Lyophilization buffer for peptide-modified liposomes

Abstract

Methods and compositions are provided for lyophilizing compositions comprising peptide-modified lipids.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 426,530, filed November 18, 2022, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with government support under Grant No. 1951301 awarded by the National Science Foundation and Grant No. W81XWH2010628 awarded by the Department of Defense. The government has certain rights in this invention. [Background technology]

[0003] Some biomolecules, including therapeutic compositions, reagents, and the like, have limited shelf life. Storing these compositions so that they retain the desired biological activity when used after storage typically requires significant expense and care. While it is possible to extend the shelf life of biomolecules by optimizing certain storage conditions, such as temperature and pH, long-term storage may still be unattainable due to storage conditions that result in structural modifications that render the biomolecule inactive. Thus, storage conditions for peptide-modified lipids (e.g., liposomes) are needed. Embodiments of the present invention address this and other important needs. Summary of the Invention

[0004] The present disclosure is based, at least in part, on the discovery that a lyophilization buffer containing cyclodextrin or a derivative thereof as a lyoprotectant maintains the size range and functionality of lyophilized lipid particles modified with binding peptides after reconstitution.

[0005] In certain embodiments, the present disclosure relates to a composition comprising a plurality of lipid particles and a liquid component, wherein the liquid component comprises water and a cyclodextrin at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component, and each lipid particle comprises a lipid and a peptide conjugate, wherein the peptide of the peptide conjugate is associated with the outer surface of the lipid particle.

[0006] In certain embodiments, the present disclosure relates to a composition comprising a plurality of lipid particles and a liquid component, wherein the liquid component comprises water and a lyoprotectant at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component, each lipid particle comprising a lipid and a plurality of peptide conjugates, wherein the peptide conjugates are selected from a platelet-binding peptide (PBP) conjugate, a von Willebrand factor-binding peptide (VBP) conjugate, and a collagen-binding peptide (CBP) conjugate, or a combination thereof, and wherein the PBP conjugate is a fibrinogen-mimetic peptide (FMP) conjugate or a P-selectin-binding peptide conjugate; wherein the plurality of PBP conjugates, VBP conjugates, and / or CBP conjugates are conjugated to the exterior surface of the particle, and wherein the PBP conjugates, VBP conjugates, and CBP conjugates, collectively, are present in less than 5 mole percent of the particle.

[0007] In certain embodiments, the present disclosure provides a method of forming a dry composition, comprising the steps of incubating any of the compositions described herein at a temperature of about 0 to about -210°C to form a cooled composition; incubating the cooled composition at a temperature of about 0 to about −60° C. and a pressure of about 0.1 to about 1.0 mTorr, thereby forming a dry composition; The present invention relates to a method, comprising:

[0008] In certain embodiments, the present disclosure relates to a composition made by any of the methods described herein.

[0009] In certain embodiments, the present disclosure relates to a method of forming a reconstituted composition, comprising contacting any of the compositions described herein with water, e.g., for less than about 30 seconds, thereby forming a reconstituted composition.

[0010] In certain embodiments, the present disclosure relates to a reconstituted composition made by any of the methods described herein.

[0011] In certain embodiments, the present disclosure relates to methods of attenuating bleeding, treating bleeding, treating vascular injury, promoting hemostasis, preventing or inhibiting platelet aggregation, promoting the aggregation of activated platelets at sites of exposed vWF and collagen, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of any of the reconstituted compositions described herein. [Brief explanation of the drawings]

[0012] [Figure 1A-1B] Figures 1A-1AA show the size range and images of lyophilized cakes of synthetic platelets lyophilized in various buffers containing Tris or HEPES as the buffer and sucrose, dextrose, or hydroxypropyl-beta-cyclodextrin (HP-β-CD) as the lyoprotectant (Post-Lyo, gray) and before lyophilization (Pre-Lyo, black). Figure 1A is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% dextrose and Tris (3 mg / mL). Figure 1B is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% dextrose and Tris (3 mg / mL). [Figure 1C] FIG. 1C is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after freeze-drying in a freeze-drying buffer of 10% dextrose and Tris (3 mg / mL). [Figure 1D-1E]Figure 1D is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a 10% dextrose and HEPES (2 mg / mL) lyophilization buffer. Figure 1E is an image of a lyophilized cake of synthetic platelets lyophilized in a 10% dextrose and HEPES (2 mg / mL) lyophilization buffer. [Figure 1F-1G] Figure 1F is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in 10% dextrose and HEPES (2 mg / mL) lyophilization buffer. Figure 1G is an image of a lyophilized cake of synthetic platelets lyophilized in 10% dextrose and HEPES (2 mg / mL) lyophilization buffer. [Figure 1H-1I] Figure 1H is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% HP-β-CD and Tris (3 mg / mL). Figure 1I is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% HP-β-CD and Tris (3 mg / mL). [Figures 1J-1K] Figure 1J is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% HP-β-CD and Tris (3 mg / mL). Figure 1K is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% HP-β-CD and Tris (3 mg / mL). [Figure 1L-1M] Figure 1L is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% HP-β-CD and HEPES (2 mg / mL). Figure 1M is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% HP-β-CD and HEPES (2 mg / mL). [Figures 1N-1O]Figure 1N is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% HP-β-CD and HEPES (2 mg / mL). Figure 1O is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% HP-β-CD and HEPES (2 mg / mL). [Figure 1P-1Q] Figure 1P is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in 5% sucrose and Tris (3 mg / mL) lyophilization buffer. Figure 1Q is an image of a lyophilized cake of synthetic platelets lyophilized in 5% sucrose and Tris (3 mg / mL) lyophilization buffer. [Figure 1R-1S] Figure 1R is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after freeze-drying in a 5% sucrose and Tris (3 mg / mL) freeze-drying buffer. Figure 1S is an image of a freeze-dried cake of synthetic platelets freeze-dried in a 5% sucrose and Tris (3 mg / mL) freeze-drying buffer. [Figure 1T-1U] Figure 1T is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a lyophilization buffer of 10% sucrose and HEPES (2 mg / mL). Figure 1U is an image of a lyophilized cake of synthetic platelets lyophilized in a lyophilization buffer of 10% sucrose and HEPES (2 mg / mL). [Figure 1V-1W] Figure 1V is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a 10% sucrose and HEPES (2 mg / mL) lyophilization buffer. Figure 1W is an image of a lyophilized cake of synthetic platelets lyophilized in a 10% sucrose and HEPES (2 mg / mL) lyophilization buffer. [Figure 1X-1Y]Figure 1X is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in 10% trehalose and HEPES (2 mg / mL) lyophilization buffer. Figure 1Y is an image of a lyophilized cake of synthetic platelets lyophilized in 10% trehalose and HEPES (2 mg / mL) lyophilization buffer. [Figure 1Z-1AA] Figure 1Z is an intensity diameter histogram showing the relative frequency of different intensity diameters for synthetic platelets before and after lyophilization in a 10% trehalose and HEPES (2 mg / mL) lyophilization buffer. Figure 1AA is an image of a lyophilized cake of synthetic platelets lyophilized in a 10% trehalose and HEPES (2 mg / mL) lyophilization buffer. [Figure 2A-2B] FIG. 1 shows intensity-diameter histogram plots of five replicate lots of lyophilized synthetic platelets (Post-Lyo, gray) and pre-lyophilized synthetic platelets (Pre-Lyo, black) containing 10% (w / v) HP-β-CD and 2 mg / mL HEPES pH 7.0 buffer. [Figures 2C-2D] FIG. 1 shows intensity-diameter histogram plots of five replicate lots of lyophilized synthetic platelets (Post-Lyo, gray) and pre-lyophilized synthetic platelets (Pre-Lyo, black) containing 10% (w / v) HP-β-CD and 2 mg / mL HEPES pH 7.0 buffer. [Figure 2E] FIG. 1 shows intensity-diameter histogram plots of five replicate lots of lyophilized synthetic platelets (Post-Lyo, gray) and pre-lyophilized synthetic platelets (Pre-Lyo, black) containing 10% (w / v) HP-β-CD and 2 mg / mL HEPES pH 7.0 buffer. [Figure 3A-3B] Figures 3A-3C characterize the synthetic platelets before freeze-drying (Pre-Lyo) and freeze-dried synthetic platelets (Post-Lyo). Figure 3A shows cryo-TEM images (13,000x magnification) of Pre-Lyo and Post-Lyo synthetic platelets. Figure 3B shows size histograms of Pre-Lyo and Post-Lyo synthetic platelets. [Figure 3C]FIG. 3C is a table of morphology statistics. [Figure 4A-1] Figures 4A-4C characterize the shelf life of freeze-dried synthetic platelets based on the physicochemical properties of particle size and zeta potential. Figure 4A shows the intensity hydrodynamic diameter histograms of freeze-dried synthetic platelets after storage and reconstitution for 28, 60, and 90 days at various temperatures, including -20°C, 4°C, ambient (room temperature, RT), and 50°C (dark gray), compared to pre-lyophilized synthetic platelets (Pre-Lyo). (Note: -20°C and 50°C are accelerated conditions, so the final time point was day 60.) [Figure 4A-2] Continued from Figure 4A. [Figure 4A-3] Continued from Figure 4A. [Figure 4A-4] Continued from Figure 4A. [Figure 4B-1] FIG. 1 summarizes the hydrodynamic diameter (nm), mean intensity diameter (nm), intensity diameter D90 (nm), PDI and zeta potential (mV) parameters before lyophilization and after lyophilization and storage at −20° C., 4° C., ambient (room temperature, RT) and 50° C. for 28, 60 and 90 days. [Figure 4B-2] Continued from Figure 4B. [Figure 4C] Figure 1 shows mean intensity hydrodynamic diameter values ​​over time (90 days) for freeze-dried synthetic platelets stored at -20° C. (filled squares), 4° C. (filled inverted triangles), ambient (room temperature, RT, filled circles), and 50° C. (filled triangles). The mean intensity diameter of the particles remains within + / - 25% of the pre-Lyo value (dashed horizontal line). [Figure 5A] Graph of the percentage of platelets bound to either synthetic platelets before lyophilization (Particle 1) or after lyophilization (Particle 1) in either 2 mg / mL HEPES or 3 mg / mL Tris + 10% (w / v) HP-β-CD (pH 7) as measured by flow cytometry. [Figure 5B]Graph of the mean Cy5 fluorescence of synthetic platelets before lyophilization (Particle 1) or after lyophilization (Particle 1) bound to platelets in either 2 mg / mL HEPES or 3 mg / mL Tris + 10% (w / v) HP-β-CD (pH 7) as measured by flow cytometry. [Figure 6A] 1 is a graph showing mouse tail bleeding times after administration of lipid particles described herein both before lyophilization and after reconstitution from a lyophilized state. [Figure 6B] 1 is a graph showing blood loss in mice following administration of lipid particles described herein, both before lyophilization and after reconstitution from the lyophilized state. [Figures 7A-7B] Figure 7A shows the zeta potential of reconstituted freeze-dried particles 1. Figure 7B shows the mean intensity diameter of reconstituted freeze-dried particles 1. [Figure 7C-7D] Figure 7C shows the particle concentration of reconstituted freeze-dried particles 1. Figure 7D shows the polydispersity index (PDI) of reconstituted freeze-dried particles 1. DETAILED DESCRIPTION OF THE INVENTION

[0013] In certain embodiments, the present disclosure relates to the use of specific buffers to effectively stabilize lipid particles (e.g., liposomes) having a polypeptide associated with their outer surface during lyophilization and to maintain the functionality and size of the lyophilized lipid particles after reconstitution. In particular, buffers comprising cyclodextrins are described herein. Accordingly, compositions are provided that include lipid particles having an associated polypeptide and a buffer comprising cyclodextrins. Methods for lyophilizing such compositions are also provided.

[0014] I. Definition In this document, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one or more elements.

[0015] The term "administering" is intended to include routes of administration that allow an agent (e.g., a composition described herein) to perform its intended function. Examples of routes of administration for bodily treatments that may be used include injection (subcutaneous, intravenous, parenteral, intraperitoneal, intrathecal, etc.), oral, inhalation, and transdermal routes. Injection may be a bolus injection or continuous infusion. Depending on the route of administration, the agent may be coated with or placed within a selected material to protect it from natural conditions that may adversely affect its ability to perform its intended function. The agent may be administered alone or in combination with a pharmaceutically acceptable carrier. The agent may also be administered as a prodrug that is converted to its active form in vivo. In some embodiments, the agent is administered orally. In other embodiments, the agent is administered via the anal and / or colorectal routes.

[0016] "About" and "approximately" generally refer to the degree of error acceptable for a measurement, given the nature or precision of the measurement. Typically, an exemplary degree of error is within 20%, preferably within 10%, and more preferably within 5% of a given value or range of values. Alternatively, particularly in biological systems, the terms "about" and "approximately" can refer to values ​​within 10-fold, preferably within 5-fold, and more preferably within 2-fold of a given value. Numerical values ​​given in this document are approximate unless otherwise stated, and can be interpreted as "about" or "approximately" unless explicitly stated.

[0017] A "conservative substitution" is the replacement of an amino acid with another amino acid that has similar physical and chemical properties, whereas a "non-conservative substitution" is the replacement of an amino acid with another amino acid that has different physical and chemical properties.

[0018] It will be understood that variations such as "comprise" or "comprises" or "comprising" are meant to imply the inclusion of a stated integer (or component) or group of integers (or components), but not the exclusion of other integers (or components) or groups of integers (or components).

[0019] As used in this document, "homology" is used synonymously with "identity."

[0020] As used herein, "homologous" refers to the similarity of subunit sequences between two polymeric molecules, e.g., between two nucleic acid molecules, e.g., between two DNA molecules or two RNA molecules, or between two polypeptide molecules. Two molecules are homologous at a subunit position if both positions are occupied by the same monomer subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, they are homologous at that position. A first region is homologous to a second region if at least one nucleotide residue position in each region is occupied by the same residue. Homology between two regions is expressed as the proportion of nucleotide residue positions in the two regions occupied by the same nucleotide residue. Homology between two sequences is a direct function of the number of matching or homologous positions. For example, if half of the positions in the sequences of two compounds (e.g., five positions in a polymer 10 subunits long) are homologous, the two sequences are 50% homologous. Two sequences share 90% homology if 90% of the positions, e.g., 9 out of 10 positions, are identical or homologous. For example, the DNA sequences 5'-ATTGCC-3' and 5'-TATGGC-3' share 50% homology.

[0021] As used in this document, "hydroxypropyl-β-cyclodextrin" or "HP-β-CD" refers to a C 63 H 112 O 42 Point to the numerator: [ka]

[0022] As used in this document, "hydroxypropyl-γ-cyclodextrin" refers to a C 72 H 128 O 48 Point to the numerator: [ka]

[0023] As used in this document, "hydroxypropyl cyclodextrin" refers to a hydroxypropyl cyclodextrin having the following structure: 39 H 66 O 31 Point to the numerator: [ka]

[0024] A "kit" is any article of manufacture (e.g., a package or container) containing at least one reagent (e.g., a probe or small molecule) for specifically detecting and / or affecting the expression of a marker. The kit may be promoted, distributed, or sold as a unit for performing the methods described herein. In certain embodiments, the kit may further include a reference standard. One of skill in the art can envision numerous such controls, including, but not limited to, common molecules. The reagents in the kit may be provided in individual containers or as a mixture of two or more reagents in a single container. Additionally, the kit may include instructional materials describing how to use the compositions in the kit.

[0025] A "mutant," "derivative," or "variant" of a polypeptide (or the DNA encoding it) is a polypeptide in which one or more amino acids (or one or more nucleotides) have been modified or changed so that the peptide (or nucleic acid) is not identical to the wild-type sequence but has homology to the wild-type polypeptide (or nucleic acid).

[0026] A "mutation" of a polypeptide (or the DNA encoding it) is an alteration or change of one or more amino acids (or one or more nucleotides) such that the peptide (or nucleic acid) is not identical to the wild-type sequence but is homologous to the wild-type polypeptide (or nucleic acid).

[0027] As used herein, "particle" is meant to include particles, spheres, capsules, and other structures having a length or diameter of about 10 nm to about 10 μm. In this application, the terms "nanosphere," "nanoparticle," "nanocapsule," "microsphere," "microparticle," "microcapsule," and "particle" are used interchangeably.

[0028] "Polypeptide" refers to a polymer of amino acid residues, related naturally occurring structural variants, and synthetic non-natural analogues thereof linked via peptide bonds, related naturally occurring structural variants, and synthetic non-natural analogues thereof. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer.

[0029] The term "protein" typically refers to large polypeptides.

[0030] The term "peptide" typically refers to a short polypeptide.

[0031] This document uses conventional notation to represent polypeptide sequences: the left-hand end of a polypeptide sequence is the amino-terminus and the right-hand end of a polypeptide sequence is the carboxyl-terminus.

[0032] A "portion" of a polypeptide means at least about 3 consecutive amino acid residues of the polypeptide. It is understood that a portion of a polypeptide can include any amino acid residue of the polypeptide.

[0033] As used in this document, a therapeutic agent that "prevents" a condition refers to a composition that, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control sample, or delays the onset of or reduces the severity of one or more symptoms of the disorder or condition relative to an untreated control sample.

[0034] A "recombinant polypeptide" is one produced by expression of a recombinant polynucleotide.

[0035] "Site" refers to the site of surface disruption, eg, injury, which disruption results in von Willebrand factor and collagen being present at that site.

[0036] The term "synergistic effect" refers to a combined effect of two or more agents described in this document that is greater than the sum of the separate effects of either agent alone.

[0037] The term "shelf life" refers to the period during which a lyophilized synthetic platelet composition can retain its physicochemical and biofunctional properties after reconstitution, e.g., within ±25%, compared to those before lyophilization. These biofunctional properties include the ability to bind to platelets, e.g., as demonstrated by flow cytometry, and the ability to provide hemostatic effects in vivo, e.g., as demonstrated by a thrombocytopenic mouse model.

[0038] The term "subject" refers to either a human or non-human animal. This term includes mammals, such as humans, primates, livestock (e.g., cows, pigs), companion animals (e.g., dogs, cats), and rodents (e.g., mice, rabbits, and rats).

[0039] "Treating" a disease in a subject or a subject having a disease refers to subjecting the subject to pharmaceutical treatment, e.g., administering a drug, to reduce or prevent the worsening of at least one symptom of the disease.

[0040] The term "therapeutic effect" refers to a local or systemic effect in animals, particularly mammals, and more particularly humans, caused by a pharmacologically active substance. This term therefore refers to any substance intended for use in animals or humans for the diagnosis, cure, mitigation, treatment, or prevention of disease, or the enhancement of desired physical or mental development and conditions. The term "therapeutically effective amount" refers to the amount of such a substance that produces some desired local or systemic effect at a reasonable risk-benefit ratio applicable to any treatment. In certain embodiments, a therapeutically effective amount of a compound depends on its therapeutic index, solubility, and the like. For example, a particular compound discovered by the methods of the present invention may be administered in an amount sufficient to produce a reasonable risk-benefit ratio applicable to such treatment.

[0041] Unless otherwise defined herein, scientific and technical terms used in this application have meanings commonly understood by those of ordinary skill in the art. Generally, the nomenclature and techniques relating to chemistry, molecular biology, cell and cancer biology, immunology, microbiology, pharmacology, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0042] Throughout this disclosure, various aspects of the invention may be expressed in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, a description of a range such as "1 to 6" should be considered to have specifically disclosed subranges, such as "1 to 3," "1 to 4," "1 to 5," "2 to 4," "2 to 6," "3 to 6," etc., as well as individual and partial numbers within that range (e.g., 1, 2, 3, 4, 5, 5.5, 6). This applies regardless of the breadth of the range.

[0043] Lyophilization buffer In some aspects of the present invention, a composition comprising lipid particles and a liquid component is provided. The liquid component may contain specific molecules, compounds, compositions, etc. useful for buffering the lyophilization of the lipid component. As used herein, the term "lyophilization buffer" refers to a composition that may be added to, combined with, or associated with a particle, molecule, or composition (e.g., a lipid particle comprising a polypeptide associated with the outer surface of the lipid particle) to be lyophilized. The buffer may stabilize the lyophilized composition so that the composition retains some, most, or all of its biological activity upon reconstitution. In some embodiments, the buffer may extend the shelf life of the lyophilized composition.

[0044] In some embodiments, a composition is provided comprising a liquid component and a plurality of lipid particles, the plurality of lipid particles comprising a plurality of polypeptides associated with the outer surface of the lipid particles. The liquid component can include elements that buffer the plurality of lipid particles during lyophilization. In some embodiments, the liquid component comprises water and a cyclodextrin. The cyclodextrin can be present at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component. In some embodiments, the cyclodextrin is a hydroxypropyl cyclodextrin. For example, in some embodiments, the cyclodextrin is a hydroxypropyl cyclodextrin (e.g., hydroxypropyl-α-cyclodextrin, hydroxypropyl-β-cyclodextrin (HP-β-CD), and hydroxypropyl-γ-cyclodextrin). In some embodiments, the liquid component comprises more than one type of hydroxypropyl cyclodextrin.

[0045] In some embodiments, the composition comprises an additional agent. For example, the compositions described herein may further comprise the zwitterionic buffer 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). The concentration of HEPES is about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.1% (w / v) to about 0.2% (w / v), about 0.2% (w / v) to about 0.5% (w / v), about 0.3% (w / v) to about 0.5% (w / v), or about 0.4% (w / v) to about 0.5% (w / v) of the lipid component. In some embodiments, the concentration of HEPES is 0.1% (w / v) to about 0.5% (w / v) of the liquid component. In some embodiments, the pH of the liquid component is about 4 to about 10, about 5 to about 10, about 6 to about 10, about 7 to about 10, about 8 to about 10, about 9 to about 10, about 5 to about 9, about 5 to about 8, about 5 to about 7, or about 5 to about 6.

[0046] In some embodiments, the composition further comprises tris(hydroxymethyl)aminomethane (Tris). The tris(hydroxymethyl)aminomethane (Tris) may be present at a concentration of about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.1% (w / v) to about 0.2% (w / v), about 0.2% (w / v), about 0.5% (w / v), about 0.3% (w / v) to about 0.5% (w / v), or about 0.4% (w / v) to about 0.5% (w / v) of the lipid component. In some embodiments, the concentration of tris(hydroxymethyl)aminomethane (Tris) is 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

[0047] In some embodiments, the composition comprises sucrose at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (w / v)).

[0048] In some embodiments, the composition comprises dextrose at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% (w / v)).

[0049] In some embodiments, the composition comprises from about 0.1% (w / v) to about 1.0% (w / v), from about 0.1% (w / v) to about 0.9% (w / v), from about 0.1% (w / v) to about 0.8% (w / v), from about 0.1% (w / v) to about 0.7% (w / v), from about 0.1% (w / v) to about 0.6% (w / v), from about 0.1% (w / v) to about 0.5% (w / v), from about 0.1% (w / v) to about 0.4% (w / v), or from about 0.1% (w / v) of the liquid component. The sodium chloride concentration is about 0.3% (w / v) to about 0.3% (w / v), about 0.2% (w / v) to about 1.0% (w / v), about 0.3% (w / v) to about 1.0% (w / v), about 0.4% (w / v) to about 1.0% (w / v), about 0.5% (w / v) to about 1.0% (w / v), about 0.6% (w / v) to about 1.0% (w / v), about 0.7% (w / v) to about 1.0% (w / v), or about 0.8% (w / v) to about 1.0% (w / v).

[0050] In some embodiments, the composition comprises ammonium sulfate at a concentration of about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.1% (w / v) to about 0.2% (w / v), about 0.2% to about 0.5% (w / v), about 0.3% (w / v) to about 0.5% (w / v), or about 0.4% (w / v) to about 0.5% (w / v) of the liquid component.

[0051] In some embodiments, the composition comprises ammonium sulfate at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

[0052] In some embodiments, the composition comprises L-histidine at a concentration of about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.1% (w / v) to about 0.2% (w / v), about 0.2% (w / v), about 0.5% (w / v), about 0.3% (w / v) to about 0.5% (w / v), or about 0.4% (w / v) to about 0.5% (w / v) of the liquid component. In some embodiments, the L-histidine is at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

[0053] In some embodiments, the composition comprises lactose monohydrate at a concentration of about 0.1% (w / v) to about 0.5% (w / v), about 0.1% (w / v) to about 0.4% (w / v), about 0.1% (w / v) to about 0.3% (w / v), about 0.1% (w / v) to about 0.2% (w / v), about 0.2% (w / v) to about 0.5% (w / v), about 0.3% (w / v) to about 0.5% (w / v), or about 0.4% (w / v) to about 0.5% (w / v) of the liquid component. In some embodiments, the lactose monohydrate is at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

[0054] In some embodiments, the composition does not include at least one of HEPES, tris(hydroxymethyl)aminomethane (Tris), sucrose, dextrose, sodium chloride, ammonium sulfate, L-histidine, and lactose monohydrate.

[0055] In some embodiments, the dry lyophilized powder components comprise 49% (w / w) to 98% (w / w) of lyoprotectant cyclodextrin and its derivatives, such as, but not limited to, hydroxypropyl-β-cyclodextrin (HP-β-CD), and 1% (w / w) to 4% (w / w) of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer, with a pH range of 5 to 9. In certain embodiments, the lyophilization buffers described herein maintain lipid particle (e.g., liposome) diameters in the range of 30 nm to 300 nm after lyophilization. In certain embodiments, these lyophilization buffers also maintain at least some of the functionality of the modified binding peptide for binding to activated platelets by flow cytometry. The use of HP-β-CD and the corresponding maintenance of size range after lyophilization of liposomes contrasts with alternative lyoprotectants, such as sucrose, dextrose, and trehalose, which result in an increase in the size range of lyophilized peptide-modified liposomes. In some embodiments, between 1% (w / w) and 4% (w / w) tris(hydroxymethyl)aminomethane (Tris) can be used.

[0056] lipid particles The compositions disclosed herein include lipid particles comprising a lipid and a peptide conjugate, wherein the peptide of the peptide conjugate is associated with the outer surface of the lipid particle. In some embodiments, the lipid of the peptide conjugate is DSPE-PEG(2k)-maleimide.

[0057] The size of the lipid particles in the composition may vary, for example, from about 1 nm to about 1000 nm, from about 1 nm to about 900 nm, from about 1 nm to about 800 nm, from about 1 nm to about 700 nm, from about 1 nm to about 600 nm, from about 1 nm to about 500 nm, from about 1 nm to about 400 nm, from about 1 nm to about 300 nm, from about 1 nm to about 200 nm, from about 1 nm to about 100 nm, from about 1 nm to about 50 nm, from about 1 nm to about 25 nm, from about 25 nm to about 100 The diameter may be about 0 nm, about 50 nm to about 1000 nm, about 100 nm to about 1000 nm, about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, or about 900 nm to about 1000 nm. In some embodiments, the lipid particles have an average diameter of about 10 nm to about 500 nm, about 50 nm to about 500 nm, about 100 nm to about 500 nm, about 200 nm to about 500 nm, about 300 nm to about 500 nm, about 400 nm to about 500 nm, about 20 nm to about 400 nm, about 20 nm to about 300 nm, about 20 nm to about 200 nm, about 20 nm to about 100 nm, or about 20 nm to about 50 nm. In some embodiments, the lipid particles have an average diameter of about 30 nm to about 300 nm.

[0058] In some embodiments, the lipid particles have a polydispersity index of about 0.1 to about 0.5, about 0.2 to about 0.5, about 0.3 to about 0.5, about 0.4 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, or about 0.1 to about 0.2. In some embodiments, the lipid particles have a polydispersity index of 0.1, 0.2, 0.3, 0.4, or 0.5 or less. In some embodiments, the lipid particles have a polydispersity index of 0.3 or less.

[0059] In some embodiments, the lipid particles have a substantially spherical morphology.

[0060] In some embodiments, the lipid particles have a net positive or net negative zeta potential.

[0061] The lipid particles comprise a peptide associated with the outer surface of the lipid particle. In some embodiments, the lipid particles comprise a phospholipid conjugated to polyethylene glycol (PEG). In some embodiments, the PEG conjugated to the phospholipid is from about 100 Da to about 10,000 Da, from about 500 Da to about 10,000 Da, from about 1,000 Da to about 10,000 Da, from about 2,000 Da to about 10,000 Da, from about 3,000 Da to about 10,000 Da, from about 4,000 Da to about 10,000 Da, from about 5,000 Da to about 10,000 Da, from about 6,000 Da to about 10,000 Da, or from about 7,000 Da to about 10,000 Da. In some embodiments, the PEG conjugated to the phospholipid has an average molecular weight of about 500 Da to about 5500 Da.

[0062] In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE). In some embodiments, the lipid particle comprises cholesterol.

[0063] In some embodiments, the lipid particle is a liposome.

[0064] peptide In some embodiments, lipid particles (e.g., liposomes) are modified with peptides. The peptide modifications of the liposomes include phospholipids conjugated to polyethylene glycol (PEG) and phospholipids conjugated to a binding peptide, which are formulated with the phospholipid-PEG, phospholipid, and optionally, lipid. These phospholipids include, for example, distearoylphosphatidylcholine (DSPC) and its derivatives, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) and its derivatives. In some embodiments, the lipid comprises cholesterol and its derivatives. In some embodiments, the phospholipid-PEG conjugate comprises PEG having a molecular weight of 1000 Da to 2000 Da.

[0065] In some embodiments, each lipid particle comprises multiple peptides associated with the outer surface of the lipid particle. For example, the lipid particle may comprise a first peptide and a second peptide. In some embodiments, the lipid particle may also comprise a third peptide. The first peptide may have affinity for a first target, the second peptide may have affinity for a second target, and the third peptide, if present, may have affinity for a third target.

[0066] In some embodiments, the polypeptide-modified lipid particle is a synthetic platelet.

[0067] In certain embodiments, the peptide is: Platelet-binding proteins (PBPs), such as P-selectin-binding peptide (DAEWVDVS (SEQ ID NO: 5)), or fibrinogen-mimetic peptides (FMPs), such as FMPs of formula (I) (also referred to herein as FMP2; cyclo-(CNPRGD{Tyr(OEt)}R-(-A) (SEQ ID NO: 1)), FMP1 (cyclo-{Pra}CNPRGD{Tyr(OEt)}RC (SEQ ID NO: 2)), linear RGD (GRGDSP (SEQ ID NO: 3)), and H12 (HHLGGAKQAGDV (SEQ ID NO: 4)); Collagen-binding peptide (CBP), such as (GPO)7 (SEQ ID NO: 8); or von Willebrand binding protein (VBP), e.g., TRYLRIHPQSWVHQI (SEQ ID NO: 6), or a combination thereof, or a conjugate thereof, or a combination of conjugates thereof. Additionally, compositions and methods for using PBP, CBP, and VBP and particles conjugated with these peptides are provided.

[0068] Platelet-binding proteins (PBPs) Fibrinogen mimetic peptide (FMP) As used herein, the terms "fibrinogen-mimetic peptide" and "activated platelet GPIIb-IIIa binding peptide" are used interchangeably in the present disclosure. In some embodiments, the fibrinogen-mimetic peptide has the formula (I): Cyclo(CNPRGD{Tyr(OEt)}R-β-A) (SEQ ID NO: 1) (referred to in this document as "FMP2"), or a salt thereof is provided herein.

[0069] In some embodiments, the fibrinogen-mimetic peptide is FMP1 having the formula: cyclo(CNPRGD{Tyr(OEt)}R-β-C) (SEQ ID NO: 2), or a salt thereof.

[0070] In some embodiments, the FMP is linear RGD (GRGDSP (SEQ ID NO: 3)).

[0071] In some embodiments, the FMP is H12 (HHLGGAKQAGDV (SEQ ID NO: 4)).

[0072] In some embodiments, the fibrinogen-mimetic peptide salt is an acetate salt or a trifluoroacetate salt. In some embodiments, the fibrinogen-mimetic peptide specifically binds to activated GPIIb-IIIa. In some embodiments, the fibrinogen-mimetic peptide inhibits platelet aggregation. In some embodiments, the fibrinogen-mimetic peptide has a concentration of less than 30 μM, for example, less than 25 μM, less than 20 μM, less than 15 μM, less than 10 μM, less than 5 μM, less than 1 μM, less than 0.5 μM, less than 0.1 μM, less than 0.05 μM, or 4.5×10 -2 Less than 4.0 × 10 -2 Less than 3.5 × 10 -2 Less than 3.0 × 10 -2 Less than 2.5 × 10 -2 Less than 2.0 × 10 -2 Less than 1.5 × 10 -2 μM, 1.0 × 10 -2 Less than 0.5 × 10 -2 Less than 0.1 × 10 -2 Less than 0.5 × 10 -3 Less than 0.1 × 10 -3 IC, e.g., less than μM 50 In certain embodiments, the fibrinogen-mimetic peptide has an IC of about 0.13 μM. 50 It has.

[0073] In some embodiments, the fibrinogen-mimetic peptides disclosed herein are conjugated to a polymer (e.g., lipid, protein, etc.). Thus, in some embodiments, a fibrinogen-mimetic peptide conjugate is provided, comprising a fibrinogen-mimetic peptide of SEQ ID NO: 1, 2, 3, or 4 conjugated to a polymer. In some embodiments, the polymer is a lipid (e.g., DSPE-PEG(2k)-maleimide). In some embodiments, the fibrinogen-mimetic peptide is conjugated to the polymer by thio-ene coupling to a thiol group on an existing or added N-terminal cysteine ​​or 3-mercaptopropionic acid. In some embodiments, the surface of a particle (e.g., synthetic platelet) comprises an FMP peptide, or a salt thereof.

[0074] In some embodiments, FMP may contain an RGD amino acid sequence motif that promotes active platelet aggregation. The RGD motif comprising FMP may contain a single repeat of the RGD motif, or may contain multiple repeats of the RGD motif, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeats of the RGD motif. Those skilled in the art will understand that conservative substitutions of specific amino acid residues in the RGD motif comprising FMP may be used, as long as the RGD motif comprising FMP retains the binding ability equivalent to that of the native RGD motif. Those skilled in the art will understand that conservative substitutions of specific amino acid residues adjacent to the RGD motif may be used, as long as the RGD motif comprising FMP retains the binding ability equivalent to that of the native RGD motif.

[0075] In some embodiments, the FMP can be a fibrinogen-mimetic peptide (FMP) described herein. In some embodiments, the FMP is of formula (I). The cyclic peptide of formula (I) has high selectivity and affinity for GPIIb-IIIa on activated platelets, but does not bind to or activate inactive platelets, and does not interact with other RGD-binding integrins. FMP can be synthesized using Fmoc-based solid-phase chemistry on Knorr resin and characterized using mass spectrometry.

[0076] Advantageously, the FMPs can each contain about 5 to about 30 amino acids. By limiting the size of the peptides to about 5 to about 30 amino acids, the FMPs can be spatially or topographically arranged on the flexible particle surface, not spatially obscuring each other, and can promote the arrest and aggregation of activated platelets at the site of injury.

[0077] P-selectin binding peptide As used herein, the term "P-selectin-binding peptide" refers to a protein or peptide that binds with high affinity (e.g., nano- to micromolar affinity) to P-selectin on platelets, e.g., a phage-display-derived peptide such as an N-terminal glycosulfopeptide mimetic of PSGL-1 (the natural ligand for P-selectin) or an EWVDV-containing peptide. In a specific embodiment, the P-selectin-binding peptide has the amino acid sequence DAEWVDVS (SEQ ID NO: 5).

[0078] In some embodiments, the P-selectin-binding peptides disclosed herein are conjugated to a polymer (e.g., lipid, protein, etc.). Thus, in some embodiments, a P-selectin-binding peptide conjugate is provided comprising the P-selectin-binding peptide of SEQ ID NO: 5 conjugated to a polymer. In some embodiments, the polymer is a lipid (e.g., DSPE-PEG(2k)-maleimide). In some embodiments, the P-selectin-binding peptide is conjugated to the polymer by thiol-ene coupling to the thiol group of an added N-terminal cysteine ​​or 3-mercaptopropionic acid. In some embodiments, the surface of a particle (e.g., synthetic platelet) comprises the P-selectin-binding peptide, or a salt thereof.

[0079] In some embodiments, the P-selectin-binding peptide may be a peptide described herein. In certain embodiments, the P-selectin-binding peptide has the amino acid sequence DAEWVDVS (SEQ ID NO: 5). Peptides having the amino acid sequence of SEQ ID NO: 5 may have high selectivity and affinity for P-selectin on activated platelets. P-selectin-binding peptides may be synthesized using FMoc-based solid-phase chemistry on Knorr resin and characterized using mass spectrometry.

[0080] Advantageously, the P-selectin-binding peptides can each contain about 5 to about 30 amino acids. By limiting the size of the peptides to about 5 to about 30 amino acids, the P-selectin-binding peptides can be spatially or topographically arranged on the flexible particle surface, not spatially obscuring each other, and can promote the arrest and aggregation of activated platelets at the site of injury.

[0081] von Willebrand binding peptide (VBP) As used herein, the term "von Willebrand binding peptide" refers to a protein or peptide that binds to von Willebrand factor with high affinity (e.g., nano- to micromolar affinity). Because von Willebrand factor has multiple binding domains, a VBP can consist of a peptide that binds to the D'D3 domain (e.g., factor FVIII-derived peptide), the A1 or A3 domain (e.g., collagen-derived / mimetic peptide), or the A1 or C4 domain (e.g., platelet GPIb or GPIIb-IIIa-derived peptide). In a specific embodiment, the VBP has the amino acid sequence TRYLRIHPQSWVHQI (SEQ ID NO: 6).

[0082] In some embodiments, the VBP disclosed herein is conjugated to a polymer (e.g., a lipid, a protein, etc.). Thus, in some embodiments, a VBP conjugate is provided comprising a VBP of SEQ ID NO: 6 conjugated to a polymer. In some embodiments, the polymer is a lipid (e.g., DSPE-PEG(2k)-maleimide). In some embodiments, the VBP is conjugated to the polymer by thiol-ene coupling to the thiol group of an added N-terminal cysteine ​​or 3-mercaptopropionic acid. In some embodiments, the surface of a particle (e.g., a synthetic platelet) comprises a VBP or a salt thereof.

[0083] In some embodiments, the VBP peptide for binding to vWF can comprise a recombinant GPIbα fragment (rGPIbα) containing the vWF-binding site (residues 1 to 302) or a short vWF-binding peptide. Using the described methods, the GPIbα fragment can be expressed in CHO cells and isolated. The short VBP can comprise the amino acid sequence TRYLRIHPQSWVHQI (SEQ ID NO: 6). A peptide having the amino acid sequence SEQ ID NO: 6 can be synthesized using solid-phase chemistry on a fluorenylmethyloxycarbonyl chloride (FMoc)-based Knorr resin and characterized by mass spectrometry. Each vWF molecule has only one binding site for this peptide; therefore, sites of vascular injury presenting multiple vWF-binding sites for multiple copies of this peptide modified on particle surfaces provide a mechanism for increased particle adhesion with increasing shear.

[0084] Collagen-binding peptide (CBP) As used herein, the term "collagen-binding peptide" refers to a protein or peptide that binds to collagen with high affinity (e.g., nano- to micromolar affinity), such as a collagen-derived sequence (e.g., GPO repeat) with helicogenic affinity for collagen, or a collagen-binding peptide obtained experimentally (e.g., phage display or isothermal titration chemistry). In a specific embodiment, the CBP has the amino acid sequence (GPO)7 (SEQ ID NO:7).

[0085] In some embodiments, the CBP disclosed herein is conjugated to a polymer (e.g., a lipid, a protein, etc.). Thus, in some embodiments, a CBP conjugate is provided comprising the CBP of SEQ ID NO: 7 conjugated to a polymer. In some embodiments, the polymer is a lipid (e.g., DSPE-PEG(2k)-maleimide). In some embodiments, the CBP is conjugated to the polymer by thiol-ene coupling to the thiol group of an added N-terminal cysteine ​​or 3-mercaptopropionic acid. In some embodiments, the surface of a particle (e.g., synthetic platelets) comprises CBP, or a salt thereof.

[0086] In some embodiments, CBPs can contain short repeats of the tripeptide GPO, which has helicogenic affinity for fibrillar collagen, such as peptides containing (GPO)7, SEQ ID NO:7. GPO trimers are based on the amino acid repeats found in native collagen structures. It has been reported that platelet activation, typically triggered by interaction with collagen through GPVI and GPIa / IIa, can also occur when platelets interact with collagen-derived peptides. This poses a potential problem for modifying synthetic particle surfaces with collagen-derived peptides for collagen binding, because in vivo, these structures could interact with resting platelets, activating them systemically and presenting a thromboembolic risk. However, the interaction of platelet receptors with collagen and the subsequent platelet activation mechanism depend on receptor clustering induced by polymeric long-chain triple-helical fibrillar collagen, rather than short collagen-mimetic peptide repeats. Indeed, it has been shown that GPO trimer repeats as high as 30-mer (10 repeats) only partially interact with platelet GPIa / IIa and GPVI integrins and are unable to activate platelets; however, they can effectively bind to fibrillar collagen via helicogenic interactions. Therefore, this small CBP can promote adhesion to fibrillar collagen but, due to the lack of a long triple helix structure, cannot activate inactive platelets. CBPs similar to VBP can also be synthesized by FMoc-based solid-phase chemistry on Knorr resin and characterized by mass spectrometry.

[0087] Synthetic Platelets and Uses In some aspects, the present disclosure relates to lipid particles that function as synthetic platelets. In certain embodiments, the particles are conjugated to multiple platelet-binding peptides (PBPs) (e.g., FMP1 peptide, FMP2 peptide, linear RGD peptide, H12 peptide, and / or P-selectin-binding peptide described herein, CBP, and VBP). For example, in some embodiments, the particles are conjugated to FMP1 peptide. In some embodiments, the particles are conjugated to FMP2 peptide. In some embodiments, the particles are conjugated to linear RGD peptide. In some embodiments, the particles are conjugated to H12 peptide. In some embodiments, the particles are conjugated to P-selectin-binding peptide. In some embodiments, the particles are conjugated to CBP. In some embodiments, the particles are conjugated to VBP. Methods of using these particles to reduce bleeding and blood loss are provided, as are compositions and methods useful for delivering therapeutic agents to the vasculature. The synthetic platelets described herein integrate platelet-mimetic adhesive and aggregation-promoting functions into a single, flexible particle. We have demonstrated that platelet-mimicking adhesion and aggregation-promoting functions can be achieved by incorporating, conjugating, or modifying flexible particles with three peptides: VBP, CBP, and PBP. First, we demonstrated that liposomes bearing VBP and CBP motifs exhibited platelet-mimicking adhesion to vWF- and collagen-coated surfaces under low to high shear in parallel plate flow chamber (PPFC) experiments in vitro. Furthermore, we demonstrated that PBP-modified liposomes pre-adhered to a surface could promote the aggregation of ADP-activated platelets on the surface, even at low platelet concentrations. Subsequently, we demonstrated that liposomes bearing all three peptides (VBP, CBP, and PBP) adhered to a vWF / collagen mixed-coated surface under high shear when introduced into a PPFC flow with a low concentration of ADP-activated platelets, and promoted the arrest and aggregation of activated platelets at the liposome adhesion site.In some embodiments, the PBP included in the synthetic platelets is a fibrinogen-mimetic peptide (FMP) described herein. In some embodiments, the FMP is FMP1, FMP2, linear RGD, H12, or a combination thereof. In some embodiments, the PBP included in the synthetic platelets is a P-selectin-binding peptide described herein.

[0088] Thus, it is an aspect of the present application that the synthetic platelets described herein may be administered, for example, intravenously, to a subject with vascular injury to reduce the subject's bleeding time. It is a further aspect of the present application that the synthetic platelets provide a nanostructure that binds to the site of vascular injury in the same way as activated platelets, increasing their rate of adhesion and aggregation to help stop bleeding.

[0089] In some embodiments, the synthetic platelets described herein can comprise a biocompatible, biodegradable, flexible particle core and multiple VBPs, CBPs, and PBPs bound, conjugated, and / or modified to a surface defined by the flexible particle core. The VBPs, CBPs, and PBPs can be spatially or topographically arranged on the flexible particle surface such that the VBPs, CBPs, and PBPs are not spatially obscured from one another and vWF and collagen can adhere to exposed vascular surfaces, sites of vascular disease, and / or sites of vascular injury, promoting the binding and aggregation of activated platelets to the particle adhesion sites.

[0090] The biocompatible, biodegradable flexible particles can be made from any biocompatible, biodegradable material that can form flexible particles to which the peptides described herein can be attached, conjugated, and / or modified. In some embodiments, the biocompatible, biodegradable flexible particles can include liposomes, hydrogels, micelles, and / or polymers, and can include or be surface-modified or engineered with VBP, CBP, and PBP.

[0091] Liposomes or hydrogels may contain lipids and / or any natural, synthetic, or semi-synthetic (i.e., modified natural) moieties, which are generally amphiphilic (i.e., containing hydrophilic and hydrophobic elements). Examples of lipids may include fatty acids, neutral lipids, phospholipids, oils, glycolipids, surfactants, fatty alcohols, waxes, terpenes, and steroids. Semi-synthetic or modified natural lipids may include natural lipids that have been chemically modified in some manner. At least one lipid may be neutral, negatively charged (i.e., anionic), or positively charged (i.e., cationic). Examples of anionic lipids may include: phosphatidic acid, phosphatidylglycerol and their fatty acid esters, amides of phosphatidylethanolamine (e.g., anandamide and methanandamide), phosphatidylserine, phosphatidylinositol and their fatty acid esters, cardiolipin, phosphatidylethylene glycol, acidic lysolipids, sulfolipids and sulfatides, saturated and unsaturated free fatty acids, and their negatively charged derivatives. Examples of cationic lipids can include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride, and common naturally occurring lipids derivatized to contain one or more basic functional groups.

[0092] Examples of other lipids that may be used, either alone or in combination, to form particles may include: Phosphocholines, such as 1-alkyl-2-acetoyl-sn-glycero-3-phosphocholines and 1-alkyl-2-hydroxy-sn-glycero-3-phosphocholines; Phosphatidylcholines having both saturated and unsaturated lipids, such as dioleoylphosphatidylcholine, dimyristoylphosphatidylcholine, dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), and diarachidonylphosphatidylcholine (DAPC); Phosphatidylethanolamines, such as dioleoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine (DPPE), and distearoylphosphatidylethanolamine (DSPE); phosphatidylserine; phosphatidylglycerol (including distearoylphosphatidylglycerol (DSPG)); phosphatidylinositol; sphingolipids, such as sphingomyelin; glycolipids, such as gangliosides GM1 and GM2; glucolipids; sulfatides; glycosphingolipids; phosphatidic acids, such as dipalmitoylphosphatidic acid (DPPA) and distearoylphosphatidic acid (DSPA); palmitic acid; stearic acid; arachidonic acid; oleic acid; lipids with polymers, such as chitin, hyaluronic acid, polyvinylpyrrolidone or poly ethylene glycol (PEG); lipids having sulfonated monosaccharides, disaccharides, oligosaccharides, or polysaccharides; cholesterol, cholesterol sulfate, and cholesterol hemisuccinate; tocopherol hemisuccinate; lipids having ether- and ester-linked fatty acids; polymeric lipids (a wide variety of which are known in the art); diacetyl phosphate; dicetyl phosphate; stearylamine; cardiolipin; phospholipids having short-chain fatty acids of about 6 to about 8 carbon atoms in length; synthetic phospholipids having asymmetric acyl chains, e.g., one acyl chain having about 6 carbon atoms and the other having about 12 carbon atoms; ceramides; non-ionic liposomes, including niosomes, such as polyoxyalkylene (e.g., polyoxyethylene) fatty acid esters, polyoxyalkylene (e.g., polyoxyethylene) fatty alcohols, polyoxyalkylene (e.g., polyoxyethylene) fatty alcohol ethers, polyoxyalkylene (e.g., polyoxyethylene) sorbitan fatty acid esters (e.g., the class of compounds known as TWEEN® (available from ICI Americas, Inc., Wilmington, Del.)), glycerol polyethylene glycol oxystearate, glycerol polyethylene glycol ricinoleate, alkyloxylated (e.g., ethoxylated) soybean sterols, alkyloxylated (e.g., ethoxylated) castor oil, polyoxyethylene-polyoxypropylene polymers, and polyoxyalkylene (e.g., polyoxyethylene) fatty acid stearates; Sterol fatty acid esters (including cholesterol sulfate, cholesterol butyrate, cholesterol isobutyrate, cholesterol palmitate, cholesterol stearate, lanosterol acetate, ergosterol palmitate, and phytosterol n-butyrate); sterol esters of sugar acids (including cholesterol glucuronide, lanosterol glucuronide, 7-dehydrocholesterol glucuronide, ergosterol gluconide, cholesterol gluconate, lanosterol gluconate, and ergosterol gluconate); Esters of sugar acids and alcohols (including lauryl glucuronide, stearoyl glucuronide, myristoyl gluconate, lauryl gluconate, myristoyl gluconate, and stearoyl gluconate); Esters of sugars and fatty acids (including sucrose laurate, fructose laurate, sucrose palmitate, sucrose stearate, glucuronic acid, gluconic acid, and polyuronic acids); Saponins (including sarsasapogenin, smilagenin, hederagenin, oleanolic acid, and digitoxigenin); Glycerol dilaurate, glycerol trilaurate, glycerol dipalmitate, glycerol and glycerol esters (including glycerol tripalmitate, glycerol distearate, glycerol tristearate, glycerol dimyristate, glycerol trimyristate); Long-chain alcohols (including n-decyl alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, and n-octadecyl alcohol); 6-(5-cholesten-3-yloxy)-1-thio- -D-galactopyranoside; Digalactosyl diglyceride; 6-(5-cholesten-3-yloxy)hexyl-6-amino-6-deoxy-1-thio- -D-galactopyranoside; 6-(5-cholesten-3-yloxy)hexyl-6-amino-6-deoxy-1-thio-α-D-mannopyranoside; 12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoic acid; N-[12-(((7′-diethylaminocoumarin-3-yl)carbonyl)methylamino)octadecanoyl]-2-aminopalmitic acid; Cholesteryl (4'-trimethylammonio) butanoate; N-succinyldioleoylphosphatidylethanolamine; 1,2-dioleoyl-sn-glycerol; 1,2-dipalmitoyl-sn-3-succinylglycerol; 1,3-dipalmitoyl-2-succinylglycerol; 1-hexadecyl-2-palmitoylglycerophosphoethanolamine and palmitoylhomocysteine; and / or any combination thereof.

[0093] Examples of biocompatible, biodegradable polymers that can be used to form the particles are: polylactide, polyglycolide, polylactide-glycolide copolymer, polylactic acid, polyglycolic acid, polylactic-glycolic acid copolymer, polycaprolactone, polycarbonate, polyesteramide, polyanhydride, polyamino acid, polyorthoester, polyacetyl, polycyanoacrylate, polyetherester, polydioxanone, polyalkylene alkylate, copolymers of polyethylene glycol and polylactide or polylactide-glycolide copolymer, biodegradable polyurethanes, and mixtures and / or copolymers thereof.

[0094] Other examples of materials that may be used to form the particles may include chitosan, polyethylene oxide, polylactic acid, polyacrylic acid, polyvinyl alcohol, polyurethane, poly N-isopropylacrylamide, polyvinylpyrrolidone (PVP), polymethacrylic acid, poly p-styrenecarboxylic acid, poly p-styrenesulfonic acid, polyvinylsulfonic acid, polyethyleneimine, polyvinylamine, polyanhydrides, poly L-lysine, poly L-glutamic acid, poly gamma-glutamic acid, polycarprolactone, polylactide, polyethylene, polypropylene, polyglycolide, polylactide-glycolide copolymer, polyamide, polyhydroxy acid, polysulfone, polyamine, polysaccharide, polyHEMA, polyanhydrides, gelatin, glycosaminoglycans (GAGs), polyhyaluronic acid, sodium polyalginate, alginic acid, albumin, hyaluronic acid, agarose, polyhydroxybutyric acid (PHB), copolymers thereof, and mixtures thereof.

[0095] The flexible particles can have a maximum length or diameter of about 100 nm to about 10 μm and a substantially spherical, discoid, and / or ellipsoid shape. The particle's physical size and shape, as well as mechanical properties, can be designed to mimic those of natural platelets, which are important in hemostasis. In some embodiments, the flexible particles can have a discoid shape with a diameter of about 2 to about 5 μm and a mechanical modulus of about 10 to about 50 kPa, which mimics the size, shape, and modulus of platelets and promotes margination and biointeraction with the vascular wall upon administration to a subject's vasculature.

[0096] In one embodiment of the present application, oblate ellipsoidal particles having a diameter of about 2 to about 5 μm and a mechanical modulus of about 10 to about 50 kPa can be prepared by first forming a polymer template. This polymer template can then be used to construct a protein / polymer shell using cross-linked layer-by-layer assembly. The polymer template can then be removed using a solvent, leaving soft, flexible proteinaceous discoidal particles having a diameter of about 2 to about 5 μm and a mechanical modulus of about 10 to about 50 kPa. The particles can then be surface-modified with VBP, CBP, and PBP at a surface density effective to promote maximum particle adhesion to exposed vWF and collagen surfaces at low to high shear stresses and promote the aggregation of activated platelets even at low (less than about 50,000 platelets per μl) platelet concentrations.

[0097] For example, spherical poly-l-lactide-glycolide copolymer (PLGA) particles with diameters of about 2 to about 3 μm can be embedded in a polyvinyl alcohol (PVA) film (e.g., about 5% w / v in water) containing 2% (v / v) glycerol as a plasticizer and biaxially stretched to twice their original length and width in an oven at about 65°C. The film can be removed from the stretcher, and the PVA can be dissolved in 15% isopropanol, followed by thorough washing with isopropanol to completely remove the PVA. This results in the recovery of flattened PLGA particles that can be resuspended in distilled water or PBS. These template particles can then be coated with layers of protein and polyelectrolyte using a layer-by-layer (LBL) method. For this purpose, the protein serum albumin (SA, e.g., human serum albumin or mouse serum albumin) and the polyelectrolyte polyallylamine hydrochloride (PAH) at a concentration of 2 mg / mL can be used for adsorption. At the pH used, albumin is negatively charged and PAH is cationic, allowing alternating layers of SA and PAH to form on PLGA template particles through electrostatic interactions. Multiple alternating layers (e.g., at least seven) can be formed on the flat template and intermittently crosslinked with glutaraldehyde for increased stability. The particles can then be exposed to a solvent mixture (e.g., 2:1 tetrahydrofuran:isopropanol) to dissolve the PLGA core, leaving behind a flexible, disc-shaped shell of soft SA / PAH layer-by-layer. The outermost layer can contain albumin, which can facilitate the attachment of the PEGylated peptides described herein.

[0098] VBP, CBP, and PBP can be conjugated to particle surfaces by reacting the peptides with maleimide-terminated lipids (e.g., maleimide-PEG-DSPE) through the thiol group on the existing or added N-terminal cysteine ​​or 3-mercaptopropionic acid. The lipid-peptide conjugates can then be incorporated into lipophilic particles, such as liposomes, using known formulation techniques.

[0099] VBP, CBP, and PBP can be conjugated to the particle surface by reacting the peptide through its N-terminus with the carboxyl terminus of a heterobifunctional PEG (e.g., maleimide-PEG-COOH). The PEG-peptide conjugate or PEGylated peptide can then be conjugated to the particle using known conjugation techniques.

[0100] The PEG molecules can have a variety of lengths and molecular weights, including, for example, PEG200, PEG1000, PEG1500, PEG2000, PEG4600, PEG10,000, or combinations thereof. In other embodiments, VBP, CBP, and PBP can be conjugated to the particle surface by PEG acrylate or PEG diacrylate molecules of various molecular weights.

[0101] In one example, VBP, CBP, and PBP can be reacted with maleimide-PEG-COOH to form Mal-PEG-peptide conjugates. SA / PAH particles with albumin as the outermost layer can then be treated with dithiothreitol (DTT) to introduce a high density of sulfhydryl (-SH) groups onto the surface. Mal-PEG-peptides can then be incubated with the DTT-treated particles, allowing the MAL termini to react with the free -SH groups, forming particles with various peptides modified on the particle surface via PEG linkers.

[0102] The relative amount of peptide conjugated to the particle surface can affect the efficiency of the particle's hemostatic activity. In some embodiments, the molar percentage of PBP, CBP, and / or VBP conjugated to the particle surface is less than 5 molar percent. In some embodiments, the molar percentage of PBP, CBP, and / or VBP is between 5% and about 0.5%, between 5% and about 1%, between 5% and about 2%, between 5% and about 3%, or between 5% and about 4%, including the lower end of the range excluding 5%. In some embodiments, the molar percentage of PBP, CBP, and / or VBP is between about 4% and about 0.1%, between about 3% and about 0.1%, between about 2% and about 0.1%, between about 1% and about 0.1%, between about 4% and about 0.5%, between about 3% and about 0.5%, between about 2% and about 0.5%, or between about 1% and about 0.5%.

[0103] The ratio of VBP to CBP conjugated to the particle surface can be about 70:30 to about 30:70, and can be adjusted accordingly to maximize adhesion under low- to high-shear conditions. In some embodiments, the ratio of VBP:CPB:PBP can be about 1:1:2 to 1:2:1 to 2:1:1. In some embodiments, the relative molar ratio of PBP:CBP:VBP is 1:5:5. In some embodiments, the relative molar ratio is between about 1:1:1 and about 1:5:1, between about 1:1:1 and about 1:1:5, between about 1:1:1 and about 1:5:5, between about 1:1:1 and about 5:1:1, between about 1:1:1 and about 5:5:1, or between about 1:1:1 and about 5:1:5. In some embodiments, the relative molar ratio of PBP:CBP:VBP is about 2:1:1, about 1:5:5, about 10:5:1, about 10:1:5, about 1:2:1, about 1:1:2, about 10:1:1, about 2:1:0, about 2:0:1, about 1:0:0, about 0:1:0, about 0:0:1, or about 0:1:1, or any ratio between any two of these ratios. It will be understood that other ratios may also be used to enhance particle adhesion and activated platelet aggregation.

[0104] In some embodiments, compositions comprising the synthetic platelets described herein may be formulated and administered to an animal, preferably a human, in need of reducing or slowing blood loss. In other embodiments, compositions comprising the synthetic platelets described herein may be formulated and administered to an animal, preferably a human, to facilitate delivery of a therapeutic agent.

[0105] In some embodiments, the synthetic platelets described herein may be provided as a pharmaceutical composition. Such a pharmaceutical composition may consist solely of the synthetic platelets in a form suitable for administration to a subject, or the pharmaceutical composition may include the synthetic platelets in addition to one or more pharmaceutically acceptable carriers, one or more additional components, one or more pharmaceutically acceptable therapeutic agents, bioactive agents, diagnostic agents, or some combination thereof. The therapeutic agent may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, e.g., in combination with a physiologically acceptable cation or anion, as is well known in the art.

[0106] As used in this document, the term "pharmaceutically acceptable carrier" means a chemical composition that can be combined with a therapeutic agent and, after such combination, can be used to administer the therapeutic agent to a subject.

[0107] As used herein, a "physiologically acceptable" ester or salt refers to an ester or salt form of a therapeutic agent that is compatible with the other ingredients of a pharmaceutical composition and is not deleterious to the subject to which the composition is administered.

[0108] In some embodiments, bioactive, diagnostic, and / or therapeutic agents may be conjugated, encapsulated, and / or contained with the synthetic platelets, thereby allowing the synthetic platelets to act as a delivery vehicle. In other embodiments, the bioactive, diagnostic, and / or therapeutic agent may simply be included in a pharmaceutical composition that includes (or does not include) the synthetic platelets and administered simultaneously with (or separately from) the administration of the synthetic platelets. The choice of bioactive, diagnostic, and / or therapeutic agent to be conjugated or encapsulated in the synthetic platelets depends on the use of the synthetic platelets and / or the condition to be treated, as well as the site and route of administration.

[0109] Bioactive agents encapsulated and / or conjugated to synthetic platelets may include any substance capable of exerting a biological effect in vitro and / or in vivo. Examples of bioactive agents include, but are not limited to, biologically active ligands, small molecules, proteins, DNA fragments, DNA plasmids, interfering RNA molecules such as siRNA, mRNA, oligonucleotides, and DNA encoding shRNA. Diagnostic agents may include any substance that may be used to image a region of interest (ROI) in a subject and / or diagnose the presence or absence of a disease or diseased tissue in a subject. Therapeutic agents may refer to any therapeutic or prophylactic agent used to treat (including prevent, diagnose, alleviate, or cure) a malady, affliction, condition, disease, or injury in a subject. It will be understood that the membrane may additionally or optionally include proteins, carbohydrates, polymers, surfactants, and / or other membrane-stabilizing materials, any or combination of which may be natural, synthetic, or semi-synthetic.

[0110] The methods of treatment using synthetic platelets described herein include administering a therapeutically effective amount of synthetic platelets to a subject in need thereof. It should be understood that the methods of treatment by delivery of synthetic platelets include not only treatment of subjects already experiencing bleeding, but also prophylactic treatment applications for subjects not yet experiencing bleeding. In a preferred embodiment, the subject is an animal. In a more preferred embodiment, the subject is a human.

[0111] In some aspects, methods of treating a subject having or suspected of having cancer are provided, comprising administering to the subject a pharmaceutical composition comprising the particles described herein. In some embodiments, the pharmaceutical composition comprises the particles and an anti-cancer therapeutic agent. In some embodiments, the particles encapsulate or are conjugated to the anti-cancer agent.

[0112] In some aspects, a method of preventing or inhibiting platelet aggregation in a subject in need thereof is provided, the method comprising administering to the subject a pharmaceutical composition comprising a fibrinogenic-mimetic peptide as described herein.

[0113] The embodiments described herein should in no way be construed as being limited to the synthetic platelets described herein, but rather should be construed to include the use of additional synthetic platelets, both known and unknown, that attenuate or reduce bleeding or blood loss.

[0114] The formulations of the pharmaceutical compositions described herein can be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods include the step of bringing the synthetic platelets into association with, or with one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into the desired single or multi-dose unit.

[0115] While the description of pharmaceutical compositions provided herein is primarily directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by those skilled in the art that such compositions are generally intended for administration to any type of animal. Modifications of pharmaceutical compositions intended for human administration to make them suitable for administration to a variety of animals are well known, and a veterinary pharmacist of ordinary skill can design and implement such modifications with no more than routine experimentation, if any. Subjects to which the pharmaceutical compositions of the present invention are contemplated include, but are not limited to, humans and other primates; animals, including commercially important animals (e.g., cows, pigs, horses, sheep, cats, dogs); and birds, including commercially important birds (e.g., chickens, ducks, geese, turkeys).

[0116] Pharmaceutical compositions useful in the methods described herein may be administered, prepared, packaged, and / or sold in formulations for parenteral, oral, rectal, vaginal, topical, transdermal, pulmonary, intranasal, buccal, ocular, or other routes of administration.

[0117] The compositions described herein may be administered by a number of routes, including, but not limited to, parenteral, oral, rectal, vaginal, topical, transdermal, pulmonary, intranasal, buccal, or ocular routes of administration. The route of administration will be readily apparent to one of skill in the art and will depend on a number of factors, including the type and severity of the disorder being treated, the type and age of the animal or human patient being treated, and the like.

[0118] Parenteral administration of a pharmaceutical composition includes any route of administration characterized by physically breaking through the tissue of a subject and administering the pharmaceutical composition through that tissue breakthrough. Parenteral administration therefore includes, but is not limited to, administering a pharmaceutical composition by injection of the composition, by applying the composition onto or through a surgical incision, by applying the composition onto or through a non-surgical wound that penetrates the tissue, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, cutaneous, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, and intra-arterial administration.

[0119] A pharmaceutical composition formulation suitable for parenteral administration comprises a therapeutic agent combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampoules or multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, and emulsions in oily or aqueous vehicles. Such formulations may further comprise one or more additional ingredients, including, but not limited to, suspending agents, stabilizers, and dispersing agents. In one embodiment of a formulation for parenteral administration, the therapeutic agent is provided in a dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

[0120] Pharmaceutical compositions useful in the methods described herein may be administered systemically as oral solid formulations, ophthalmic formulations, suppositories, aerosols, topical formulations, or other similar formulations. In addition to compounds such as heparin sulfate, or bioequivalents thereof, such pharmaceutical compositions may contain pharmaceutically acceptable carriers and other ingredients known to enhance and facilitate administration.

[0121] The pharmaceutical compositions described herein may also be formulated to provide delayed, sustained, or controlled release. Generally, controlled release preparations are pharmaceutical compositions that can release synthetic platelets at a desired or required rate to maintain constant activity for a desired or required period of time.

[0122] The pharmaceutical compositions described herein may be prepared, packaged, or sold in bulk, as a single unit dose, or as a plurality of single unit doses. As used herein, a "unit dose" refers to a discrete amount of a pharmaceutical composition containing a predetermined amount of an active. The amount of active is generally equal to the dose administered to a subject or a convenient fraction of that dose (e.g., one-half or one-third of that dose).

[0123] The relative amounts of the components in the pharmaceutical compositions of the present invention will vary depending on the identity, size, and condition of the subject being treated, as well as the route by which the composition is administered. As a non-limiting example, the composition may contain from 0.1% to 100% (w / w) synthetic platelets.

[0124] The synthetic platelet compositions described herein may be administered to deliver a dose of about 1 ng / kg / day to about 100 mg / kg / day. In one embodiment, a dose may be administered to result in a synthetic platelet concentration in the mammal's blood of about 0.01 μg / mL to about 625 μg / mL. The exact dose administered will vary depending on numerous factors, including, but not limited to: the type of animal, the amount of bleeding being treated, the type of bleeding being treated, the type of wound being treated, the age of the animal, and the route of administration. Preferably, the synthetic platelet dose will vary from about 1 μg to about 50 mg per kilogram of animal body weight. More preferably, the dose will vary from about 10 μg to about 15 mg per kilogram of animal body weight. Even more preferably, the dose will vary from about 100 μg to about 10 mg per kilogram of animal body weight.

[0125] Pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to known techniques and may contain, in addition to the therapeutic agent, additional ingredients (e.g., dispersing agents, wetting agents, or suspending agents described herein). Such sterile injectable formulations may be prepared using a non-toxic parenterally-acceptable diluent or solvent (e.g., water or 1,3-butanediol). Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution, and fixed oils (e.g., synthetic mono- or di-glycerides).

[0126] As used herein, "additional ingredients" includes, but is not limited to, one or more of the following: excipients; surfactants; dispersing agents; inert diluents; granulating and disintegrating agents; binders; lubricants; sweeteners; flavoring agents; coloring agents; preservatives; physiologically degradable compositions such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersing or wetting agents; emulsifying agents; analgesics; buffers; salts; thickening agents; fillers; emulsifiers; antioxidants; antibiotics; antifungal agents; stabilizers; and pharmaceutically acceptable polymeric or hydrophobic materials. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Genaro, ed., 1985, Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., the contents of which are incorporated herein by reference.

[0127] The pharmaceutical composition can be administered to the animal as needed. The pharmaceutical composition can be administered to the animal as frequently as several times a day, or less frequently (e.g., once a day, once a week, once every two weeks, once a month, etc.), or even less frequently, such as once every few months, or even once a year or less frequently. The frequency of administration is readily apparent to those skilled in the art and depends on many factors, including, but not limited to, the type and severity of the disease being treated, the type and age of the animal, etc.

[0128] Freeze-drying method In one aspect of the present invention, a method of forming a dry composition is provided, comprising the steps of: incubating a composition at a temperature of about 0 to about −210° C. to form a cooled composition; and incubating the cooled composition at a temperature of about 0 to about −60° C. and a pressure of about 0.1 to about 1.0 mTorr, thereby forming a dry composition. In some embodiments, the temperature to which the composition is cooled is about 0 to about −250° C., about 0 to about −200° C., about 0 to about −150° C., about 0 to about −100° C., about 0 to about −50° C., about −50 to about −250° C., about −100 to about −250° C., about −150 to about −250° C., about −150 to about −250° C., or about −200 to about −250° C. In some embodiments, the cooled composition is subjected to a temperature of about 0 to about −100° C., about 0 to about −90° C., about 0 to about −80° C., about 0 to about −70° C., about 0 to about −60° C., about 0 to about −50° C., about 0 to about −40° C., about 0 to about −30° C., about 0 to about −20° C., about 0 to about −10° C., about −10 to about −100° C., about −20 to about −100° C., about −30 to about −100° C., about −40 to about −100° C., about −50 to about −100° C., about −60 to about −100° C., about −70 to about −100° C., about −80 to about −100° C., or about −90 to about −100° C. In some embodiments, the cooled composition is subjected to the above-described temperatures at a pressure of about 0.1 to about 1.0 mTorr. In some embodiments, the cooled composition is subjected to a temperature at a pressure of about 0.1 to about 0.9 mTorr, about 0.1 to about 0.8 mTorr, about 0.1 to about 0.7 mTorr, about 0.1 to about 0.6 mTorr, about 0.1 to about 0.5 mTorr, about 0.1 to about 0.4 mTorr, about 0.1 to about 0.3 mTorr, about 0.1 to about 0.2 mTorr, about 0.2 to about 0.9 mTorr, about 0.3 to about 0.9 mTorr, about 0.4 to about 0.9 mTorr, about 0.5 to about 0.9 mTorr, about 0.6 to about 0.9 mTorr, about 0.7 to about 0.9 mTorr, or about 0.8 to about 0.9 mTorr.

[0129] In some embodiments, the lipid particles in the dry composition have a diameter of from about 1 nm to about 1000 nm. In some embodiments, the lipid particles in the dry composition have a diameter of about 100 nm to about 1000 nm, about 100 nm to about 900 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 400 nm, about 100 nm to about 300 nm, about 100 nm to about 200 nm, about 100 nm to about 100 nm, about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, or about 900 nm to about 1000 nm.

[0130] In some embodiments, the lipid particles in the dry composition have an average diameter of about 1 nm to about 1000 nm. In some embodiments, the lipid particles in the dry composition have an average diameter of about 100 nm to about 1000 nm, about 100 nm to about 900 nm, about 100 nm to about 800 nm, about 100 nm to about 700 nm, about 100 nm to about 600 nm, about 100 nm to about 500 nm, about 100 nm to about 400 nm, about 100 nm to about 300 nm, about 100 nm to about 200 nm, about 100 nm to about 100 nm, about 200 nm to about 1000 nm, about 300 nm to about 1000 nm, about 400 nm to about 1000 nm, about 500 nm to about 1000 nm, about 600 nm to about 1000 nm, about 700 nm to about 1000 nm, about 800 nm to about 1000 nm, or about 900 nm to about 1000 nm.

[0131] In some embodiments, the lipid particles in the dry composition have a spherical morphology. In some embodiments, the lipid particles in the dry composition have a polydispersity index of about 0.1 to about 0.5, about 0.2 to about 0.5, about 0.3 to about 0.5, about 0.4 to about 0.5, about 0.1 to about 0.4, about 0.1 to about 0.3, or about 0.1 to about 0.2. In some embodiments, the lipid particles have a polydispersity index of 0.1, 0.2, 0.3, 0.4, or 0.5 or less. In some embodiments, the lipid particles have a polydispersity index of 0.3 or less.

[0132] In some embodiments, the lipid particles in the dry composition have a net positive or net negative zeta potential. In some embodiments, the lipid particles in the dry composition have a shelf life of at least two years at ambient conditions. For example, in some embodiments, the lipid particles in the dry composition have a shelf life of at least two years at storage conditions of -20°C to 4°C or up to 50°C. In some embodiments, the lipid particles in the dry composition carry a load of one or more therapeutic agents.

[0133] The following examples are put forth so as to provide those of skill in the art with a complete disclosure and description of how to make and use the assay, screening and treatment methods of the present invention, and are not intended to limit the scope of what the inventors regard as their invention.

[0134] [Example] [Example 1] Method for freeze-drying synthetic platelets overview To develop a method for freeze-drying synthetic platelets, we first investigated freezing, primary drying, and secondary drying parameters based on the appearance of the freeze-dried cake and ease of reconstitution. Next, we examined the freeze-drying volume and lipid concentration based on size and charge measurements using an Anton Paar Litesizer™. The composition of the freeze-drying buffer was then investigated, including lyoprotectants and their concentrations, buffering agents and their concentrations, and buffer pH, which were determined by both physicochemical and functional characteristics. Freeze-drying parameters were evaluated based on physical and functional characterization of the freeze-dried synthetic platelets for size by dynamic light scattering (DLS), charge by zeta potential, morphology and size by cryo-scanning electron microscopy (cryo-TEM), and platelet-binding functionality by flow cytometry. Method reproducibility was assessed across freeze-dried batches with a target percent coefficient of variation (%CV) of less than 25%.

[0135] Materials and Methods Synthetic platelet preparations Synthetic platelet preparations, Particle 1, containing Cy5 dye containing platelet-binding peptides, collagen-binding peptides, and vWF-binding peptides (PBP, CBP, and VBP, respectively), were manufactured using a standard lipid film rehydration method for liposome preparation, followed by extrusion through 200 nm and then 100 nm pore-size filters. Various lyoprotectants and buffers were added during the lipid film rehydration step, and in some cases, additional lyoprotectants were added after extrusion. The individual reagent components of Particle 1 and their relative amounts are summarized in Tables 1 and 2. The equipment and other items used during the manufacture and characterization of lyophilized Particle 1 are summarized in Tables 3 and 4. Triplicate batches were generated for each lyophilization group to obtain inter-lot means and standard deviations. In addition, all samples were analyzed in triplicate to obtain intra-lot means and standard deviations. The inter-lot standard deviations were used to validate sample size using a power analysis formula, which justified the sample size for the triplicate lots.

[0136] [Table 1]

[0137] [Table 2]

[0138] [Table 3]

[0139] [Table 4]

[0140] Freeze-drying cycle development First, freeze-drying parameters, including freezing temperature, primary drying temperature and pressure, and secondary drying temperature and pressure, were tested for the appearance, yield, and ease of reconstitution of the freeze-dried synthetic platelet cake. The freeze-dried synthetic platelet cakes resulting from this study are summarized in Table 5. Insufficient freezing resulted in sample boiling and product loss, while secondary drying temperatures higher than room temperature resulted in freeze-dried synthetic platelet cakes that were difficult to reconstitute. Using a temperature gradient for secondary drying resulted in freeze-dried synthetic platelet cakes that were easier to reconstitute. A freeze-drying run performed with pre-cooling of the freeze-dryer shelves to achieve a temperature gradient from -45°C to 25°C enabled distinct primary and secondary drying stages. Both the primary and secondary drying stages were set with a freeze-dryer condenser temperature of -45°C and a vacuum of 0.2 mTorr for 960 minutes. The 1-hour primary drying stage was achieved by pre-cooling the freeze-dryer shelf temperature to -60°C and allowing the freeze-dryer system to naturally reach 0°C after approximately 1 hour of natural heat transfer at an approximate rate of +1°C / min. This pre-cooling of the freeze-dryer shelves was completed by removing the shelves, placing them in a -80°C freezer for 2 hours, and then placing them back into the Lyovapor L-200 Pro (TL036) freeze-dryer immediately before loading the shell-frozen samples. Once the shelf temperature reached 0°C and continued to warm to ambient (approximately 25°C), this stage was considered secondary drying. Secondary drying is intended to remove any residual water by more rapidly sublimating water molecules with increasing temperature [(Iyer et al., J. Pharmaceutical Sciences (2016) 105(5): 1684-1692)]. This freeze-drying process was then repeated in five separate runs to demonstrate process reproducibility.

[0141] [Table 5]

[0142] Freeze-dried volume and concentration The freezing capacity and concentration of the lyophilized samples were determined by shell-freezing various concentrations and volumes of SP in 10% (w / v) trehalose in 4 mg / mL HEPES, as shown in Table 6. Shell-freezing was achieved by immersing the glass vials containing the SP in liquid nitrogen (-196°C) for 5 minutes at an angle of approximately 45° while rotating at approximately 30 rpm [Wang et al. (2019) Advanced Drug Delivery Reviews 151-152: 56-71]. Each lyophilized frozen batch was resuspended in cell culture-grade water and analyzed for size and charge to determine the freezing concentration and volume.

[0143] [Table 6]

[0144] Physicochemical and functional characterization of freeze-dried particles 1 Particle size by Litesizer Particle 1 in each lyoprotectant and rehydration buffer combination was diluted in the respective buffer to a 0.1 mg / mL solution for Litesizer analysis, and particle size was measured using a refractive index of 1.34 and a viscosity of 0.00099 Pa·s.

[0145] Zeta potential charge Particle 1 in each lyoprotectant and rehydration buffer combination was diluted in the respective buffer to a 0.1 mg / mL solution for Litesizer analysis, and zeta potential analysis was measured in millivolts (mV) using a refractive index of 1.34 and a viscosity of 0.00099 pa·s.

[0146] Morphology and size by cryo-TEM Cryo-TEM analysis of particle 1 was performed at various lyoprotectant and buffer concentrations to assess morphology using plunge-frozen cryo-TEM samples. Samples were prepared by diluting particle 1 to 0.5 mg / mL in cell culture-grade water, adsorbing onto glow-discharged lacey carbon-coated copper grids for 1 min, blotting with filter paper for 8 s at a power of 10, and plunge-frozen in liquid ethane (below the devitrification temperature of -137 °C). Samples were imaged at magnifications ranging from 9,000x to 30,000x. Particle diameters in cryo-TEM images were measured using the online software ImageJ.

[0147] Functionality by flow cytometry The functionality of the synthetic platelets to bind to activated platelets was assessed using flow cytometry. Briefly, platelet-rich plasma (PRP) from healthy human donors was diluted two-fold with Tyrode's buffer (137 mmol / L NaCl, 12 mmol / L NaHCO3, 2.0 mmol / L KCl, 0.3 mmol / L Na2HPO4, 1 mmol / L MgCl2, 5 mmol / L HEPES, 5 mmol / L glucose, pH 7.3), supplemented with 0.03 units / mL apyrase, and centrifuged at 100 × g for 15 minutes at 25°C to pellet any contaminating white and red blood cells.

[0148] PRP was supplemented with 1 μg / mL prostacyclin for 5 min and centrifuged at 600 × g for 15 min at 25°C to wash the platelets.

[0149] The platelet pellet was gently resuspended in Tyrode's buffer and allowed to equilibrate on the benchtop for 20-30 min before flow cytometry staining.

[0150] The washed platelets were dispensed in triplicate into flow cytometry tubes and incubated with FITC anti-CD62P, 5 μM TRAP agonist, and each Cy5-labeled synthetic platelet preparation at 25° C. for 20 minutes.

[0151] Platelets were read by a flow cytometer, gating the platelet population using side scatter (SSC) on the y-axis and forward scatter (FCS) on the x-axis, all on a log plot, until 50,000 counts per sample were measured. The degree of activation in the platelet population was assessed by FITC staining, and particle binding to platelets was assessed by Cy5 fluorescence levels and % positive platelets.

[0152] Functionality in a mouse model of thrombocytopenia For the functional thrombocytopenia (TCP) mouse model, platelet counts of wild-type C57 / BL6J mice were obtained by retro-orbital (RO) blood collection of 0.1 mL for platelet counting using a HemaVet 950. Dosage calculations were performed, and mice were intraperitoneally injected with 0.2 μg / g anti-CD42b (anti-GPIbα) antibody. 18 hours after antibody injection, platelet counts were obtained again to monitor for the induction of thrombocytopenia (an average of approximately 75% decrease in platelet count). SP doses were administered at 0.1, 1.0, and 10.0 mg / kg. Fifteen minutes after treatment administration, the mice's tails were transversely incised 1 mm from the tip using a sharp surgical blade and immersed in 1200 μL of warm (37°C) saline. The time required for bleeding (bleeding time) was recorded. Blood loss was also recorded using the hemoglobin reagent method.

[0153] Data analysis All unique sample configurations were run in at least triplicate to obtain the mean and standard deviation (Equation 1). To validate the method, this standard deviation (σ) was used along with a Z-score of 1.96 (95% confidence interval) and an error margin of 25% (E) to calculate the appropriate sample size (n) using Cochran's power analysis equation shown in Equation 3. Statistical significance was determined using one-way analysis of variance (GraphPad Prism). All results with a p-value ≤ 0.05 were considered significant.

[0154]

number

[0155] result Size and charge of particle 1 in various lyophilization buffers Particle 1 formulations with HEPES and Tris buffers containing various lyoprotectants including dextrose, sucrose, trehalose, and HP-β-CD were examined for size and charge after lyophilization / reconstitution on a Litesizer 500. The results for Particle 1 formulations in HEPES and Tris are summarized in Tables 7 and 8, respectively.

[0156] [Table 7]

[0157] [Table 8]

[0158] [Table 9]

[0159] Across five lyophilization batches, particle 1 in 2 mg / mL HEPES + 10% (w / v) HP-β-CD (pH 7) buffer was highly reproducible with %CVs below the acceptance standard of 25% for all size parameters and charge, as shown in Table 9. Intensity diameter histograms of synthetic platelets before and after lyophilization overlapped or improved after lyophilization (Figures 2A-E).

[0160] Morphology and size by cryo-TEM Cryo-TEM was used to characterize the morphology and size of Particle 1. After lyophilization / reconstitution, it retained its size, spherical morphology, and unilamellarity, as shown in Figures 3A-3C.

[0161] Physicochemical stability under storage at various temperatures The physicochemical stability of Particle 1 in lyophilization buffer 2 mg / mL HEPES + 10% (w / v) HP-β-CD (pH 7) was evaluated at various storage temperatures: −20°C, 4°C, room temperature (approximately 25°C), and 50°C for up to 3 months. Particle 1 was stored as a lyophilized powder at the temperatures described above, and the particle size distribution and zeta potential were analyzed using a Litesizer immediately after rehydration with water at 28, 60, and 90 days. Figures 4A–C show that the size and charge of Particle 1 were maintained within ±25% of their pre-lyophilization values, suggesting stability under all storage conditions tested.

[0162] Functionality of activated platelet binding by flow cytometry The functionality of activated platelet binding for Particle 1 containing lyophilization buffers 2 mg / mL HEPES or 3 mg / mL Tris + 10% (w / v) HP-β-CD (pH 7) was evaluated by flow cytometry. As shown in Figure 5A, the percentage of activated platelets bound to Particle 1 was maintained in both the HEPES and Tris HP-β-CD buffer groups compared to Particle 1 before lyophilization. Similarly, as shown in Figure 5B, the mean Cy5 fluorescence from platelet-bound Particle 1 after lyophilization was maintained compared to the particles before lyophilization for both the HEPES and Tris HP-β-CD buffer groups. Therefore, the functionality of activated platelet binding for lyophilized synthetic platelets was maintained after lyophilization.

[0163] Hemostatic function in a mouse model of thrombocytopenia The hemostatic functionality of Particle 1, containing lyophilization buffer 2 mg / mL HEPES + 10% (w / v) HP-β-CD (pH 7), was evaluated in a thrombocytopenic (TCP) mouse model. As shown in Figure 6A, the bleeding times of TCP mice administered Post-Lyo Particle 1 at 1 mg / kg and 10 mg / kg were comparable to those of Pre-Lyo Particle 1 at 1 mg / kg, but were improved compared with those administered at 10 mg / kg. Similarly, as shown in Figure 6B, the blood loss (μL) of TCP mice administered Post-Lyo Particle 1 at 1 mg / kg and 10 mg / kg was comparable to those of Pre-Lyo Particle 1 at 1 mg / kg, but was improved compared with those administered at 10 mg / kg. Thus, the hemostatic functionality of lyophilized synthetic platelets is maintained after lyophilization.

[0164] Lyo-cake reconstitution studies The objective of this study was to evaluate the physicochemical and visual properties of particles 1 lyophilized in 2 mg / mL HEPES + 10% (w / v) HP-β-CD (pH 7) after reconstitution in water at various time points. Figure 7A shows the zeta potential, Figure 7B shows the mean intensity diameter, Figure 7C shows the particle concentration, and Figure 7D shows the polydispersity index over time for lyophilized particles 1 after reconstitution. All parameters were within acceptable limits (represented as dotted lines) at all time points after reconstitution. No visible aggregates were observed in any of the samples.

[0165] conclusion In conclusion, we investigated both the freeze-drying method and buffer to maintain the size range and functionality of synthetic platelets after freeze-drying. This buffer maintained the size range of 50 nm to 200 nm when immediately reconstituted or stored at various temperatures from -20 to 50°C for up to 3 months, and maintained its ability to bind activated platelets as determined by flow cytometry and reduce bleeding in a thrombocytopenic mouse model. Additionally, we validated this freeze-drying process and showed it to be highly reproducible with a %CV well below 25%.

[0166] Incorporation by Reference All publications, patents, and patent applications mentioned herein are incorporated by reference in their entirety as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.

[0167] Also, all polynucleotide and polypeptide sequences that reference an accession number associated with an entry in the public databases maintained, for example, by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or by the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov, are incorporated by reference in their entirety.

[0168] equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. A composition comprising a plurality of lipid particles and a liquid component, wherein the liquid component comprises water and a cyclodextrin at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component, and each lipid particle comprises a lipid and a peptide conjugate, wherein the peptide of the peptide conjugate is associated with the outer surface of the lipid particle.

2. 2. The composition of claim 1, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxypropyl-γ-cyclodextrin, hydroxypropyl-α-cyclodextrin and / or hydroxypropyl cyclodextrin.

3. 2. The composition of claim 1, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin (HP-β-CD).

4. 4. The composition of claim 1, further comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the lipid component, and wherein the pH of the liquid component is about 5 to about 9.

5. The composition of any one of claims 1 to 3, wherein the composition does not contain 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

6. 6. The composition of any one of claims 1 to 5, further comprising tris(hydroxymethyl)aminomethane (Tris) at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

7. The composition of any one of claims 1 to 5, wherein the composition does not contain tris(hydroxymethyl)aminomethane (tris).

8. 8. The composition of any one of claims 1 to 7, further comprising sodium chloride at a concentration of about 0.1% (w / v) to about 1% (w / v) of the liquid component.

9. The composition of any one of claims 1 to 7, wherein the composition does not contain sodium chloride.

10. 10. The composition of any one of claims 1 to 9, further comprising ammonium sulfate at a concentration of about 0.11% (w / v) to about 0.5% (w / v) of the liquid component.

11. The composition of any one of claims 1 to 9, wherein the composition does not contain ammonium sulfate.

12. 12. The composition of any one of claims 1 to 11, further comprising L-histidine at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

13. The composition of any one of claims 1 to 11, wherein the composition does not contain L-histidine.

14. 14. The composition of any one of claims 1 to 13, further comprising lactose monohydrate at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

15. The composition of any one of claims 1 to 13, wherein the composition does not contain lactose monohydrate.

16. The composition of any one of claims 1 to 15, wherein the composition does not contain sucrose.

17. The composition of any one of claims 1 to 16, wherein the composition does not contain dextrose.

18. The composition of any one of claims 1 to 4, wherein the composition consists essentially of lipid particles, cyclodextrin, HEPES, and water.

19. 19. The composition of any one of claims 1 to 18, wherein the peptide conjugate is a platelet-binding peptide (PBP) conjugate, a von Willebrand factor-binding peptide (VBP) conjugate, or a collagen-binding peptide (CBP) conjugate, or a combination thereof, and the PBP conjugate is a fibrinogen-mimetic peptide (FMP) conjugate or a P-selectin-binding peptide conjugate; and wherein a plurality of PBP conjugates, VBP conjugates, and / or CBP conjugates are conjugated to the outer surface of the lipid particle, and the PBP conjugates, VBP conjugates, and CBP conjugates are collectively present in less than 5 mole percent of the lipid particle.

20. 1. A composition comprising a plurality of lipid particles and a liquid component, wherein the liquid component comprises water and a lyoprotectant at a concentration of about 1% (w / v) to about 20% (w / v) of the liquid component, each lipid particle comprising a lipid and a plurality of peptide conjugates, wherein the peptide conjugates are selected from a platelet-binding peptide (PBP) conjugate, a von Willebrand factor-binding peptide (VBP) conjugate, and a collagen-binding peptide (CBP) conjugate, or a combination thereof, and wherein the PBP conjugate is a fibrinogen-mimetic peptide (FMP) conjugate or a P-selectin-binding peptide conjugate; wherein the plurality of PBP conjugates, VBP conjugates, and / or CBP conjugates are conjugated to the outer surface of the particle, and wherein the PBP conjugates, VBP conjugates, and CBP conjugates are collectively present in less than 5 mole percent of the particle.

21. 21. The composition of claim 20, wherein the lyoprotectant is cyclodextrin, sucrose, dextrose, or trehalose, or a combination thereof.

22. 21. The composition of claim 20, wherein the lyoprotectant is a cyclodextrin.

23. 21. The composition of claim 20, wherein the lyoprotectant is hydroxypropyl-β-cyclodextrin (HP-β-CD), hydroxypropyl-γ-cyclodextrin, hydroxypropyl-α-cyclodextrin and / or hydroxypropyl cyclodextrin.

24. 23. The composition of claim 22, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin (HP-β-CD).

25. 25. The composition of any one of claims 20 to 24, further comprising 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the lipid component, and wherein the pH of the liquid component is about 5 to about 9.

26. 25. The composition of any one of claims 20 to 24, wherein the composition does not contain 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES).

27. 27. The composition of any one of claims 20 to 26, further comprising tris(hydroxymethyl)aminomethane (Tris) at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

28. The composition of any one of claims 20 to 26, wherein the composition does not contain tris(hydroxymethyl)aminomethane (tris).

29. 29. The composition of any one of claims 20 to 28, further comprising sodium chloride at a concentration of about 0.1% (w / v) to about 1% (w / v) of the liquid component.

30. The composition of any one of claims 20 to 28, wherein the composition does not contain sodium chloride.

31. 31. The composition of any one of claims 20 to 30, further comprising ammonium sulfate at a concentration of about 0.11% (w / v) to about 0.5% (w / v) of the liquid component.

32. The composition of any one of claims 20 to 30, wherein the composition does not contain ammonium sulfate.

33. 33. The composition of any one of claims 20 to 32, further comprising L-histidine at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

34. The composition of any one of claims 20 to 32, wherein the composition does not contain L-histidine.

35. 35. The composition of any one of claims 20 to 34, further comprising lactose monohydrate at a concentration of about 0.1% (w / v) to about 0.5% (w / v) of the liquid component.

36. 35. The composition of any one of claims 20 to 34, wherein the composition does not contain lactose monohydrate.

37. The composition of any one of claims 20 to 35, wherein the composition does not contain sucrose.

38. The composition of any one of claims 20 to 36, wherein the composition does not contain dextrose.

39. 26. The composition of any one of claims 20 to 25, wherein the composition consists essentially of lipid particles, cyclodextrin, HEPES, and water.

40. 21. The composition of claim 19 or claim 20, wherein the PBP, VBP, and / or CBP or combination thereof are spatially or topographically arranged on the surface of the lipid particle such that the PBP, VBP, and / or CBP or combination thereof do not spatially obscure each other.

41. The composition of any one of claims 19 to 40, wherein PBP, VBP and / or CBP or a combination thereof is conjugated to the surface of the lipid particle via a PEG linker.

42. 42. The composition of claim 19, wherein the lipid particles comprise DSPE-PEG2k-PBP, DSPE-PEG2k-VBP, or DSPE-PEG2k-CBP.

43. The PBP conjugate comprises DSPE-PEG2k-FMP, wherein FMP has the formula (I): Cyclo-(CNPRGD{Tyr(OEt)}R-β-A) formula (I) 43. The composition according to any one of claims 19 to 42, wherein

44. The composition of any one of claims 19 to 43, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

2.

45. The composition of any one of claims 19 to 44, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO:

4.

46. The composition of any one of claims 19 to 45, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

5.

47. 47. The composition of any one of claims 19 to 46, wherein the PBP is flanked by additional amino acid sequences.

48. 48. The composition of any one of claims 19 to 47, wherein the VBP comprises SEQ ID NO:

6.

49. 49. The composition of claim 48, wherein the VBP is flanked by additional amino acid sequences.

50. 50. The composition of any one of claims 19 to 49, wherein the CBP comprises SEQ ID NO:

7.

51. 51. The composition of claim 50, wherein the CBP is flanked by additional amino acid sequences.

52. 52. The composition of any one of claims 19 to 51, wherein the lipid particle comprises a plurality of PBP conjugates, a plurality of VBP conjugates, and a plurality of CBP conjugates.

53. 53. The composition of any one of claims 19 to 52, wherein the molar percentage of the PBP conjugate is between 0.05% and 5.00% molar percent of the lipid particle.

54. 53. The composition of any one of claims 19 to 52, wherein the molar percentage of the FMP conjugate is 0.05% to 5.00% molar percent of the lipid particle.

55. 55. The composition of any one of claims 19 to 54, wherein the molar percentage of the CBP conjugate is between 0.10% and 1.50% molar percent of the lipid particle.

56. 56. The composition of any one of claims 19 to 55, wherein the molar percentage of the VBP conjugate is between 0.10% and 1.50% molar percent of the lipid particle.

57. 57. The composition of any one of claims 19 to 56, wherein the molar percentage of the PBP conjugate is about 1.00% of the lipid particle, the molar percentage of the CBP conjugate is about 0.50% of the lipid particle, and the molar percentage of the VBP conjugate is about 0.50% of the lipid particle.

58. 58. The composition of any one of claims 19 to 57, wherein the lipid particle comprises both a VBP conjugate and a CBP conjugate; and the ratio of VBP to CBP provided on the surface of the lipid particle is from about 70:30 to about 30:

70.

59. 59. The composition of any one of claims 19 to 58, wherein the relative molar ratio of PBP:CBP:VBP is 2:1:

1.

60. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 1:5:

5.

61. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 10:5:

1.

62. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

5.

63. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

1.

64. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 1:2:

1.

65. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

2.

66. 57. The composition of any one of claims 19 to 56, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

1.

67. 58. The composition of any one of claims 19 to 57, wherein the CBP and / or VBP is not conjugated to the surface of the lipid particle.

68. 48. The composition of any one of claims 19 to 47, wherein the relative molar ratio of PBP:CBP is 2:

1.

69. 48. The composition of any one of claims 19 to 47, wherein the relative molar ratio of PBP:VBP is 2:

1.

70. 70. The composition of any one of claims 1 to 69, wherein the lipid comprises a phospholipid.

71. 70. The composition of any one of claims 1 to 69, wherein the lipid comprises a phospholipid conjugated to polyethylene glycol (PEG).

72. 72. The composition of claim 71, wherein the PEG conjugated to the phospholipid has an average molecular weight of about 500 Da to about 5500 Da.

73. 73. The composition of any one of claims 1 to 72, wherein the lipid comprises cholesterol, distearoylphosphatidylcholine (DSPC) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

74. 74. The composition of any one of claims 1 to 73, wherein the lipid particles have a diameter of about 1 to 1000 nm.

75. 75. The composition of any one of claims 1 to 74, wherein the lipid particles have a diameter of about 30 to 300 nm.

76. 76. The composition of any one of claims 1 to 75, in the form of a liposome.

77. 77. The composition of any one of claims 1 to 76, wherein the lipid particles are substantially spherical.

78. 78. The composition of any one of claims 1 to 77, wherein the lipid particles have a polydispersity index of 0.3 or less.

79. 79. The composition of any one of claims 1 to 78, wherein the lipid particles have a net positive or net negative zeta potential.

80. 80. The composition of any one of claims 1 to 79, wherein the particles promote platelet aggregation.

81. 81. The composition of any one of claims 1 to 80, wherein the particles bind to activated platelets.

82. 82. The composition of any one of claims 1 to 81, wherein the particles bind to ADP or TRAP-activated platelets.

83. 1. A method of forming a dry composition, comprising: incubating the composition of any one of claims 1 to 82 at a temperature of about 0 to about -210°C to form a cooled composition; incubating the cooled composition at a temperature of about 0 to about −60° C. and a pressure of about 0.1 to about 1.0 mTorr, thereby forming a dry composition; A method comprising:

84. 84. The method of claim 83, further comprising cooling the composition of any one of claims 1 to 46 at a rate of between -1.0°C / sec and -0.1°C / sec prior to incubating the composition.

85. 85. The method of claim 83 or 84, wherein the lipid particles in the dry composition have a diameter of from about 1 nm to about 1000 nm.

86. 86. The method of any one of claims 83 to 85, wherein the lipid particles in the dry composition have an average diameter of from about 1 nm to about 1000 nm.

87. 87. The method of any one of claims 83 to 86, wherein the lipid particles in the dry composition have a spherical morphology.

88. 88. The method of any one of claims 83 to 87, wherein the lipid particles in the dry composition have a polydispersity index of 0.3 or less.

89. 89. The method of any one of claims 83 to 88, wherein the lipid particles in the dry composition have a net positive or net negative zeta potential.

90. 90. The method of any one of claims 83 to 89, wherein the lipid particles in the dry composition have a shelf life of at least 2 years at ambient conditions.

91. 91. The method of any one of claims 83 to 90, wherein the lipid particles in the dry composition have a shelf life of at least 2 years at storage conditions of -20°C to 4°C.

92. 92. The method of any one of claims 83 to 91, wherein the lipid particles in the dry composition have a shelf life of at least 2 years at storage conditions up to 50°C.

93. 93. The method of any one of claims 83 to 92, wherein the lipid particles in the dry composition carry a load of one or more therapeutic agents.

94. 94. A composition made by the method of any one of claims 83 to 93.

95. 95. A method of forming a reconstituted composition, comprising contacting the composition of claim 94 with water, e.g., for less than about 30 seconds, thereby forming a reconstituted composition.

96. 96. The method of claim 95, wherein the reconstituted composition comprises lipid particles having a diameter of about 1 nm to about 1000 nm.

97. 97. The method of claim 96, wherein the lipid particles have an average diameter of about 30 nm to about 300 nm.

98. 98. The method of claim 96 or claim 97, wherein the lipid particles have a substantially spherical morphology.

99. 99. The method of any one of claims 96 to 98, wherein the lipid particles have a polydispersity index of 0.3 or less.

100. 100. A reconstituted composition made by the method of any one of claims 95 to 99.

101. 101. A method of attenuating bleeding, treating bleeding, treating vascular injury, promoting hemostasis, preventing or inhibiting platelet aggregation, promoting aggregation of activated platelets at sites where vWF and collagen are exposed, or treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of the reconstituted composition of claim 100.

102. 102. The method of claim 101, wherein the reconstituted composition is administered intravenously.

103. 103. The method of claim 101 or claim 102, wherein the reconstituted composition is administered intravenously as a bolus dose or injected into a saline or dextrose bag.

104. 102. The method of claim 101, wherein the reconstituted composition is aerosolized and administered by inhalation, e.g., orally or intranasally, or both.

105. 102. The method of claim 101, wherein the reconstituted composition is administered intraosseously.

106. 102. The method of claim 101, wherein the reconstituted composition is administered transdermally.

107. 107. A method according to any one of claims 101 to 106 for treating bleeding, wherein the bleeding is non-compressible bleeding, for example resulting from trauma.

108. 107. A method of attenuating bleeding according to any one of claims 101 to 106, wherein the bleeding is due to surgery.

109. 107. The method of any one of claims 101 to 106, for attenuating bleeding, wherein the bleeding is due to thrombocytopenia, postpartum hemorrhage, traumatic brain injury, von Willebrand's disease, Glanzmann's thrombasthenia, or Bernard-Soulier syndrome.

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