Platelet-binding proteins and conjugates thereof, particles containing them, and uses thereof

Abstract

The present invention relates to platelet-binding peptides and conjugates thereof, and particles comprising these and other peptides and conjugates. Additionally, compositions and methods for using these peptides and particles are provided.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 426,521, filed November 18, 2022, which is incorporated herein by reference in its entirety.

[0002] government support This invention was made with support from the National Science Foundation under Grant No. 1951301. The Government has certain rights in this invention. [Background technology]

[0003] The management of uncontrolled bleeding requires a multifactorial approach. Uncontrolled bleeding occurs in a variety of clinical indications, including (but not limited to) trauma, surgery, thrombocytopenia, and postpartum hemorrhage. Currently, the standard treatment for these indications is the transfusion of human donor platelets in combination with other blood product therapies, such as packed red blood cells (RBCs), plasma, and / or whole blood. Platelets are the blood cells that primarily form primary hemostatic clots at the site of bleeding injury and also promote secondary hemostasis to fix and stabilize the clot and control bleeding. Overwhelming evidence from previous clinical studies has demonstrated the benefit of donor platelet transfusion to prevent or treat bleeding. However, donor platelets have limited availability and portability, special storage requirements, a high risk of contamination, and a very short shelf life, presenting logistical challenges to widespread access and sustained use. Furthermore, transfusion carries significant risks, including hospital-acquired infection, immunosuppression, transfusion-associated acute lung injury, and even death.

[0004] Non-platelet substitutes, including red blood cells modified with the platelet-binding Arg-Gly-Asp (RGD) sequence, albumin-based fibrinogen-coated microcapsules, and liposome systems, have been investigated as coagulants, but these products suffer from toxicity, thrombosis, and limited efficacy. RGD peptides can bind to beta-3 integrins, including glycoprotein (GP) IIb-IIIa, on platelets. Furthermore, certain cyclic RGD peptides exhibit selective and potent binding to activated GPIIb / IIIa proteins over inactive forms and other beta-3 integrins. The original cyclic peptide referenced in this document (first reported by Cheng et al., J. Med. Chem. 1994, 37, 1, 1-8), which was further modified to enable bioconjugation with lipids, is cyclized by a Cys-Cys disulfide chemical structure. However, this cyclic RGD peptide is not chemically stable.

[0005] Furthermore, non-platelet substitutes with surface modifications containing multiple peptides or proteins may exert multiple hemostatic functions, including binding to a variety of injury-associated cells (e.g., platelets) and proteins (e.g., collagen and von Willebrand factor). Summary of the Invention

[0006] The present invention is based, at least in part, on the discovery that the chemical stability of cyclic RGD peptides can be significantly improved by modifying the peptide chemical structure, replacing the terminal Cys-Cys cyclization (disulfide bond) with a beta-alanine-mediated NC cyclization. In the new chemical structure, the cyclization bond, the sequence around the RGD motif, the number of amino acids, and the size of the ring structure are altered. Despite these modifications, this peptide still exhibits the same inhibitory effects as Cheng et al. (In Human PRP / ADP Platelet Aggregation, IC 50 = 0.22 μM) (see U.S. Patent No. 9,107,845, which is incorporated herein in its entirety), the peptide structure revealed its antithrombotic activity (IC 50= 0.13 μM). Furthermore, this peptide structure is amenable to chemical coupling to maleimide-functionalized lipids, proteins, polymers, etc. through thiol-ene coupling to the freed thiol group on the N-terminal cysteine. Depending on the physicochemical properties of these bioconjugates, they may self-assemble into particles. Particles containing the novel GPIIb-IIIa peptide alone or in combination with other motifs may promote platelet aggregation and be used as hemostatic agents (as opposed to the antithrombotic applications described above).

[0007] The present invention is also based, at least in part, on the discovery that the presence of certain peptides on the surface of synthetic platelets results in more efficient hemostatic activity (i.e., efficient clotting). Furthermore, optimizing the molar percentage of the peptides disclosed herein decorating the particles can significantly increase the hemostatic activity of the particles. In certain embodiments, the present disclosure relates to particles comprising a plurality of platelet-binding peptide (PBP) conjugates, von Willebrand factor-binding peptide (VBP) conjugates, or collagen-binding peptide (CBP) conjugates, or combinations thereof, wherein the PBP conjugates are fibrinogen-mimetic peptide (FMP) conjugates or P-selectin-binding peptide conjugates, and the plurality of PBP conjugates, VBP conjugates, and / or CBP conjugates are conjugated to the outer surface of the particle, and the PBP conjugates, VBP conjugates, and CBP conjugates are present, collectively, in less than 5 molar percent of the particle.

[0008] In certain embodiments, the present disclosure provides a fibrinogen-mimetic peptide (FMP) of formula (I): Cyclo(CNPRGD{Tyr(OEt)}R-β-A) Formula (I), or a salt thereof. In certain embodiments, the present disclosure relates to a peptide conjugate comprising a peptide conjugated to a polymer, wherein the peptide is a fibrinogen-mimetic peptide and is any of the FMPs described herein. In certain embodiments, the present disclosure relates to a particle comprising a plurality of platelet-binding peptide (PBP) conjugates, wherein the PBP conjugates include any of the peptide conjugates described herein. In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising any of the fibrinogen-mimetic peptides described herein and a carrier. In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising any of the peptide conjugates described herein and a carrier. In certain embodiments, the present disclosure relates to a method of preventing or inhibiting platelet aggregation in a subject in need thereof, the method comprising administering to the subject a composition comprising any of the fibrinogen-mimetic peptides (FMPs) described herein. In certain embodiments, the present disclosure relates to a method of preventing or inhibiting platelet aggregation in a subject in need thereof, the method comprising administering to the subject any of the fibrinogen-mimetic peptides described herein or any of the pharmaceutical compositions comprising an FMP described herein.

[0009] In certain embodiments, the present disclosure relates to a pharmaceutical composition comprising any of the particles described herein and a carrier.

[0010] In certain embodiments, the present disclosure relates to a method for promoting the aggregation of activated platelets at a site with exposed vWF and collagen, the method comprising administering to the site any of the particles described herein or any of the pharmaceutical compositions comprising the particles described herein.

[0011] In certain embodiments, the present disclosure relates to a method of reducing bleeding, treating vascular injury, or promoting hemostasis in a subject, comprising administering to the site of bleeding or vascular injury any of the particles described herein, or any of the pharmaceutical compositions comprising the particles described herein. [Brief explanation of the drawings]

[0012] [Figure 1] Figures 1A-1B show the dose-dependent inhibition of terminal aggregation of human platelets by light transmission aggregometry for both 1A) cyclo{Pra}CNPRGD{Tyr(OEt)}RC (FMP1) and 1B) cyclo(CNPRGD{Tyr(OEt)}R-β-A) (FMP2). Figure 1C) shows the log dose-response (% inhibition of aggregation) curves, which show IC50 = 536.6 μM for FMP1 and IC50 = 0.13 μM for FMP2. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001. [Figure 2] Figure 2A shows evidence of FMP1 dimerization / trimerization by MALDI-TOF mass spectrometry, likely due to cleavage and reformation of cyclic disulfide bonds with free sulfhydryls on adjacent peptides, suggesting poor stability. Figure 2B shows no evidence of FMP2 dimerization / trimerization. [Figure 3] 3A-3B show that MALDI-TOF mass spectrometry confirmed the conjugates of 3A) FMP1 and DSPE-PEG(2000)-azide, and 3B) FMP2 and DSPE-PEG(2000)-maleimide. [Figure 4] Figure 4 shows the effective diameters of synthetic platelet particles in nanometers (nm) measured by dynamic light scattering after 15 weeks of storage in buffer at room temperature. Particle 1 = synthetic platelet preparation with 15 mol% FMP1, Particle 2 = synthetic platelet preparation with 2.5 mol% FMP1, Particle 3 = synthetic platelet preparation with 15 mol% FMP2, Particle 4 = synthetic platelet preparation with 2.5 mol% FMP2. Particles with FMP2 are smaller on average than those containing FMP1, likely due to less particle aggregation. [Figure 5]Figure 5 shows the final platelet aggregation percentage (%, normalized to simulated thrombocytopenic platelet-rich plasma (tPRP) group) of synthetic platelet particles containing FMP1 versus FMP2 peptides. Particles with FMP2 showed enhanced platelet aggregation to the same extent as particles with FMP1. Particle size (effective diameter 200 nm vs. 100 nm measured by DLS) did not affect platelet aggregation ability. NA = no agonist. Error bars for particles containing 15% 200 nm FMP are n = 4. [Figure 6] Figures 6A-6E show flow cytometry evaluation of rhodamine B-labeled synthetic platelet particles containing 15 mol% FMP2 (particle 3) and FMP1 (particle 1) conjugated to activated human washed platelets compared to control particles (CP, liposomes without peptide modification). 6A-6B) Gating of platelet populations by forward scatter and size scatter, 6C) Gating of platelets using CD41a-positive events, 6D) Histogram of rhodamine B-positive events for TRAP-activated platelets, and 6E) Histogram of rhodamine B-positive events for ADP-activated platelets. 6D-6E) Tables show mean platelet activation by mean rhodamine B (YG582-A) and CD62P-labeled (R660-A) levels. [Figure 7-1] Figures 7A-7Y show representative DLS intensity diameter relative frequency histograms for synthetic platelet particle preparations: 7A) Particle 1, 7B) Particle 2, 7C) Particle 3, 7D) Particle 4, 7E) Particle 5, 7F) Particle 6, 7G) Particle 7, 7H) Particle 8, 7I) Particle 9, 7J) Particle 10, 7K) Particle 11, 7L) Particle 12, 7M) Particle 14, 7N) Particle 15, 7O) Particle 16, 7P) Particle 18, 7Q) Particle 19, 7R) Particle 20, 7S) Particle 21, 7T) Particle 22, 7U) Particle 23, 7V) Particle 24, 7W) Particle 26, 7X) Particle 27, and 7Y) Particle 28. [Figure 7-2] This is a continuation of Figure 7. [Figure 7-3] This is a continuation of Figure 7. [Figure 7-4] This is a continuation of Figure 7. [Figure 7-5] This is a continuation of Figure 7. [Figure 7-6] This is a continuation of Figure 7. [Figure 7-7] This is a continuation of Figure 7. [Figure 8] 8A-8C show representative cryo-TEM images and particle size histograms of 8A) particle 5, 8B) particle 11, and 8C) particle 12. [Figure 9] Figure 9 shows the functional analysis of various synthetic platelet preparations (Cy-5 labeled) bound to activated human platelets using flow cytometry at a platelet:particle ratio of approximately 1:1000. [Figure 10] Figures 10A-10B show platelet aggregation profiles in normal plasma (+) and platelet-depleted plasma (-) in the presence of various synthetic platelet preparations. Platelet:particle ratios are approximately 1:10. Results are reported as 10A) final % aggregation and 10B) maximum % aggregation normalized to the TCP defect in the absence of particles. [Figure 11] Figures 11A-11D show functional analysis of various synthetic platelet preparations using BioFlux, showing 11A) platelet binding area (% coverage), 11B) platelet binding rate, 11C) particle binding area (% coverage), and 11D) particle binding rate to collagen / vWF-coated surfaces under flow with low platelet counts. Positive control (+) is a normal number of platelets. Negative control (-) is a low number of platelets with vehicle (buffer). [Figure 12] Figures 12A-12B show 12A) blood loss and 12B) bleeding time after tail amputation in thrombocytopenic mice treated with various synthetic platelet preparations (particles 5, 12, 20, 22, and 28) at various doses ranging from 0.1 to 10 mg / kg. Baseline represents bleeding in mice with normal platelet counts. Negative control (-) is treatment with vehicle (buffer). Positive control (+) is treatment with allogeneic platelets. Statistically significant differences compared to the negative control group are indicated by *p<0.05. Numbers within the bars represent the n value for each group. DETAILED DESCRIPTION OF THE INVENTION

[0013] detail In this document, it has been determined that modifying the peptide chemical structure and replacing the terminal Cys-Cys cyclization (disulfide bond) with a β-alanine-mediated NC cyclization significantly improves the chemical stability of cyclic Arg-Gly-Asp (RGD) peptides. Accordingly, the present invention relates, in part, to a platelet-binding protein (PBP) of formula (I) and a conjugate comprising a PBP of formula (I). Furthermore, in certain embodiments, the present invention relates to a particle comprising: 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 in this document as FMP2; cyclo(CNPRGD{Tyr(OEt)}R-β-C) (SEQ ID NO:1)), FMP1 (cyclo(CNPRGD{Tyr(OEt)}R-β-C) (SEQ ID NO:2)), linear RGD (GRGDSP (SEQ ID NO:3)), and H12 (HHLGGAKQAGDV (SEQ ID NO:4)); Collagen-binding peptides (CBPs), such as (GPO)7 (SEQ ID NO:8); or Von Willebrand binding protein (VBP), such as 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 to these peptides are 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] A "kit" is any article of manufacture (e.g., 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 of the invention. The kit may be promoted, distributed, or sold as a unit for performing a method of the invention. The kit may include one or more reagents necessary to express a composition useful in a method of the invention. 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. The kit may further include instructional materials describing how to use the compositions in the kit.

[0022] 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).

[0023] 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).

[0024] 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.

[0025] "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.

[0026] The term "protein" typically refers to large polypeptides.

[0027] The term "peptide" typically refers to a short polypeptide.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] A "recombinant polypeptide" is a polypeptide produced by expression of a recombinant polynucleotide.

[0032] "Site" refers to the site of surface disruption, eg, injury, that results in von Willebrand factor and collagen being present at that site.

[0033] The term "synergistic effect" refers to a combined effect of two or more agents described in this document that may be greater than the sum of the separate effects of either agent alone.

[0034] 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).

[0035] "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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] In some embodiments, the FMP is linear RGD (GRGDSP (SEQ ID NO:3)).

[0042] In some embodiments, the FMP is H12 (HHLGGAKQAGDV (SEQ ID NO:4)).

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] P-selectin binding peptide As used herein, the term "P-selectin binding peptide" refers to a protein or peptide that binds with high affinity (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).

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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 (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., a factor FVIII-derived peptide), the A1 or A3 domain (e.g., a collagen-derived / mimetic peptide), or the A1 or C4 domain (e.g., a platelet GPIb or GPIIb-IIIa-derived peptide). In a specific embodiment, the VBP has the amino acid sequence TRYLRIHPQSWVHQI (SEQ ID NO:6).

[0053] 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.

[0054] 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 of 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 decorated on particle surfaces provide a mechanism for increased particle adhesion with increasing shear.

[0055] 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 (nano- to micromolar affinity), e.g., a collagen-derived sequence (e.g., GPO repeats) with helicogenic affinity for collagen, or a collagen-binding peptide obtained experimentally (e.g., by phage display or isothermal titration chemistry). In a specific embodiment, the CBP has the amino acid sequence (GPO)7 (SEQ ID NO:7).

[0056] 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.

[0057] In some embodiments, CBP can comprise a peptide containing short repeats of the tripeptide GPO, which has helicogenic affinity for fibrillar collagen, such as (GPO)7SEQ 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.

[0058] Synthetic platelets In some aspects, the present disclosure relates to 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, CBP, and VBP, as described herein). 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.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] 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.

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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%.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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 fibrogenic mimetic peptide as described herein.

[0084] The embodiments described herein should in no way be construed as being limited to the synthetic platelets described herein, but should be construed to include the use of additional synthetic platelets, both known and unknown, that reduce or decrease bleeding or blood loss.

[0085] 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.

[0086] 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).

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] 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).

[0094] 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.

[0095] 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.

[0096] 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).

[0097] 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.

[0098] 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. [Example]

[0099] Example 1: Technical Study Overview A. Research Objectives and Background: The purpose of this study was to compare the efficacy of a new fibrinogen-mimetic peptide (FMP) peptide sequence with previously used sequences. The original FMP peptide sequence is cyclo{Pra}CNPRGD{Tyr(OEt)}RC trifluoroacetate. The new peptide sequence is cyclo(CNPRGD{Tyr(OEt)}R-β-A) trifluoroacetate.

[0100] [ka] FMP2 peptide: cyclo(CNPRGD{Tyr(OEt)}R-β-A) MW: 1061.18 g / mol Isoelectric point: 9.2

[0101] [ka] FMP1 peptide: cyclo{Pra}CNPRGD{Tyr(OEt)}RC. MW: 1204.35 g / mol Isoelectric point: 8.1

[0102] B. Method: The properties of the FMP2 peptide and peptide-modified particles (synthetic platelets) were compared with those of the older FMP1 peptide. First, Chang et al. [1] showed that a cyclic RGD peptide could selectively bind to activated platelets and prevent the aggregation of human platelets, as measured by transmitted light aggregometry. This protocol was used to investigate the IC of FMP1 and FMP2. 50For comparison of values, adaptations were performed. Specifically, whole human blood was collected from aspirin-naive healthy donors via venipuncture into a syringe containing 3.8% w / v sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifuging the whole blood at 150 × g for 15 minutes in a tabletop centrifuge. Platelet aggregation experiments were performed on a Bio / Data PAP-8E platelet aggregation profiler by stimulating 225 μL of tPRP with 25 μL of 5 or 10 μM adenosine 5'-diphosphate (ADP) (or saline as a negative control). 20 μL of saline containing 0.0001 μM to 250 μM FMP1 or FMP2 (by order of magnitude) was added to 25% diluted PRP, and the final aggregation percentage was recorded. In GraphPad Prism®, a nonlinear log dose-response fit was applied to the data and the IC of each peptide was calculated by calculating the intercept at y = 50. 50 The acceptance criteria for this assay were FMP2 IC 50 ≦ FMP1 IC 50 The purpose was to show that.

[0103] Next, signs of peptide dimerization / trimerization (indicative of low stability) were investigated using matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF) analysis using a Bruker UltraFlex III MALDI-TOF / TOF mass spectrometer (S / N 247420 00194). Samples were prepared using the sandwich matrix method by sandwiching 5 μL of a 20 mg / mL solution in methanol with the matrix trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB, 25 mg / mL). Samples were measured in both positive reflectron mode (1000–10,000 m / z range) and positive linear mode (500–5000 m / z range).

[0104] The FMP peptides were then conjugated to functionalized lipid PEG molecules to generate lipopeptides. FMP1 was conjugated to DSPE-PEG(2000)-azide via the alkyne moiety of propargylglycine using copper-catalyzed azide-alkyne click chemistry, and the conjugation was confirmed using MALDI-TOF. FMP2 was conjugated to DSPE-PEG(2000)-maleimide via the sulfhydryl moiety to form a stable thioether bond, and the conjugation was confirmed using MALDI-TOF.

[0105] These lipid-peptide conjugates were formulated into synthetic platelet particles (Table 1) using standard lipid film rehydration and extrusion methods. Briefly, DSPC, cholesterol, DSPE-PEG(2000)-FMP1 or DSPE-PEG(2000)-FMP2, DSPE-PEG(2000)-VBP, DSPE-PEG(2000)-CBP, DSPE-mPEG(1000), and DSPE-rhodamine B were dissolved in 1:1 chloroform / methanol and mixed at various molar percentages. The organic solvent was removed under vacuum (Rotovap) to form a thin lipid film, followed by additional drying under a stream of nitrogen. The lipid film was then reconstituted with saline, followed by at least 10 freeze-thaw cycles and at least five extrusions through either 100 nm or 200 nm pore size filters (10 mL Lipex Extruder) to obtain particles of the desired size.

[0106] [Table 1]

[0107] The resulting particles were characterized for their physicochemical properties, i.e., particle size and surface charge, using a Zetasizer (Anton Paar LiteSizer™ 500). Synthetic platelets containing FMP2 were evaluated to ensure that particle size and charge were comparable (within ±20%) to those containing FMP1 and that changes in surface chemistry did not cause particle aggregation over time (15 weeks). Furthermore, the functionality of these peptide-modified particles was investigated using two methods: platelet aggregation assays and flow cytometry. For both assays, whole human blood was collected from healthy aspirin-naive donors by venipuncture into a syringe containing 3.8% w / v sodium citrate. For platelet aggregation assays, platelet-rich plasma (PRP) was obtained by centrifuging the whole blood at 150 × g for 15 min in a tabletop centrifuge. Half of the PRP was saved, and platelet-free plasma (PFP) was obtained by centrifuging the remaining PRP at 13,000 × g for 5 min. The platelet count of PRP was determined using a Coulter Counter (Multisizer 3 Coulter Counter, Beckman Coulter) and adjusted to 50,000 platelets / μL to generate thrombocytopenic platelet-rich plasma (tPRP). Platelet aggregation experiments were performed on a Bio / Data PAP-8E platelet aggregation profiler by stimulating 225 μL of tPRP with 25 μL of 5 or 10 μM adenosine 5'-diphosphate (ADP) (or saline as a negative control). Various synthetic platelet particle formulations were added to tPRP containing resting platelets (without ADP) versus tPRP with activated platelets (with ADP). Final aggregation percentages were reported and normalized to the final aggregation percentage of ADP-activated tPRP from the same donor without synthetic platelet particles (Equation 1). This step was necessary to provide a more accurate representation of the final aggregation improvement due to donor-to-donor blood variability.

[0108]

number

[0109] For flow cytometry, platelet-rich plasma (PRP) was obtained by centrifuging whole blood in a tabletop centrifuge at 150 × g for 15 minutes. To obtain gel-filtered platelets, an Econo-Column® chromatography column was filled approximately 50% with Sepharose CL-2B beads and used to obtain gel-filtered platelets. The column was washed with HEPES Tyrode's buffer and then with supplemented HEPES Tyrode's buffer (1% BSA, 5 mM D-glucose). PRP was diluted with acid citrate dextrose (0.3% v / v). PRP was added to the column, and an aliquot was collected. The cloudy solution was mixed and used for sample preparation. Samples were prepared with 5 μL of FITC anti-CD41a and 5 μL of AlexaFluor 647 anti-CD62P. Platelets were activated (or left unactivated) with 5 μM TRAP or 10 μM ADP for 20 minutes at 25°C. The samples were then diluted to 1 mL with Tyrode's solution (Modified I, Boston BioProducts, Inc.) and transferred to a FACS tube. Samples were evaluated on a BD-LSR1 instrument. The laser parameters and voltages were set as follows: FSC: 625, SSC: 375, B525: 520, R660: 650, YG582: 694, YG610: 490. Two lasers were set for rhodamine B, and YG582 was used for analysis. Data analysis was performed using FlowJo™ and GraphPad Prism®. Samples were gated for the total cell population and then for the single cell population. FITC-CD41a-stained cells were gated to determine the platelet population. Within this population, the mean number of rhodamine B-labeled particles bound to total platelets (CD41a staining) and activated platelets (AlexaFluor 647 anti-CD62P) was determined. Mean fluorescence and standard deviation (CV) were determined using FlowJo™ software, and data analysis was performed using GraphPad Prism®. Overall, these assays demonstrated that the functionality of FMP2-modified synthetic platelets (i.e., binding to GPIIb / IIIa on activated platelets and enhancing activated platelet aggregation) was retained.To account for both lot-to-lot and intra-lot and assay variability, the physicochemical and functional properties of three separate manufacturing lots of each synthetic platelet product were evaluated. Functional testing was performed on at least three different days using blood from different donors. The mean and standard deviation for each assay run were calculated in Microsoft Excel® or GraphPad Prism®. Statistically significant differences were determined using a two-tailed Welch t-test and 95% confidence intervals in GraphPad Prism software. Results were considered statistically significant with a p-value of <0.05.

[0110] C. Results: Functional evaluation of peptides The ability of each peptide to inhibit platelet aggregation was tested over a concentration range from 0.0001 μM to 250 μM, and the IC 50 The IC of FMP2 was determined (Figures 1A-1B). 50 The IC of the FMP1 peptide was approximately 0.13 μM. 50 was approximately 536.6 μM.

[0111] MALDI-TOF characterization of peptides and lipid-peptide conjugates Mass spectral results for FMP1 versus FMP2 are shown in Figures 2A-2B. FMP1 showed evidence of dimerization / trimerization, likely due to cleavage of the cyclic disulfide bond and its reformation with free sulfhydryls on adjacent peptides. No dimerization / trimerization was observed in the FMP2 sample, suggesting improved stability. Furthermore, mass spectral results for the lipid-peptide conjugates are shown in Figures 3A-3B. The results confirm that FMP1 and FMP2 are effectively conjugated to DSPE-PEG(2000). A summary of the actual measured peaks versus the expected theoretical molecular weights is shown in Table 2, showing that all peptide and lipid-peptide conjugates are within 10% of the expected theoretical values. Overall, MALDI-TOF confirmed that both FMP1 and FMP2 were conjugated to their respective lipids.

[0112] [Table 2]

[0113] Physicochemical characterization of synthetic platelets Size and zeta potential characterization of synthetic platelet particle formulations was recorded immediately after production (Table 3) and over a 15-week storage period at 25°C (Figure 4). Particles produced with both 2.5 mol% (Particle 4) and 15 mol% (Particle 3) FMP2 maintained their size within the desired range (150-250 nm diameter), whereas particles produced with both 2.5 mol% (Particle 2) and 15 mol% (Particle 1) FMP1 exhibited values ​​higher than the expected starting size range (diameter >250 nm), possibly indicating particle aggregation. Welch's t-test for all formulations included in the study showed that Particle 2 and Particle 4 were not significantly different (p = 0.0542), while Particle 1 and Particle 3 formulations were significantly different (p = 0.0100). The polydispersity index (PDI) of Particle 4 was greater than 0.3, suggesting particle aggregation.

[0114] [Table 3]

[0115] Functional evaluation of synthetic platelet particles using platelet aggregation assays The percent final platelet aggregation (calculated using Equation 1) is summarized in Figure 5. The synthetic platelet preparation containing FMP2 showed increased final aggregation compared to the tPRP sample without synthetic platelets, and the level of increase was similar to that of particles containing FMP1 (p = 0.3986, not significant).

[0116] Functional evaluation of synthetic platelet particles by flow cytometry The gating strategy and fluorescence histograms for the ADP- and TRAP-activated samples are shown in Figures 6A–6E. Flow cytometry results showed low binding of control particles (CP, lipid particles without peptide modification) to non-activated, ADP-activated, and TRAP-activated platelets. Synthetic platelet particle formulations containing FMP1 (particle 1) and FMP2 (particle 3) showed significant binding to TRAP- and ADP-activated platelets (p < 0.05 compared to the CP group).

[0117] D. Summary / Conclusion A summary of these characterization studies, including experimental methods, test articles, pass / fail criteria, and results, is summarized in Table 4 below.

[0118] [Table 4] TIFF2025538487000008.tif177165

[0119] Overall, this data suggests that FMP2 inhibits platelet aggregation at concentrations up to three orders of magnitude lower than FMP1, indicating that changes in chemical structure significantly improved the peptide's potency. FMP2 also exhibits greater stability than FMP1, as indicated by the lack of dimer / trimer formation. Synthetic platelet particles incorporating FMP2 exhibit comparable and, in some cases, improved physicochemical and functional properties compared to particles containing FMP1.

[0120] reference [1] Cheng, S. et al., Craig, “Design and Synthesis of Novel Cyclic RGD-Containing Peptides as Highly Potent and Selective Integrin αIIbβ3 Antagonists. J. Med. Chem., 37(1):1-8 (1994). [2] Yee, D. et al., "Aggregometry detects platelet hyperreactivity in healthy individuals," Blood, 106:2723-2729 (2005).

[0121] Example 2: Various synthetic platelet preparations A. Research Objectives and Background Various synthetic platelet product compositions were evaluated based on size, surface charge, stability, and functionality. Briefly, the molar ratios of the components were varied as shown in Table 5. Physicochemical characterization of the synthetic platelet products consisted of dynamic light scattering and zeta potential measurements using an Anton Parr Litesizer 500, size and morphology assessed by cryo-TEM, and concentration and yield determined by a Biotek Synergy M5 fluorescent plate reader. The stability of selected formulations from the physicochemical analysis was assessed by size and charge using an Anton Parr Litesizer 500 on samples under simulated storage and shipping conditions (room temperature, 4°C, cycling from 4°C to room temperature, and ice packs with and without agitation). Functionality characterization of selected formulations from the physicochemical analysis was assessed by binding to activated platelets in flow cytometry using a BD Accuri B6 cytometer, platelet aggregation assay using a BioData PAP-8E LTA, collagen / vWF binding and platelet recruitment under flow using a BioFlux, clot formation time in ROTEM-NATEM, and in vivo bleeding time / blood loss assessment in a tail cut model in thrombocytopenic mice.

[0122] [Table 5] TIFF2025538487000010.tif52164

[0123] B. Method manufacturing Synthetic platelet particles were prepared using a standard lipid film rehydration and extrusion method. Briefly, DSPC, cholesterol, DSPE-PEG(2000)-PBP (FMP2, lRGD, Fg-H12, or P-selectin), DSPE-PEG(2000)-VBP, DSPE-PEG(2000)-CBP, DSPE-mPEG(1000), and DSPE-Cy5 were dissolved in 1:1 chloroform / methanol and mixed at various molar percentages. The organic solvent was removed under vacuum (Rotovap) to form a thin lipid film, followed by additional drying under a stream of nitrogen. The lipid film was then reconstituted with saline, followed by at least 10 freeze-thaw cycles and at least five extrusions through either 100 nm or 200 nm pore size filters (10 mL Lipex Extruder) to obtain particles of the desired size.

[0124] Duplicate batches were manufactured using each particle formulation optimization group to obtain average lot-to-lot means and standard deviations. In addition, all samples were analyzed in triplicate to obtain within-lot means and standard deviations.

[0125] Physicochemical analysis Particles were diluted with cell culture-grade water to a 1 mg / mL solution for size and charge analysis. The effective hydrodynamic diameter, polydispersity index, intensity- and number-normalized mean diameter, and zeta potential were acquired and evaluated using an Anton Parr Litesizer 500. For morphology analysis using cryo-TEM, samples prepared at 0.5 mg / mL in cell culture-grade water were adsorbed onto glow-discharge-treated coated copper grids for 2 minutes, blotted with filter paper, and flash-frozen in liquid ethane below the devitrification temperature of -137°C. Samples were imaged at magnifications ranging from 13,000x to 60,000x, and particle diameters were measured using the online software ImageJ.

[0126] In vitro functionality by flow cytometry The active platelet-binding functionality of the synthetic platelet preparations was evaluated by flow cytometry. Human whole blood was obtained from healthy donors by venipuncture and collected in vacutainers containing 3.8% sodium citrate. To obtain platelet-rich plasma (PRP), whole blood was centrifuged at 120 × g for 15 minutes at 25°C (no brake). PRP 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), and 0.03 units / mL apyrase was added. Contaminating red blood cells and white blood cells were pelleted by centrifugation at 100 × g for 15 minutes at 25°C. Prostacyclin (1 μg / mL) was added to PRP for 5 minutes, followed by centrifugation at 600 × g for 15 minutes at 25°C to wash the platelets. The platelet pellet was gently resuspended in Tyrode's buffer and equilibrated on a benchtop for 20–30 minutes before flow cytometry staining. The washed platelets were aliquoted into three flow cytometry tubes and incubated with Alexa Fluor 647 anti-CD62P, 5 μM TRAP agonist, and each synthetic platelet preparation (platelet:particle ratio approximately 1:1000) for 20 minutes at 25°C.

[0127] FITC anti-GP1b(α) was then added to the platelets and incubated for 1 minute before reading on the flow cytometer. Platelets were analyzed on a BD Accuri B6 cytometer by gating for the platelet population (side scatter (SSC) on the y-axis, forward scatter (FCS) on the x-axis, both on a logarithmic plot) until 50,000 counts per sample were measured. Gating on the fluorescent signal was performed using either FITC or Cy5 on the y-axis and FSC on the x-axis, both on a logarithmic plot. Flow cytometry data were analyzed with FlowJo software to obtain fluorescent counts for Cy5-labeled synthetic platelets and FITC-labeled platelets.

[0128] In vitro functionality by agglutination testing The active platelet aggregation functionality of the synthetic platelet products was assessed by transmitted light platelet aggregometry. Human whole blood was obtained by venipuncture from healthy donors and collected in vacutainers containing 3.8% sodium citrate. To obtain platelet-rich plasma (PRP), the whole blood was centrifuged at 120 × g for 15 minutes at 25°C (no brake). Platelet aggregation was performed on a Bio / Data PAP-8E platelet aggregation profiler by stimulating 225 μL of 25% diluted PRP with 25 μL of 10 μM adenosine 5'-diphosphate (ADP) (or saline as a negative control). 20 μL of each synthetic platelet sample was added to the 25% diluted PRP, and the final aggregation percentage was recorded. The final aggregation percentage was reported and normalized to the final aggregation percentage of particle-free TRAP / ADP-activated (tPRP) from the same donor (Equation 1).

[0129]

number

[0130] In vitro functionality with BioFlux The collagen / vWF binding and platelet recruitment functions of the synthetic platelet preparations were evaluated using BioFlux plates (0–200 dyn / cm). 2 ) was performed by adding 50 μL of equine Chronolog collagen (100 μg / mL) to the outlet well at 30 dyn / cm 2 Flow was initiated at 400 rpm for 30 seconds to coat the plate with collagen from outlet to inlet. Flow was then stopped and the plate was incubated at 37°C for 1 hour. 600 μL of 0.1% (w / v) BSA was then added to the inlet wells, followed by saline, at 30 dyn / cm. 2The wells were then washed with 0.1% (w / v) BSA solution for 5–10 minutes. Whole blood was obtained from healthy human donors by venipuncture and collected in vacutainers containing 3.8% sodium citrate. To obtain platelet-rich plasma (PRP), the whole blood was centrifuged at 120 × g for 15 minutes at 25°C (no brake). To label platelets with calcein-AM, 2 μL of calcein-AM (stock concentration 1 mg / mL) was added per mL of PRP solution and incubated for 20 minutes at room temperature. Platelets and Cy5-labeled synthetic platelet particles were added at a platelet:particle ratio of 1:100. Platelet-particle binding was recorded as % of the covered area and calculated over the first minute to obtain the binding rate.

[0131] In vitro functionality with ROTEM-NATEM The enhanced clotting function of synthetic platelet preparations was evaluated by ROTEM using the STARTEM technique. Human whole blood was obtained by venipuncture from healthy donors and collected in vacutainers containing 3.8% sodium citrate. Platelet-rich plasma (PRP) and red blood cell fractions were obtained by centrifuging the whole blood at 150 × g for 15 minutes. Platelet-free plasma (PFP) was obtained by further centrifuging half of the PRP at 13,000 × g for 5 minutes. PRP, PFP, and red blood cells were remixed in a ratio to obtain simulated thrombocytopenic whole blood (tWB) with 10% of the normal platelet count. 20 μL of STARTEM reagent and 2 μL of synthetic platelet particles were added to a ROTEM cup, followed by 300 μL of tWB, resulting in a final platelet:SP ratio of 1:10. The collected endpoints were clot formation time (CFT), clot formation kinetics A10, and alpha angle. All treatment group values ​​were normalized to the tWB condition without synthetic platelets to minimize the effect of donor-to-donor variability in platelet / clotting function.

[0132] In vivo functionality in thrombocytopenic mice The hemostatic function of synthetic platelet preparations was evaluated in thrombocytopenic (TCP) mice after tail amputation. Platelet counts from wild-type C57 / BL6J mice were obtained by collecting 0.1 mL of retro-orbital blood for platelet counting using a HemaVet 950. After dose calculations, mice were intraperitoneally injected with anti-CD42b (anti-GPIbα) antibody at 0.2 μg / g. Eighteen hours after antibody injection, platelet counts were retaken to monitor the induction of thrombocytopenia (an average approximately 75% decrease in platelet count). Synthetic platelet preparations were injected retro-orbitally at 0.1, 1.0, and 10 mg / kg and 5 mL / kg. Fifteen minutes after treatment administration, a 1 mm section from the tip of the mouse's tail was amputated with a sharp surgical blade and immersed in 1200 μL of warm (37°C) saline. The time it took for bleeding to stop (bleeding time) was recorded. Total blood loss was also measured using a standard hemoglobin assay in saline.

[0133] C. Results Size and Charge by Litesizer The hydrodynamic diameter and zeta potential of synthetic platelet formulations were evaluated using a Litesizer 500 particle analyzer. The Litesizer 500 obtained the hydrodynamic diameter, intensity diameter, polydispersity index (PDI), and zeta potential of all formulations using backscattering at 0.1 mg / mL samples (Table 6 and Figures 7A-7Y). Some particle formulations with high peptide content (greater than 3 mol% of the total lipid content) exhibited physicochemical instability. Furthermore, some particle formulations with FMP2 alone or FMP2 without CBP also exhibited physicochemical instability.

[0134] [Table 6]

[0135] Morphology and size by cryo-TEM The morphology and size of several synthetic platelet preparations were evaluated by cryo-TEM. As shown in Table 7 and Figures 8A-8C, all three preparations evaluated had a monolayer percentage of 85% or greater. The particle size distribution and PDI values ​​measured by cryo-TEM were comparable to Litesizer measurements of hydrodynamic and intensity diameters.

[0136] [Table 7]

[0137] In vitro functionality by flow cytometry The percentage of the activated platelet population that tested positive for Cy5-labeled particle binding for the various synthetic platelet preparations is summarized in Figure 9. Overall, preparations containing the platelet-binding peptide FMP2 showed higher binding to activated platelets compared to preparations containing lRGD, Fg-H12, or P-selectin-binding peptides.

[0138] In vitro functionality by agglutination testing The final and maximum aggregation percentages of PRP samples with platelet deficiency (i.e., thrombocytopenia, TCP) containing various synthetic platelet preparations, compared to samples without synthetic platelets, were calculated and recorded as shown in Figures 10A-10B. In this aggregation assay, only particle 12 demonstrated an increase in both the final aggregation percentage (115%) and the maximum aggregation percentage (108%) compared to the platelet-deficient sample. For all preparations, the increase in aggregation was not significantly different from the platelet-deficient sample.

[0139] In vitro functionality with ROTEM-NATEM A summary of the ROTEM-NATEM analysis of synthetic platelet products in human whole blood (WB) with 10% platelets (i.e., platelet-deficient) is summarized in Table 8. Particles 12 and 20 showed significant (p<0.05) rescue in coagulation kinetic parameters, including clot formation time (CFT), amplitude of the clotting curve at 10 minutes (A10), and alpha angle, compared to buffer-treated samples. Particle 23 showed a significant delay in coagulation kinetics compared to the buffer group (p<0.05).

[0140] [Table 8]

[0141] In vitro functionality with BioFlux Binding of synthetic platelet particles to collagen / vWF surfaces and recruitment of platelets to the surface were assayed using BioFlux. Platelet and particle binding areas (% coverage) and binding rates for various synthetic platelet preparations are summarized in Figures 11A-11D. In this assay, only particle 12 showed a significant increase in platelet recruitment (both coverage area and binding rate) onto the collagen / vWF surface compared to the negative control (vehicle, *p<0.05 and **p<0.01). Particle 12 also showed significant adhesion to the vWF / collagen surface.

[0142] In vivo function in thrombocytopenic mice Several synthetic platelet preparations were evaluated for hemostatic efficacy after tail amputation in thrombocytopenic mice. Blood loss and bleeding time were recorded, as summarized in Figures 12A-12D. All preparations tested showed a significant reduction in total blood loss at doses of 0.1-10 mg / kg (*p<0.05). Bleeding time was also reduced in animals treated with Particle 5, Particle 12, and Particle 20. Overall, the greatest effect was observed at doses of 0.5-5 mg / kg.

[0143] D. Summary / Conclusion The results of these physicochemical and functional characterization studies are summarized in Table 9 below.

[0144] [Table 9] TIFF2025538487000016.tif32162

[0145] Overall, the choice of platelet-binding peptide and the ratio between the various peptides in the formulation influence the physicochemical and functional properties of the synthetic platelet formulation.

[0146] References [1] Danei, M. Dehghankhold, S. Ataei, F. Hasanzadeh Davarani, R. Javanmard, A. Dokhani, S. Khorasani and MR Mozafari. Impact of Particle Size and Polydispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems. Pharmaceutics (2018) Vol 10, Issue 57, 1-17. [2] FDA Guidance for Liposomal Drug Products. US Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). April 2018. [3] Wang, Y. Grainger, DW Lyophilized liposome-based parenteral drug development:Reviewing complex product design strategies and current regulatory environments. Advanced Drug Delivery Reviews (2019) Vol 151-152. pg 56-71. [4] Tang, X. M. J. Pikal, Design of freeze-drying processes for pharmaceuticals:practical advice, Pharm. Res. 21 (2004) 191-200. [5] Sylvester, B. A. Porfire, M. Achim, L. Rus, I. Tomuja, A step forward towards the development of stable freeze-dried liposomes:a quality by design approach (QbD), Drug Dev. Ind. Pharm. 44 (2018) 385-397. [6] Iyer, Lavanya K et al. “Process and Formulation Effects on Protein Structure in Lyophilized Solids Using Mass Spectrometric Methods.” Journal of pharmaceutical sciences vol. 105,5 (2016):1684-1692. doi:10.1016 / j.xphs.2016.02.033. [7] Danaei, M. M. Dehghankhold, S. Ataei, F. Hasanzadeh Davarani, R. Javanmard, A. Dokhani, S. Khorasani and M. R. Mozafari. Impact of Particle Size and Poly dispersity Index on the Clinical Applications of Lipidic Nanocarrier Systems. Pharmaceutics. 10 (57) (2018) 1-17.

[0147] Incorporation by reference All publications, patents, and patent applications mentioned in this document are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application (including any definitions contained herein) will control.

[0148] Additionally, polynucleotide and polypeptide sequences that refer to accession numbers corresponding to entries in public databases (e.g., databases such as those maintained by The Institute for Genomic Research (TIGR) on the World Wide Web at tigr.org and / or the National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov) are also incorporated by reference in their entirety.

[0149] 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 particle comprising a plurality of platelet-binding peptide (PBP) conjugates, von Willebrand factor-binding peptide (VBP) conjugates, or collagen-binding peptide (CBP) conjugates, or combinations thereof, wherein the PBP conjugates are fibrinogen-mimetic peptide (FMP) conjugates or P-selectin-binding peptide conjugates, and the plurality of PBP conjugates, VBP conjugates, and / or CBP conjugates are conjugated to the outer surface of the particle, and the PBP conjugates, VBP conjugates, and CBP conjugates are collectively present at less than 5 mol% of the particle.

2. The particle of claim 1, wherein the PBP, VBP, and / or CBP, or combinations thereof, are spatially or topographically arranged on the particle surface such that the PBP, VBP, and / or CBP, or combinations thereof, do not spatially obscure each other.

3. 3. The particle according to claim 1, wherein PBP, VBP, and / or CBP, or a combination thereof, is conjugated to the particle surface via a PEG linker.

4. 4. The particle of claim 1, wherein the particle comprises DSPE-PEG2k-PBP, DSPE-PEG2k-VBP, or DSPE-PEG2k-CBP.

5. 5. The particle of claim 1, wherein the PBP conjugate comprises DSPE-PEG2k-FMP, wherein FMP is of formula (I): (Chem.1) Cyclo(CNPRGD{Tyr(OEt)}R-β-A) Formula (I)

6. The particle of any one of claims 1 to 5, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

2.

7. The particle of any one of claims 1 to 5, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

3.

8. The particle of any one of claims 1 to 6, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

4.

9. The particle of any one of claims 1 to 7, wherein the PBP conjugate comprises the amino acid sequence of SEQ ID NO:

5.

10. 10. The particle of claim 5, wherein the PBP is flanked by additional amino acid sequences.

11. 11. The particle of any one of claims 1 to 10, wherein the VBP comprises SEQ ID NO:

6.

12. The particle of claim 11 , wherein the VBP is flanked by additional amino acid sequences.

13. 13. The particle of any one of claims 1 to 12, wherein the CBP comprises SEQ ID NO:

7.

14. 14. The particle of claim 13, wherein the CBP is flanked by additional amino acid sequences.

15. The particle of any one of claims 1 to 14, wherein the particle comprises a plurality of PBP conjugates, a plurality of VBP conjugates, and a plurality of CBP conjugates.

16. 16. The particle of any one of claims 1 to 15, wherein the molar percentage of the PBP conjugate is between 0.05% and 5.00% molar percent of the particle.

17. 17. The particle of any one of claims 1 to 16, wherein the molar percentage of the FMP conjugate is between 0.05% and 5.00% molar percent of the particle.

18. 18. The particle of any one of claims 1 to 17, wherein the molar percentage of the CBP conjugate is between 0.10% and 1.50% molar percent of the particle.

19. 19. The particle of any one of claims 1 to 18, wherein the molar percentage of the VBP conjugate is between 0.10% and 1.50% molar percent of the particle.

20. 20. The particle of any one of claims 1 to 19, wherein the molar percentage of the PBP conjugate is about 1.00% of the particle, the molar percentage of the CBP conjugate is about 0.50% of the particle, and the molar percentage of the VBP conjugate is about 0.50% of the particle.

21. 21. The particle of any one of claims 1 to 20, wherein the particle comprises both a VBP conjugate and a CBP conjugate; and the ratio of VBP to CBP provided on the particle surface is from about 70:30 to about 30:

70.

22. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 2:1:

1.

23. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 1:5:

5.

24. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 10:5:

1.

25. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

5.

26. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

1.

27. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 1:2:

1.

28. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

2.

29. 22. The particle of any one of claims 1 to 21, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

1.

30. 11. The particle according to any one of claims 1 to 10, wherein the CBP and / or VBP is not conjugated to the surface of the particle.

31. 11. The particle of claim 1, wherein the relative molar ratio of PBP:CBP is 2:

1.

32. 11. The particle of claim 1, wherein the relative molar ratio of PBP:VBP is 2:

1.

33. 33. The particle of any one of claims 1 to 32, further comprising a lipid.

34. 34. The particle of claim 33, wherein the lipid comprises a phospholipid.

35. 34. The particle of claim 33, wherein the lipid comprises a phospholipid conjugated to polyethylene glycol (PEG).

36. 36. The particle of claim 35, wherein the average molecular weight of the PEG conjugated to the phospholipid is from about 500 Da to about 5500 Da.

37. 34. The particle of claim 33, wherein the lipid comprises cholesterol, distearoylphosphatidylcholine (DSPC), or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

38. 38. The particle of any one of claims 1 to 37, in the form of a liposome.

39. 39. The particle of any one of claims 1 to 38, wherein the particle has a diameter of about 1 to 1000 nm.

40. 39. The particle of any one of claims 1 to 38, wherein the particle has a diameter of about 30 to 300 nm.

41. 41. The particle of any one of claims 1 to 40, wherein the particle is substantially spherical.

42. 42. The particles of any one of claims 1 to 41, wherein the particles have a polydispersity index of 0.3 or less.

43. 43. The particle of any one of claims 1 to 42, wherein the particle has a net positive or net negative zeta potential.

44. 43. The particle of any one of claims 1 to 42, wherein the particle has a surface charge of less than -5mV.

45. 45. The particle of any one of claims 1 to 44, wherein the particle is stable under storage in an aqueous medium for at least 12 weeks.

46. 46. ​​The particle of any one of claims 1 to 45, wherein the particle promotes platelet aggregation.

47. 47. The particle of any one of claims 1 to 46, wherein the particle binds to activated platelets.

48. 48. The particle of any one of claims 1 to 47, wherein the particle binds to ADP or TRAP-activated platelets.

49. Fibrinogen-mimetic peptides (FMPs) of formula (I): (Case 2) Cyclo(CNPRGD{Tyr(OEt)}R-β-A) Formula (I) Or its salt.

50. 50. The fibrinogen-mimetic peptide of claim 49, wherein the fibrinogen-mimetic peptide is an acetate salt of formula (I), such as a trifluoroacetate salt of formula (I).

51. 51. The fibrinogen-mimetic peptide of claim 49 or 50, wherein the fibrinogen-mimetic peptide specifically binds to activated GPIIb-IIIa on platelets.

52. 52. The fibrinogen-mimetic peptide of any one of claims 49 to 51, wherein the fibrinogen-mimetic peptide inhibits platelet aggregation.

53. IC of fibrinogen-mimetic peptides 50 53. The fibrinogen-mimetic peptide of any one of claims 49 to 52, wherein the fibrinogen-mimetic peptide has a saturation of about 0.13 μM.

54. 54. A peptide conjugate comprising a peptide conjugated to a polymer, wherein the peptide is a fibrinogen-mimetic peptide according to any one of claims 49 to 53.

55. 55. The peptide conjugate of claim 54, wherein the polymer is a lipid.

56. 56. The peptide conjugate of claim 55, wherein the lipid comprises DSPE-PEG(2k).

57. 57. The peptide conjugate of any one of claims 54 to 56, wherein FMP is conjugated to the polymer by thiol-ene coupling to the thiol group of the N-terminal cysteine.

58. 58. A particle comprising a plurality of platelet-binding peptide (PBP) conjugates, wherein the PBP conjugates comprise the peptide conjugate of any one of claims 55 to 57.

59. 59. The particle of claim 58, further comprising a plurality of von Willebrand factor binding peptide (VBP) conjugates, or collagen binding peptide (CBP) conjugates, or a combination thereof.

60. 60. The particle of claim 58 or 59, wherein the PBP, VBP, and / or CBP, or combinations thereof, are spatially or topographically arranged on the particle surface such that the PBP, VBP, and / or CBP, or combinations thereof, do not spatially obscure each other.

61. 61. The particle of any one of claims 58 to 60, wherein PBP, VBP, and / or CBP, or a combination thereof, is conjugated to the particle surface via a PEG linker.

62. 62. The particle of any one of claims 58 to 61, wherein the particle comprises DSPE-PEG2k-PBP, DSPE-PEG2k-VBP, or DSPE-PEG2k-CBP.

63. 63. The particle of any one of claims 59 to 62, wherein the PBP conjugate further comprises the amino acid sequence of SEQ ID NO:

3.

64. 63. The particle of any one of claims 59 to 62, wherein the PBP conjugate further comprises the amino acid sequence of SEQ ID NO:

4.

65. 63. The particle of any one of claims 59 to 62, wherein the PBP conjugate further comprises the amino acid sequence of SEQ ID NO:

5.

66. 66. The particle of any one of claims 58 to 65, wherein the PBP is flanked by additional amino acid sequences.

67. 67. The particle of any one of claims 59 to 66, wherein the VBP comprises SEQ ID NO:

6.

68. 68. The particle of claim 67, wherein the VBP is flanked by additional amino acid sequences.

69. 69. The particle of any one of claims 59 to 68, wherein the CBP comprises SEQ ID NO:

7.

70. 70. The particle of claim 69, wherein the CBP is flanked by additional amino acid sequences.

71. 71. The particle of any one of claims 59 to 70, wherein the particle comprises a plurality of PBP conjugates, a plurality of VBP conjugates, and a plurality of CBP conjugates.

72. 72. The particle of any one of claims 58 to 71, wherein the molar percentage of the PBP conjugate is between 0.05% and 5.00% molar percent of the particle.

73. 72. The particle of any one of claims 58-71, wherein the molar percentage of the FMP conjugate is between 0.05% and 5.00% molar percent of the particle.

74. 74. The particle of any one of claims 59 to 73, wherein the molar percentage of the CBP conjugate is between 0.10% and 1.50% molar percent of the particle.

75. 75. The particle of any one of claims 59 to 74, wherein the molar percentage of the VBP conjugate is between 0.10% and 1.50% molar percent of the particle.

76. 76. The particle of any one of claims 59 to 75, wherein the molar percentage of the PBP conjugate is about 1.00% of the particle, the molar percentage of the CBP conjugate is about 0.50% of the particle, and the molar percentage of the VBP conjugate is about 0.50% of the particle.

77. 77. The particle of any one of claims 59 to 76, wherein the particle comprises both a VBP conjugate and a CBP conjugate; and the ratio of VBP to CBP provided on the particle surface is from about 70:30 to about 30:

70.

78. 78. The particle of any one of claims 59 to 77, wherein the relative molar ratio of PBP:CBP:VBP is 2:1:

1.

79. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 1:5:

5.

80. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 10:5:

1.

81. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

5.

82. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

1.

83. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 1:2:

1.

84. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 1:1:

2.

85. 76. The particle of any one of claims 59 to 75, wherein the relative molar ratio of PBP:CBP:VBP is 10:1:

1.

86. 67. The particle of any one of claims 59 to 66, wherein no CBP or VBP is conjugated to the surface of the particle.

87. 67. The particle of any one of claims 59 to 66, wherein the relative molar ratio of PBP:CBP is 2:

1.

88. 67. The particle of any one of claims 59 to 66, wherein the relative molar ratio of PBP:VBP is 2:

1.

89. 89. The particle of any one of claims 59 to 88, further comprising a lipid.

90. 90. The particle of claim 89, wherein the lipid comprises a phospholipid.

91. 90. The particle of claim 89, wherein the lipid comprises a phospholipid conjugated to polyethylene glycol (PEG).

92. 92. The particle of claim 91, wherein the average molecular weight of the PEG conjugated to the phospholipid is from about 500 Da to about 5500 Da.

93. 90. The particle of claim 89, wherein the lipid comprises cholesterol, distearoylphosphatidylcholine (DSPC) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE).

94. 94. The particle of any one of claims 58 to 93, in the form of a liposome.

95. 95. The particle of any one of claims 58 to 94, wherein the particle has a diameter of about 1 to 1000 nm.

96. 95. The particle of any one of claims 58 to 94, wherein the particle has a diameter of about 30 to 300 nm.

97. 97. The particle of any one of claims 58 to 96, wherein the particle is substantially spherical.

98. 98. The particle of any one of claims 58 to 97, wherein the particle has a polydispersity index of 0.3 or less.

99. 99. The particle of any one of claims 58 to 98, wherein the particle has a net positive or net negative zeta potential.

100. 99. The particle of any one of claims 58 to 98, wherein the particle has a surface charge of less than -5mV.

101. 101. The particle of any one of claims 58 to 100, wherein the particle is stable under storage in an aqueous medium for at least 12 weeks.

102. 102. The particle of any one of claims 58 to 101, wherein the particle promotes platelet aggregation.

103. 103. The particle of any one of claims 58 to 102, wherein the particle binds to activated platelets.

104. 104. The particle of any one of claims 58 to 103, wherein the particle binds to ADP or TRAP-activated platelets.

105. A pharmaceutical composition comprising the fibrinogen-mimetic peptide of any one of claims 49 to 53 and a carrier.

106. 58. A pharmaceutical composition comprising the peptide conjugate of any one of claims 54 to 57 and a carrier.

107. A pharmaceutical composition comprising a particle according to any one of claims 1 to 48 and 58 to 104 and a carrier.

108. 108. The pharmaceutical composition of any one of claims 105 to 107, further comprising a therapeutic agent for the treatment of a disease or condition.

109. 109. The pharmaceutical composition of claim 108, wherein the particles encapsulate a therapeutic agent.

110. 110. The pharmaceutical composition of claim 108 or 109, wherein the therapeutic agent is an antibacterial, antiviral, or antifungal agent.

111. 110. The pharmaceutical composition of claim 108 or 109, wherein the therapeutic agent is an anticancer agent.

112. 110. The pharmaceutical composition of claim 108 or 109, wherein the therapeutic agent is a hemostatic agent.

113. 113. The pharmaceutical composition of any one of claims 105 to 112, wherein the carrier is a pharmaceutically acceptable carrier.

114. 54. A method for preventing or inhibiting platelet aggregation in a subject in need thereof, comprising administering to the subject a composition comprising a fibrinogen-mimetic peptide (FMP) according to any one of claims 49 to 53.

115. 104. A method for preventing or inhibiting platelet aggregation in a subject in need thereof, comprising administering to the subject a fibrinogen-mimetic peptide according to any one of claims 49 to 53, or a pharmaceutical composition according to claim 105.

116. A method for promoting activated platelet aggregation at a site where vWF and collagen are exposed, comprising administering to the site a particle described in any one of claims 1 to 48 and 58 to 104, or a pharmaceutical composition described in any one of claims 107 to 113.

117. 114. A method of reducing bleeding in a subject, comprising administering to the site of bleeding a particle described in any one of claims 1 to 48 and 58 to 104, or a pharmaceutical composition described in any one of claims 107 to 113.

118. 114. A method of treating vascular injury in a subject, comprising administering to the site of vascular injury in the subject a particle described in any one of claims 1 to 48 and 58 to 104, or a pharmaceutical composition described in any one of claims 107 to 113.

119. 119. The method of claim 118, wherein the vascular injury is non-compressible bleeding due to trauma, bleeding due to surgery, or thrombocytopenia.

120. 114. A method of promoting hemostasis in a subject in need thereof, comprising administering to the subject a particle described in any one of claims 1 to 48 and 58 to 104, or a pharmaceutical composition described in any one of claims 107 to 113.

121. 112. A method of treating cancer in a subject in need thereof, comprising administering to the subject the pharmaceutical composition of claim 111.

122. 122. The method of claim 121, wherein the subject has or is at risk of having thrombocytopenia.

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