, about 0.1% to about 99.5%, about 0.5%> to about 90%>, or the like, of structures in combination with a pharmaceutically acceptable carrier.
  • the administration may be localized (e.g., to a particular region, physiological system, tissue, organ, or cell type) or systemic, depending on the condition to be treated.
  • the composition may be administered through parental injection, implantation, orally, vaginally, rectally, buccally, pulmonary, topically, nasally, transdermally, surgical administration, or any other method of administration where access to the target by the composition is achieved.
  • parental modalities examples include intravenous, intradermal, subcutaneous, intracavity, intramuscular, intraperitoneal, epidural, or intrathecal.
  • implantation modalities include any implantable or injectable drug delivery system. Oral administration may be useful for some treatments because of the convenience to the patient as well as the dosing schedule.
  • compositions described herein may be given in dosages, e.g., at the maximum amount while avoiding or minimizing any potentially detrimental side effects.
  • the compositions can be administered in effective amounts, alone or in a combinations with other compounds.
  • a composition when treating cancer, a composition may include the structures described herein and a cocktail of other compounds that can be used to treat cancer.
  • a composition when treating conditions associated with abnormal lipid levels, a composition may include the structures described herein and other compounds that can be used to reduce lipid levels (e.g., cholesterol lowering agents).
  • a therapeutically effective amount means that amount of a material or composition comprising an inventive structure that is effective for producing some desired therapeutic effect in a subject at a reasonable benefit/risk ratio applicable to any medical treatment. Accordingly, a therapeutically effective amount may, for example, prevent, minimize, or reverse disease progression associated with a disease or bodily condition. Disease progression can be monitored by clinical observations, laboratory and imaging investigations apparent to a person skilled in the art.
  • a therapeutically effective amount can be an amount that is effective in a single dose or an amount that is effective as part of a multi-dose therapy, for example an amount that is administered in two or more doses or an amount that is administered chronically.
  • the effective amount of any one or more structures described herein may be from about 10 ng/kg of body weight to about 1000 mg/kg of body weight, and the frequency of
  • administration may range from once a day to once a month. However, other dosage amounts and frequencies also may be used as the invention is not limited in this respect.
  • a subject may be administered one or more structure described herein in an amount effective to treat one or more diseases or bodily conditions described herein.
  • An effective amount may depend on the particular condition to be treated.
  • the effective amounts will depend, of course, on factors such as the severity of the condition being treated; individual patient parameters including age, physical condition, size and weight; concurrent treatments; the frequency of treatment; or the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • a maximum dose be used, that is, the highest safe dose according to sound medical judgment.
  • the selected dosage level will depend upon a variety of factors including the activity of the particular inventive structure employed, the route of administration, the time of
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required.
  • the physician or veterinarian could start doses of the structures described herein employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and then gradually increasing the dosage until the desired effect is achieved.
  • a structure or pharmaceutical composition described herein is provided to a subject chronically.
  • Chronic treatments include any form of repeated
  • a chronic treatment involves administering a structure or pharmaceutical composition repeatedly over the life of the subject.
  • chronic treatments may involve regular administrations, for example one or more times a day, one or more times a week, or one or more times a month.
  • a suitable dose such as a daily dose of a structure described herein will be that amount of the structure that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above.
  • doses of the structures described herein for a patient when used for the indicated effects, will range from about 0.0001 to about 100 mg per kg of body weight per day.
  • the daily dosage may range from 0.001 to 50 mg of compound per kg of body weight, or from 0.01 to about 10 mg of compound per kg of body weight.
  • lower or higher doses can be used.
  • the dose administered to a subject may be modified as the physiology of the subject changes due to age, disease progression, weight, or other factors.
  • the effective daily dose of the active compound may be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • instructions and methods may include dosing regimens wherein specific doses of compositions, especially those including structures described herein having a particular size range, are administered at specific time intervals and specific doses to achieve reduction of cholesterol (or other lipids) and/or treatment of disease while reducing or avoiding adverse effects or unwanted effects.
  • kits any of the above-mentioned compositions useful for diagnosing, preventing, treating, or managing a disease or bodily condition packaged in kits, optionally including instructions for use of the composition. That is, the kit can include a description of use of the composition for participation in any disease or bodily condition, including those associated with abnormal lipid levels. The kits can further include a description of use of the compositions as discussed herein. The kit also can include instructions for use of a combination of two or more compositions described herein. Instructions also may be provided for administering the composition by any suitable technique, such as orally, intravenously, or via another known route of drug delivery.
  • kits described herein may also contain one or more containers, which can contain components such as the structures, signaling entities, and/or biomolecules as described.
  • the kits also may contain instructions for mixing, diluting, and/or administrating the compounds.
  • the kits also can include other containers with one or more solvents, surfactants, preservatives, and/or diluents (e.g., normal saline (0.9% NaCl), or 5% dextrose) as well as containers for mixing, diluting or administering the components to the sample or to the patient in need of such treatment.
  • compositions of the kit may be provided as any suitable form, for example, as liquid solutions or as dried powders.
  • the composition provided is a dry powder
  • the powder may be reconstituted by the addition of a suitable solvent, which may also be provided.
  • liquid form may be
  • the solvent will depend on the particular inventive structure and the mode of use or administration. Suitable solvents for compositions are well known and are available in the literature.
  • the kit in one set of embodiments, may comprise one or more containers such as vials, tubes, and the like, each of the containers comprising one of the separate elements to be used in the method.
  • one of the containers may comprise a positive control in the assay.
  • the kit may include containers for other components, for example, buffers useful in the assay.
  • a "subject" or a “patient” refers to any mammal (e.g., a human), for example, a mammal that may be susceptible to a disease or bodily condition such as a disease or bodily condition associated with abnormal lipid levels.
  • a disease or bodily condition such as a disease or bodily condition associated with abnormal lipid levels.
  • subjects or patients include a human, a non-human primate, a cow, a horse, a pig, a sheep, a goat, a dog, a cat or a rodent such as a mouse, a rat, a hamster, or a guinea pig.
  • the invention is directed toward use with humans.
  • a subject may be a subject diagnosed with a certain disease or bodily condition or otherwise known to have a disease or bodily condition.
  • a subject may be diagnosed as, or known to be, at risk of developing a disease or bodily condition.
  • a subject may be diagnosed with, or otherwise known to have, a disease or bodily condition associated with abnormal lipid levels, as described herein.
  • a subject may be selected for treatment on the basis of a known disease or bodily condition in the subject.
  • a subject may be selected for treatment on the basis of a suspected disease or bodily condition in the subject.
  • the composition may be administered to prevent the development of a disease or bodily condition.
  • the presence of an existing disease or bodily condition may be suspected, but not yet identified, and a composition of the invention may be administered to diagnose or prevent further development of the disease or bodily condition.
  • a "biological sample,” as used herein, is any cell, body tissue, or body fluid sample obtained from a subject.
  • body fluids include, for example, lymph, saliva, blood, urine, and the like.
  • Samples of tissue and/or cells for use in the various methods described herein can be obtained through standard methods including, but not limited to, tissue biopsy, including punch biopsy and cell scraping, needle biopsy; or collection of blood or other bodily fluids by aspiration or other suitable methods.
  • the present example demonstrates the inhibition of intercellular communication by contacting cells with synthetic nanostructure.
  • HDL NPs target SR- Bl, manipulate cellular cholesterol homeostasis, and modulate the uptake of exosomes by disrupting lipid rafts.
  • HDL NP Synthesis Biomimetic high-density lipoprotein-like nanoparticles (HDL NPs) were synthesized and characterized as previously described [Yang et al. 2013; Luthi et al. 2012; Thaxton et al. 2009]. Briefly, citrate stabilized 5 nm diameter gold nanoparticles (AuNP, Ted Pella) were used as a template for surface chemical modification. Purified human apolipoprotein Al (apoA-I) was incubated with a solution of AuNPs (80 nM) at 5-fold molar excess (400 nM, final) for 1 hour at room temperature (RT) with gentle stirring.
  • a solution of AuNPs 80 nM
  • 5-fold molar excess 400 nM, final
  • the phospholipids, 1-2- dipalmitoyl-sn-glycero-3-phosphocholine and 1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine-N-[3-(2-pyridyldithio)propionate] were added at 250 molar excess relative to [AuNP] in a mixture of ethanol and water (1 :4), and allowed to incubate at RT for 4 hours with gentle stirring.
  • the HDL NPs were then purified and concentrated using tangential flow filtration.
  • A375 melanoma cells (ATCC) and RAW 264.7 macrophages (ATCC) were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/ streptomycin.
  • DMEM Dulbecco's Modified Eagle Medium
  • HMVECs endothelial cell growth medium
  • Promocell a cell line.
  • Cells were incubated at 37 °C and in a humidified 5% C0 2 environment.
  • the GFP-SR-Bl plasmid [Neculai et al. 2013] was stably transfected in the A375 cells using Lipofectamine 2000 (Life Technologies) and transfectants were selected using Geneticin (Life Technologies) followed by fluorescent associated cell sorting (FACS).
  • Exosome isolation and labeling A375 melanoma exosomes were isolated from conditioned media using differential ultracentrifugation [Thery et al. 2006]. In brief, cells were cultured in exosome deficient media for 72 hours at which point the cell culture media was collected and centrifuged at 2000 x g to remove dead cells and debris. Next, larger vesicles and cell debris were removed by centrifugation at 10,000 x g for 30 minutes. Exosomes were then pelleted by centrifugation at 100,000 x g for 70 minutes, and subsequently washed in PBS by another 100,000 x g centrifugation step for 70 minutes. Exosomes were re-suspended in PBS.
  • the amount of apo A-I was calculated for hHDL assuming that 70% of the total protein is apo A-I [Rader et al. 2009].
  • the amount of apo A-I is equivalent for hHDL and HDL NP and, because each hHDL and HDL NP has approximately three copies of apo A-I [Huang et al. 2011], the dose of particles is assumed equivalent.
  • Exosome uptake assays The cellular uptake of exosomes was measured by fluorescence microscopy and flow cytometry after cell treatments. A375 cells, HMVECs and RAW 264.7 macrophages were treated with fluorescent exosomes at a concentration of 1 ⁇ g/ml (exosomal protein). For fluorescence microscopy experiments, cells were plated on coverslips coated with 0.1% gelatin. Exosome uptake was measured over the course of 24 hours using a BD LSR
  • Cholesterol and cholesterol ester quantification The total cholesterol and cholesteryl ester content of hHDLs and HDL NPs was measured using an Amplex Red cholesterol detection assay (Life Technologies). The free cholesterol content of each sample was measured in the absence and presence of cholesterol esterase to determine the free cholesterol and total cholesterol, respectively. Cholesteryl ester amount was determined by subtracting the free cholesterol from total cholesterol measurement. To determine the free and esterified cholesterol content of hHDL and HDL NPs before cell incubation we followed the protocol supplied by the manufacturer.
  • the free and esterified cholesterol content of the hHDL and HDL NP acceptors was measured after incubating with cultured A375 melanoma cells in serum free media and HDL NP (50 nM, final) or hHDL (50 nM, final) for 24 hours. After the treatment interval, the culture media was collected and centrifuged to rid the media of cells and cell debris. The total cholesterol and free cholesterol was then determined from conditioned media samples using the Amplex Red assay.
  • Cholesterol efflux assay A375 cells were cultured in DMEM containing 1 ⁇ / ⁇ ⁇ [1,2,- 3 H] cholesterol (Perkin-Elmer) overnight to label the cellular cholesterol pool. Cells were then washed in PBS and resuspended in serum free media. Human HDL or HDL NPs were added to the culture media and allowed to incubate for 6 hours. Cell culture media was then collected and subjected to liquid scintillation counting. The percentage of cholesterol efflux was determined by using the formula counts media/(counts cells + counts media) x 100. Efflux of cholesterol in the absence of an acceptor was also measured and interpreted along with other results.
  • the membranes were incubated with d primary antibodies (diluted in blocking solution) overnight at 4 °C, was washed 3 times in 0.1%> TBST (10 minutes/wash) and incubated with the appropriate HRP-conjugated secondary antibody in blocking buffer for 1 hour at room temperature. The membranes were then washed in 0.1% TBST (3 x 10 min) and developed with ECL kit (GE Healthcare).
  • Lipid raft labeling A375 lipid rafts were labeled using cholera toxin subunit b (CTx-B) conjugates with Alexafluor 488 or Alexafluor 647 to (Life Technologies) at a final concentration 1 ⁇ g/ml, for 30 minutes at 37 °C [Svensson et al. 2013]. The cells were then washed in PBS. And visualized using fluorescence microscopy.
  • Cx-B cholera toxin subunit b
  • Fluorescence microscopy Fluorescence microscopy was performed using an AIR confocal microscope with assistance from the Northwestern University Center for Advanced Microscopy. Images were analyzed using NIS Elements (Nikon) and ImageJ (NIH) software. Live cell confocal fluorescence microscopy to assess lipid raft dynamics was performed with a Nikon Eclipse Tl microscope equipped with an Andor iXon Ultra 897 camera and analyzed using Metamorph software (Molecular Devices).
  • High-density lipoproteins are dynamic natural nanostructures that function to sequester, transport, and deliver cholesterol [McMahon et al. 2011]. High-density lipoprotein- like nanoparticles are synthesized as described above. Comparison of HDL NPs to certain spherical hHDL species reveals similarities with regard to size, shape, surface chemistry, and negative surface charge [Luthi et al. 2012; McMahon & Thaxton 2014; Luthi et al. 2015]. Functionally, hHDLs bind SR-B1, which mediates the bi-directional flux of free cholesterol and the influx of esterified cholesterol to cells [Luthi et al. 2012; Van Eck et al.
  • HDL NPs have been shown to mediate bi-directional free cholesterol flux through SR-Bl [Yang et al. 2013; Luthi et al. 2012; Thaxton et al. 2009], the gold nanoparticle core of HDL NPs occupies the same physical space as esterified cholesterol and triglycerides in spherical hHDL. Occupying this space renders HDL NPs incapable of delivering to cells a payload of cholesteryl ester [Yang et al. 2013].
  • HDL NPs induce cholesterol efflux from melanoma cells at levels that exceed those observed for hHDL (Fig. 2b).
  • Cholesterol efflux is at least in part mediated by specific targeting of the SR-Bl receptor by hHDLs and HDL NPs, as treatment with Blocks Lipid Transport 1 (BLT-1), an inhibitor of SR-Bl -mediated cholesterol flux [Nieland et al. 2002], resulted in reduced efflux to both hHDLs and HDL NPs (Fig. 2b).
  • BLT-1 Blocks Lipid Transport 1
  • hHDLs and HDL NPs have increased free cholesterol (percent of total measured cholesterol); however, there is no measurable esterified cholesterol in HDL NPs versus hHDLs (Fig. 2a).
  • cell viability assays to ascertain whether treatment by HDL NPs reduced A375 cell viability. Data demonstrate that HDL NP treatment does not result in reduced viability (Fig. 3) even at doses above those that inhibit cellular exosome uptake (vide infra) at time points up to 72 hours.
  • cholesterol and cholesteryl ester-poor HDL NPs are not inherently toxic to A375 melanoma cells, target SR-Bl, and differentially modulate cholesterol flux through this receptor.
  • fluorescence confocal microscopy was used to visualize lipid rafts in A375 melanoma cells by labeling the rafts with cholera toxin subunit b (CTx-B) conjugated to Alexafluor-647.
  • Cx-B cholera toxin subunit b
  • SR-Bl stably expressing a green fluorescent protein-SR-Bl (GFP-SR-B1) fusion protein in the A375 cells.35
  • GFP-SR-B1 green fluorescent protein-SR-Bl
  • HDL NPs comprise a 5 nm diameter gold core and have the size, shape, and surface chemistry consistent with some hHDL species [Luthi et al. 2012].
  • these particles are capable of binding SR-Bl, resulting in the efflux of free cholesterol from cells, yet are unable to deliver esterified cholesterol. Therefore, we measured exosome uptake and SR-Bl clustering after treating A375 cells with: agents having an identical gold nanoparticle core, but with passive surface chemistry (polyethylene glycol nanoparticles, PEG NPs); the blocking Ab targeting SR-Bl [Gantman et al. 2010]; the small molecule inhibitor of free and esterified cholesterol flux through SR-Bl, BLT-1 [Nieland et al. 2002]; and siRNA targeting melanoma cell SR-Bl expression.
  • HDL NPs are the only targeted single-entity agent that leads to clustering of GFP-SR-B1 and potent inhibition of cellular exosome uptake.
  • Treatment of the cells with the PEG nanoparticle; hHDL SR-Bl blocking antibody; siRNA that reduces SR-Bl expression; or BLT-1 did not result in the inhibition of exosome uptake or clustering of the receptor (Fig. 1 la-1). Comparing the data obtained with the other agents to that for HDL NP alone demonstrates that occupying SR-Bl and modulating free and esterified cholesterol flux by the HDL NP particle functions to cluster SR- Bl and disrupt cellular exosome uptake.
  • melanoma exosomes are known to target endothelial and macrophage cells leading to activation of an angiogenic response [Hood et al. 2009], and modulation of the immune system [Filipazzi et al. 2012]. Therefore, we chose an endothelial cell line, human dermal microvascular endothelial cells (HMVECs), as a proof-of-concept system to assess SR-Bl expression and exosome uptake.
  • HMVECs human dermal microvascular endothelial cells
  • HMVECs Like A375 cells, HMVECs express SR-Bl (Fig. 4). Human dermal microvascular endothelial cells (HMVECs) were treated with Dil labeled A375 exosomes and analyzed using fluorescence microscopy with and without HDL NP treatment. Data demonstrate a decrease in cellular fluorescence suggesting that exosome uptake is blocked in HDL NP -treated HMVECs. Treatment with hHDL had minimal effect at decreasing exosome uptake. RAW 264.7 macrophages also express SR-B1 [Matveev et al.
  • HDL NPs decreased the uptake of exosomes in RAW 264.7 macrophages as demonstrated by fluorescence microscopy.
  • HDL NPs are a targeted and functional nanoconjugate that inhibit cellular vesicle uptake.
  • HDL NPs tightly bind to SR-B1 localized to lipid rafts and modulate free and esterified cholesterol flux through this receptor and the HDL NPs are responsible for clustering and stagnating SR-B1 at the cell membrane and dramatically reducing cellular exosome uptake.
  • HDL NPs are a targeted nanoparticle that may inhibit intercellular communication.
  • the present example demonstrates the preparation of synthetic nanostructures with an agent and without an agent and the loading of vesicles with these nanostructures.
  • HDL NPs were synthesized using 5nm citrate stabilized colloidal gold nanoparticles (BBI Solutions) incubated with five-fold molar excess human apolipoprotein AI (Meridian Life Sciences) for one hour with shaking.
  • Other lipids varied depending on the type of particle.
  • Fig 12A shows Rhodamine fluorescence from all groups; rhodamine fluorescence plotted on x-axis. Only exosomes from cells treated with the rhodamine HDL NPs display high rhodamine fluorescence (fourth panel).
  • Fig. 12B shows Exo-FITC stains all exosomes present on the beads. Fluorescence is present for all groups treated with exosomes but not beads with no exosomes, confirming that exosomes were present in all samples yet only the rhodamine exosomes contain the rhodamine tracer.
  • Exosomes with biotin HDL NPs were incubated with a Cy5-streptavidin antibody and analyzed by flow cytometry using the ExoFlow kit to confirm presence of biotin on the exterior of the exosome (Figure 13).
  • Streptavidin beads coated with biotinylated CD81 antibody were incubated with control exosomes (top row), HDL NP exosomes (middle row), and biotinylated HDL NP exosomes (bottom row). All beads were incubated with each type of exosome individually, then washed and incubated with a Cy5-streptavadin antibody. Beads were washed again and analyzed via flow cytometry. The beads naturally have a high affinity for streptavidin, and all groups were highly fluorescent for Cy5 (right group in scatter plot), with a small proportion with lower fluorescence, containing less non-specific interactions (left group).
  • Umemoto, T. et al. Apolipoprotein Al and high-density lipoprotein have antiinflammatory effects on adipocytes via cholesterol transporters: ATP-binding cassette A-l, ATP- binding cassette G-l, and scavenger receptor B-l . Circulation research 112, 1345-1354 (2013).

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Abstract

La présente invention concerne des nanostructures, des compositions et des procédés pour traiter des états liés à la vésicule ou liés à l'exosome. Dans certains cas, les nanostructures et/ou compositions peuvent être utilisées pour traiter des cancers, des troubles neurologiques, des troubles rhumatologiques, des troubles viraux ou d'autres maladies ou états au moins en partie par régulation de l'absorption de vésicule. L'invention concerne également des procédés d'analyse, d'imagerie et de modulation de vésicules et de processus de vésicules cellulaires.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2018168779A1 (fr) * 2017-03-14 2018-09-20 国立大学法人大阪大学 Biomarqueur de la bronchopneumopathie chronique obstructive
US10967072B2 (en) 2016-04-27 2021-04-06 Northwestern University Short interfering RNA templated lipoprotein particles (siRNA-TLP)
US11285106B2 (en) 2010-01-19 2022-03-29 Northwestern University Synthetic nanostructures including nucleic acids and/or other entities

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20060029655A1 (en) * 2001-06-25 2006-02-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Method for preparation of vesicles loaded with biological material and different uses thereof
US7018841B1 (en) * 1997-04-15 2006-03-28 Regents Of The University Of Michigan Compositions and methods for the inhibition of neurotransmitter uptake of synaptic vesicles
US20110268750A1 (en) * 2008-03-18 2011-11-03 Universite Montpellier 2 Sciences Et Techniques Chimeric polynucleotides and polypeptides enabling secretion of a polypeptide of interest in association with exosomes and use thereof for the production of immunogenic compositions
WO2012142568A2 (fr) * 2011-04-15 2012-10-18 Luc Montagnier Transmission à distance de signaux électromagnétiques induisant des nanostructures amplifiables dans une séquence adn spécifique
US20130034599A1 (en) * 2010-01-19 2013-02-07 Northwestern University Synthetic nanostructures including nucleic acids and/or other entities
US20130164740A1 (en) * 2011-12-27 2013-06-27 Abbott Laboratories Cellular Analysis of Body Fluids
US20140038901A1 (en) * 2011-04-01 2014-02-06 Cornell University Circulating exosomes as diagnostic/prognostic indicators and therapeutic targets of melanoma and other cancers

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7018841B1 (en) * 1997-04-15 2006-03-28 Regents Of The University Of Michigan Compositions and methods for the inhibition of neurotransmitter uptake of synaptic vesicles
US20060029655A1 (en) * 2001-06-25 2006-02-09 Yissum Research Development Company Of The Hebrew University Of Jerusalem Method for preparation of vesicles loaded with biological material and different uses thereof
US20110268750A1 (en) * 2008-03-18 2011-11-03 Universite Montpellier 2 Sciences Et Techniques Chimeric polynucleotides and polypeptides enabling secretion of a polypeptide of interest in association with exosomes and use thereof for the production of immunogenic compositions
US20130034599A1 (en) * 2010-01-19 2013-02-07 Northwestern University Synthetic nanostructures including nucleic acids and/or other entities
US20140038901A1 (en) * 2011-04-01 2014-02-06 Cornell University Circulating exosomes as diagnostic/prognostic indicators and therapeutic targets of melanoma and other cancers
WO2012142568A2 (fr) * 2011-04-15 2012-10-18 Luc Montagnier Transmission à distance de signaux électromagnétiques induisant des nanostructures amplifiables dans une séquence adn spécifique
US20130164740A1 (en) * 2011-12-27 2013-06-27 Abbott Laboratories Cellular Analysis of Body Fluids

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11285106B2 (en) 2010-01-19 2022-03-29 Northwestern University Synthetic nanostructures including nucleic acids and/or other entities
US10967072B2 (en) 2016-04-27 2021-04-06 Northwestern University Short interfering RNA templated lipoprotein particles (siRNA-TLP)
WO2018168779A1 (fr) * 2017-03-14 2018-09-20 国立大学法人大阪大学 Biomarqueur de la bronchopneumopathie chronique obstructive

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