PLGA-PSP nanoparticle and application thereof

Abstract

The present invention relates to a PLGA-PSP nanoparticle, the PLGA-PSP nanoparticle comprises a plurality of PSP conjugates, each PSP conjugate is represented by the following structural formula: PLGA-PEG-PSP, PLGA represents a polylactic acid-glycolic acid copolymer forming the core part of the nanoparticle, PEG represents polyethylene glycol for connecting PLGA and PSP, PSP represents a platelet specific polypeptide, and the PLGA-PEG-PSP is used for connecting PLGA with PSP. The polypeptide is located on the surface of the nanoparticle and comprises one or more, such as two or three, of collagen binding peptide (CBP), vWF binding peptide (VBP) and fibrinogen mimetic peptide (FMP). Research results show that the PLGA-PSP nanoparticles can significantly induce adhesion and aggregation of platelets at an injured part, so that hemostasis is promoted, the bleeding amount is reduced, and the bleeding time is shortened.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to PLGA-PSP nanoparticles and applications thereof. Background Art

[0002] Traumatic bleeding is a common clinical medical problem, accounting for 30%-40% of all trauma-related deaths. Severe trauma can also easily lead to infection and even threaten the patient's life. Platelets play a crucial role in thrombosis, physiological hemostasis, immune defense, and promoting tissue wound healing. They are widely used to treat and prevent bleeding disorders caused by thrombocytopenia or platelet dysfunction, particularly traumatic bleeding, surgical bleeding, and bleeding in patients undergoing bone marrow transplantation, leukemia, and chemotherapy and radiotherapy for cancer. However, with the growing demand for platelets, the existing blood supply system has become unable to meet clinical needs. Platelets are short-lived and easily activated after ex vivo, losing their hemostatic activity. They are difficult to store stably at room temperature outside the body for long periods of time, making them unable to meet the urgent and large-scale demand for treating traumatic bleeding in extreme situations such as natural disasters and war. Platelet substitutes are considered an effective way to address the platelet shortage. Therefore, the development of novel biomimetic platelets holds critical clinical significance for the treatment of traumatic bleeding.

[0003] Traumatic bleeding damages the vascular lining, exposing the subendothelial matrix. The GP Ib / IX / V complex on the platelet surface interacts with von Willebrand factor (vWF) secreted by endothelial cells, leading to reversible platelet attachment to damaged endothelial cells. Simultaneously, GP Ia-IIa interacts with collagen, enabling platelets to slowly roll at the site of vascular injury. Once platelets are activated, the integrins GP IIb / IIIa (integrin α2bβ3) on the platelet surface undergo a conformational change. Activated GP IIb / IIIa irreversibly bind to fibrinogen in the extracellular matrix, allowing platelets to stably adhere to the site of vascular injury. Platelets then recruit other locally activated platelets to the site of injury for aggregation, ultimately achieving effective hemostasis at the wound.

[0004] Studies have shown that liposomes modified with fibrinogen functional polypeptide fragments (arginyl-glycyl-aspartic acid, RGD) peptides can effectively target platelet activation, significantly improve hemostasis efficiency, and shorten the bleeding time of the mouse tail. Functionally integrated platelet-like nanoparticles (PLN) can shorten the bleeding time of the mouse tail by 65%, but there is a possibility of inducing pulmonary microthrombosis ADDIN EN.CITE ADDIN EN.CITE.DATA. The literature Blood.2023 Jun 8;141(23):2891-2900 reported that synthetic platelet nanoparticles reduced the blood loss of VWD-2B mice by 35%, but organic solvents such as chloroform and methanol were used in the synthesis process, which may have certain cytotoxicity. Preventive use of SynthoPlate TM SynthoPlate can effectively correct the hemostatic defect in thrombocytopenic mice, but TM The distearoylphosphatidylethanolamine (DSPE) used is a phospholipid naturally present in cell membranes. Excessive consumption may cause a burden on lipid metabolism. Moreover, since DSPE itself is difficult to dissolve and is usually soluble in organic solvents such as ether, chloroform and dichloromethane, it may have certain toxicity due to the presence of organic solvents. The self-assembling short peptide RGD is highly favored in hemostatic applications due to its strong modifiability, low cost, excellent viscoelasticity, high biocompatibility and low immunoreactivity. Research from the Yale University Biomedical Engineering Laboratory also pointed out that RGD-modified poly(lactic-co-glycolic acid) (PLGA) nanoparticles can target and connect to activated platelets, causing large platelet aggregation and shortening the bleeding time at the injured femoral artery in rats. At the same time, these nanoparticles can also effectively shorten the bleeding time at the injured site in blunt and blast injuries. However, the cyclic RGD contains one or two ring structures and has poor biodegradability. RGD lacks platelet specificity and can bind to different integrins on the surfaces of multiple cells, which has potential off-target side effects. RGD may partially activate resting platelets, thereby causing systemic thrombotic risks.

[0005] Therefore, in the context of a long-term shortage of platelet supply, there is an urgent need in the art to develop a hemostatic material that can effectively stop bleeding, has good safety, high stability, and can be stored for a long time. Summary of the Invention

[0006] As described in the background technology section, there is an urgent need in the art for a hemostatic material that can effectively stop bleeding, has good safety, high stability, and can be stored for a long time.

[0007] Therefore, in a first aspect, the present invention provides a PLGA-PSP nanoparticle comprising a plurality of PSP conjugates, each of which is represented by the following formula: PLGA-PEG-PSP, in: The PLGA represents poly(lactic-co-glycolic acid), which constitutes the core of the nanoparticles. The PEG represents polyethylene glycol, which is used to connect the PLGA and the PSP. The PSP represents a platelet-specific polypeptide, located on the surface of the nanoparticle, and includes one or more, for example, two or three, of collagen-binding peptide (CBP), vWF-binding peptide (VBP) and fibrinogen mimetic peptide (FMP).

[0008] In a second aspect, the present invention provides the use of the PLGA-PSP nanoparticles in the preparation of a medicament for preventing or treating: a) bleeding, such as arterial bleeding, venous bleeding and / or capillary bleeding; b) Vascular damage, such as vascular damage caused by trauma or surgery; c) Bleeding disorders caused by thrombocytopenia or platelet dysfunction.

[0009] The present invention has at least one or more of the following technical effects: 1. The present invention links VBP, CBP, and FMP to the surface of PLGA-PSP nanoparticles, enabling them to mimic the adhesion and aggregation properties of natural platelets. Upon vascular injury, the PLGA-PSP nanoparticles specifically bind to exposed collagen and VWF via CBP and VBP, respectively. FMP then binds to activated platelet GP IIb-IIIa receptors via fibrinogen, targeting the site of vascular injury and promoting platelet recruitment and aggregation, thereby promoting hemostasis and reducing bleeding volume and duration. Research results demonstrated that the PLGA-PSP nanoparticles provided by the present invention achieved rapid hemostasis for venous, visceral, and arterial bleeding in both normal and thrombocytopenic mice. Furthermore, the PLGA-PSP nanoparticles promoted the adhesion and aggregation of activated platelets without promoting platelet activation, thus preventing changes in the body's coagulation function.

[0010] 2. The vesicle-like structure of the PLGA-PSP nanoparticles of the present invention can highly mimic the vesicle-like structure of natural platelets in terms of structure and function; PLGA-PSP nanoparticles have no effect on resting platelets and do not induce spontaneous platelet aggregation. They only significantly promote platelet aggregation in the presence of platelet stimulants.

[0011] 3. The PLGA-PSP nanoparticles of the present invention exhibit a vesicle-like structure, presenting as monodispersed, regular spheres with no agglomeration, and are stable for long-term storage. Transmission electron microscopy results show that after six months of conventional storage at 4°C, the PLGA-PSP nanoparticles retain a relatively intact vesicle-like structure, with no significant changes in morphology or particle size.

[0012] 4. The PLGA-PSP nanoparticles of the present invention use PLGA. Since PLGA is a biodegradable copolymer formed by the copolymerization of lactic acid and glycolic acid monomers, and no organic solvents such as chloroform and methanol are used in the synthesis of PLGA-PSP, the PLGA-PSP nanoparticles of the present invention have good biosafety. Studies have shown that PLGA-PSP nanoparticles do not cause inflammation and microthrombosis in tissues and organs, and are safe for human use. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other implementation plans can be obtained based on these drawings without paying any creative work.

[0014] Figure 1 This is the ultraviolet absorption spectrum of the PLGA-PSP nanoparticles of the present invention.

[0015] Figure 2 This is the particle size distribution diagram of the PLGA-PSP nanoparticles of the present invention.

[0016] Figure 3 Figures 1 and 2 are transmission electron micrographs of the PLGA-PSP nanoparticles of the present invention, wherein Figure A is a transmission electron micrograph of freshly synthesized PLGA-PSP nanoparticles, and Figure B is a transmission electron micrograph of PLGA-PSP nanoparticles stored at 4°C for 6 months.

[0017] Figure 4 The results show the platelet adhesion inducing ability of the PLGA-PSP nanoparticles of the present invention.

[0018] Figure 5 The results show the ability of the PLGA-PSP nanoparticles of the present invention to induce platelet aggregation, wherein Figure A shows the aggregation of platelets in PRP under resting conditions, and Figure B shows the aggregation of platelets in PRP in the presence of platelet stimulant ADP.

[0019] Figure 6The results of the hemostatic ability of the PLGA-PSP nanoparticles of the present invention on venous bleeding in traumatic mice are shown, wherein Figure A shows the hemostatic time of tail vein bleeding in normal mice, Figure B shows the amount of tail vein bleeding in normal mice, Figure C shows the hemostatic time of tail vein bleeding in platelet-reduced mice, and Figure D shows the amount of tail vein bleeding in platelet-reduced mice.

[0020] Figure 7 The results of the hemostatic ability of the PLGA-PSP nanoparticles of the present invention on visceral bleeding in traumatic mice are shown, wherein Figure A shows the hemostatic time of liver bleeding in normal mice, Figure B shows the amount of liver bleeding in normal mice, Figure C shows the hemostatic time of liver bleeding in thrombocytopenic mice, and Figure D shows the amount of liver bleeding in thrombocytopenic mice.

[0021] Figure 8 The results of the hemostatic ability of the PLGA-PSP nanoparticles of the present invention on arterial bleeding in traumatic mice are shown, wherein Figure A shows the hemostatic time of femoral artery bleeding in normal mice, Figure B shows the amount of femoral artery bleeding in normal mice, Figure C shows the hemostatic time of femoral artery bleeding in thrombocytopenic mice, and Figure D shows the amount of femoral artery bleeding in thrombocytopenic mice.

[0022] Figure 9 The figure shows histological staining of PLGA-PSP nanoparticles of the present invention injected into mice via the tail vein. DETAILED DESCRIPTION

[0023] Hereinafter, the present invention will be described in detail. It should be understood that the following description is only for illustration of the present invention and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be subject to the appended claims. In addition, those skilled in the art will understand that the technical solutions of the present invention may be modified without departing from the spirit and purpose of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.

[0025] In the context of the present invention, many embodiments use the expressions "comprising", "including" or "consisting essentially / mainly of..." The expressions "comprising", "including" or "consisting essentially / mainly of..." should be understood as open-ended expressions, indicating that in addition to the elements, components, assemblies, method steps, etc. specifically listed after the expression, other elements, components, assemblies, method steps, etc. are also included. In addition, in this document, the expressions "comprising", "including" or "consisting essentially / mainly of..." may also be understood as closed-ended expressions in some cases, indicating that only the elements, components, assemblies, method steps specifically listed after the expression are included, and no other elements, components, assemblies, method steps are included. In this case, the expression is equivalent to the expression "consisting of..."

[0026] It should be noted that, for the numerical values ​​or numerical ranges mentioned herein, unless the context clearly states otherwise, they are all qualified by the term "about". Herein, for a numerical value, the term "about" means ±5% of the numerical value, such as ±4%, ±3%, ±2%, or ±1%. For a numerical range, the term "about" means ±5% of the upper and lower limits of the numerical range, such as ±4%, ±3%, ±2%, or ±1%.

[0027] In this document, ordinal numbers such as "first," "second," and "third" are sometimes used to modify or qualify elements, components, parts, method steps, solutions, solvents, temperatures, systems, etc. It should be noted that, in this document, these terms are only used to distinguish the elements, components, parts, method steps, solutions, solvents, temperatures, systems, etc. that they modify or qualify, and are not intended to limit their order or importance.

[0028] In response to the safety issues of existing platelet-like nanoparticles, such as causing inflammation, microthrombi, and potential toxicity caused by residual organic solvents, the present invention aims to provide a new hemostatic material to solve at least one of the above safety issues.

[0029] In a first aspect, the present invention provides a PLGA-PSP nanoparticle comprising a plurality of PSP conjugates, each of which is represented by the following structural formula: PLGA-PEG-PSP, in: The PLGA represents poly(lactic-co-glycolic acid), which constitutes the core of the nanoparticles. The PEG represents polyethylene glycol, which is used to connect the PLGA and the PSP. The PSP represents a platelet-specific polypeptide, located on the surface of the nanoparticle, and includes one or more, for example, two or three, of collagen-binding peptide (CBP), vWF-binding peptide (VBP) and fibrinogen mimetic peptide (FMP).

[0030] As can be seen from the above, the PLGA-PSP nanoparticles of the present invention contain PSP conjugates, and each of the PSP conjugates is represented by the formula PLGA-PEG-PSP.

[0031] The PSP conjugate comprises PLGA. PLGA is a hydrophobic material that forms the core of the nanoparticles. PLGA is a biodegradable copolymer formed by copolymerization of lactic acid and glycolic acid monomers. After PLGA is injected into the body, it gradually degrades into its comonomer molecules, lactic acid and glycolic acid. These metabolites can be excreted from the body through normal metabolic pathways and can ultimately be metabolized into CO2 and H2O, leaving no toxic residues. PLGA can efficiently load small molecule drugs, proteins, and nucleic acids, with a drug loading rate significantly higher than that of phospholipid carriers and greater stability.

[0032] In one embodiment, the degree of polymerization of the polyglycolic acid in the PLGA is 30-90, for example, 30, 32, 35, 38, 40, 42, 45, 48, 50, 52, 55, 58, 60, 62, 65, 68, 70, 72, 75, 78, 80, 82, 85, 88 or 90, preferably 30-70.

[0033] In another embodiment, the degree of polymerization of the polylactic acid in the PLGA is 50-100, for example, 50, 52, 55, 58, 60, 62, 65, 68, 70, 72, 75, 78, 80, 72, 85, 88, 90, 92, 95, 98 or 100, preferably 50-90.

[0034] By controlling the degree of polymerization of polyglycolic acid and polylactic acid within the above range, the formation of a nanoscale vesicle-like structure that mimics the structure of platelets can be ensured.

[0035] The PSP conjugate further comprises PEG. PEG is a hydrophilic segment used to link the PLGA and the PSP. In one embodiment, the PEG has a degree of polymerization (DP) of 40-130, such as 40, 42, 45, 48, 50, 52, 55, 58, 60, 62, 65, 68, 70, 72, 75, 78, 80, 82, 85, 88, 90, 92, 95, 98, 100, 102, 105, 108, 110, 112, 115, 118, 120, 122, 125, 128, or 130, preferably 50-80. In a preferred embodiment, the PEG is PEG 3400. By controlling the DP of PEG within the above range, the steric hindrance of the binding of the polypeptide to the PLGA can be reduced without affecting the spatial structure of the polypeptide.

[0036] The PSP conjugate further comprises a platelet-specific polypeptide (PSP). These functional polypeptides are located on the surface of the nanoparticles and include one or more, for example, two or three, of collagen-binding peptide (CBP), vWF-binding peptide (VBP) and fibrinogen mimetic peptide (FMP).

[0037] Collagen-binding peptides (CBPs) are a class of polypeptides that specifically bind to collagen, a major component of the vascular subendothelial matrix. When exposed to blood during vascular injury, CBPs trigger platelet adhesion and thrombosis. CBPs recognize conformational epitopes on collagen (e.g., hydrophobic regions on the α-chain of collagen) through their specific amino acid sequences (e.g., containing GPO repeats), thereby blocking platelet binding to collagen (e.g., inhibiting platelet surface receptor GPVI or integrin α2β1).

[0038] In one embodiment, the CBP comprises or consists of the amino acid sequence [GPO] 7. In a further embodiment, in addition to the amino acid sequence [GPO] 7, the CBP further comprises 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) natural or unnatural amino acids on one or both sides of the amino acid sequence.

[0039] In another embodiment, the CBP comprises a variant that has at least 75%, 80%, 85%, 90%, 95%, 98% or 100% amino acid sequence identity to the amino acid sequence [GPO] 7 and still has the function of the amino acid sequence [GPO] 7. In a further embodiment, the CBP comprises, in addition to the variant, an additional 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9 or 10) natural amino acids and / or unnatural amino acids on one or both sides of the variant.

[0040] In a preferred embodiment, the CBP is a linear peptide. In a most preferred embodiment, the CBP is [GPO]7.

[0041] VWF-binding peptides (VBPs) specifically bind to von Willebrand factor (vWF), a key coagulation protein in plasma that mediates platelet adhesion to damaged vascular endothelium and stabilizes factor VIII under high shear stress. Most VBPs bind to the A1 region of vWF, blocking its interaction with platelet glycoprotein Ib (GPIb) (inhibiting platelet adhesion); a smaller number target the A3 region, blocking vWF binding to collagen. VBPs can inhibit vWF-mediated platelet aggregation (antithrombotic) and reduce thrombosis initiation by blocking the vWF-collagen interaction (applicable to arterial thrombosis).

[0042] In one embodiment, the VBP comprises or consists of the amino acid sequence TRYLRIHPQSWVHQI. In a further embodiment, the VBP comprises the amino acid sequence TRYLRIHPQSWVHQI and further comprises 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) natural or unnatural amino acids on one or both sides of the amino acid sequence.

[0043] In another embodiment, the VBP comprises a variant that has at least 75%, 80%, 85%, 90%, 95%, 98% or 100% amino acid sequence identity to the amino acid sequence TRYLRIHPQSWVHQI and still has the function of the amino acid sequence TRYLRIHPQSWVHQI. In a further embodiment, the VBP comprises, in addition to the variant, an additional 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9 or 10) natural amino acids and / or unnatural amino acids on one or both sides of the variant.

[0044] In a preferred embodiment, the VBP is a linear peptide. In a most preferred embodiment, the VBP is TRYLRIHPQSWVHQI.

[0045] Fibrinogen-mimetic peptides (FMPs) are a class of peptides that mimic the functional domains of fibrinogen. Fibrinogen is a key protein in blood coagulation, activating platelet integrin αIIbβ3 (GPIIb / IIIa) through its RGD sequence (e.g., Arg-Gly-Asp on the γ chain). FMPs contain either RGD or KQAGDV sequences and competitively bind to the platelet αIIbβ3 receptor. FMPs come in two types: inhibitory FMPs and activating FMPs. Inhibitory FMPs block fibrinogen binding to platelets, resulting in antiplatelet aggregation (antithrombotic) properties, while activating FMPs promote platelet cross-linking, thereby achieving hemostasis (used in bleeding disorders). The FMPs described in this invention are activating FMPs.

[0046] In one embodiment, the FMP comprises or consists of the amino acid sequence HHLGGAKQAGDV. In a further embodiment, the FMP comprises the amino acid sequence HHLGGAKQAGDV and further comprises 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) natural and / or unnatural amino acids on one or both sides of the amino acid sequence.

[0047] In another embodiment, the FMP comprises a variant that has at least 75%, 80%, 85%, 90%, 95%, 98%, or 100% amino acid sequence identity to the amino acid sequence HHLGGAKQAGDV and still has the function of the amino acid sequence HHLGGAKQAGDV. In a further embodiment, the FMP comprises, in addition to the variant, an additional 3-10 (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) natural or unnatural amino acids on one or both sides of the variant.

[0048] In a preferred embodiment, the FMP is a linear peptide. In a most preferred embodiment, the FMP is HHLGGAKQAGDV.

[0049] In a preferred embodiment, the FMP is a linear peptide. The advantage of a linear FMP is that it has less steric hindrance and more readily forms a stable spherical structure upon binding to PLGA. This facilitates stable binding to platelet αIIbβ3 receptors, triggering platelet aggregation. Using a linear FMP enhances the platelet aggregation-inducing activity of PLGA-PSP nanoparticles. In a most preferred embodiment, the FMP is HHLGGAKQAGDV.

[0050] For the natural amino acids or non-natural amino acids mentioned above, herein, the natural amino acids can be selected from leucine, isoleucine, valine, methionine (methionine), phenylalanine, tryptophan, threonine, lysine, histidine, glycine, alanine, serine, aspartic acid, asparagine, glutamic acid, glutamine, proline, arginine, tyrosine or cysteine, and the non-natural amino acids are selected from artificially synthesized amino acids such as D-alanine, D-serine, norleucine, cyclohexylalanine, fluorophenylalanine, azidolysine, alkynylalanine, alkenylglycine, homoarginine or homoserine.

[0051] In a preferred embodiment, the PLGA-SPS nanoparticles contain multiple species of CBP, VBP, and FMP, such as two or more species, more preferably three species.

[0052] Therefore, in one embodiment, the PLGA-PSP nanoparticles comprise three of PLGA-PEG-CBP, PLGA-PEG-VBP, and PLGA-PEG-FMP. In a preferred embodiment, the PLGA-PSP nanoparticles comprise PLGA-PEG-CBP, PLGA-PEG-VBP, and PLGA-PEG-FMP in a molar ratio of 1:1:1.

[0053] In one embodiment, the PLGA-PSP nanoparticles have a vesicle-like structure. More specifically, the PLGA-PSP nanoparticles of the present invention have a vesicle-like structure, are monodisperse, regular spheres, lack agglomeration, and are injectable. The vesicle-like structure of the PLGA-PSP nanoparticles of the present invention can highly mimic the vesicle structure and hemostatic function of natural platelets in terms of structure and function.

[0054] In one embodiment, the PLGA-PSP nanoparticles have a particle size distribution of 50-200 nm, for example, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm, and have an average particle size of 120 nm.

[0055] The PLGA-PSP nanoparticles of the present invention are highly stable and can be stored stably for a long time. Transmission electron microscopy results show that after conventional storage at 4°C for 6 months, the PLGA-PSP nanoparticles still have a relatively complete vesicle-like structure, and their morphology and particle size do not change significantly.

[0056] Thus, in one embodiment, the PLGA-PSP nanoparticles retain a substantially intact vesicle-like structure after storage at 4° C. for at least 6 months, and their particle size distribution remains between 50 and 200 nm, for example, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 100 nm, 110 nm, 120 nm, 130 nm, 140 nm, 150 nm, 160 nm, 170 nm, 180 nm, 190 nm or 200 nm, and their average particle size is 120 nm.

[0057] In addition, the PLGA-PSP nanoparticles of the present invention have no effect on resting platelets and do not induce spontaneous platelet aggregation. They significantly promote platelet aggregation only in the presence of a platelet stimulant.

[0058] The PLGA-PSP nanoparticles of the present invention can be synthesized by first reacting PLGA-PEG-NHS with VBP, CBP, and FMP, respectively, specifically by allowing the ester group on the NHS side of PLGA-PEG-NHS to undergo an amidation reaction with the terminal amino group of VBP, CBP, or FMP to form an amide bond, thereby synthesizing PLGA-PEG-CBP, PLGA-PEG-VBP, and PLGA-PEG-FMP; then, the PLGA-PEG-CBP, PLGA-PEG-VBP, and PLGA-PEG-FMP are mixed in a specific ratio to form the PLGA-PSP nanoparticles of the present invention. The present invention does not use organic solvents such as chloroform and methanol during the synthesis of PLGA-PSP, thus having good biosafety, and the synthesis process is simple, making it suitable for large-scale production.

[0059] The present invention enables PLGA-PSP nanoparticles to mimic the adhesion and aggregation properties of natural platelets by attaching one or more, for example, two or three, of VBP, CBP, and FMP to their surfaces. When a blood vessel is injured, the administered PLGA-PSP nanoparticles specifically bind to exposed collagen and VWF via CBP and VBP, respectively. FMP then binds to activated platelet GP IIb-IIIa receptors via fibrinogen, targeting the site of vascular injury and promoting platelet recruitment and aggregation, thereby promoting hemostasis and reducing bleeding volume and duration.

[0060] The PLGA-PSP nanoparticles of the present invention comprise a plurality of PSP conjugates, each of which has PLGA and PSP connected together by a flexible PEG segment.

[0061] Because VBP, CBP, and FMP are attached to the surface of the PLGA-PSP nanoparticles of the present invention, they can mimic the adhesion and aggregation properties of natural platelets. When a blood vessel is damaged, the administered PLGA-PSP nanoparticles specifically bind to exposed collagen and VWF via CBP and VBP, respectively. FMP then binds to activated platelet GP IIb-IIIa receptors via fibrinogen, targeting the site of vascular injury and promoting platelet recruitment and aggregation, thereby promoting hemostasis and reducing bleeding volume and duration.

[0062] In addition, since the PLGA-PSP nanoparticles of the present invention use PLGA instead of distearoylphosphatidylethanolamine (DSPE), the defects associated therewith no longer exist. Moreover, since PLGA can be gradually degraded into co-monomer molecules lactic acid and glycolic acid, these metabolites can be excreted from the body through normal metabolic pathways and can ultimately be metabolized into CO2 and H2O without toxic residues, and therefore can be used safely.

[0063] In a second aspect, the present invention provides use of the PLGA-PSP nanoparticles of the first aspect of the present invention in the preparation of a medicament for preventing or treating: a) bleeding, such as arterial bleeding, venous bleeding and / or capillary bleeding; b) Vascular damage, such as vascular damage caused by trauma or surgery; c) Bleeding disorders caused by thrombocytopenia or platelet dysfunction.

[0064] In a third aspect, the present invention provides a method for reducing or alleviating blood loss in a bleeding animal, the method comprising administering the PLGA-PSP nanoparticles according to the first aspect of the present invention to the bleeding animal to promote hemostasis.

[0065] In one embodiment, the exsanguinated animal is an exsanguinated human.

[0066] In one embodiment, the administration can be implemented by various approaches, including but not limited to parenteral administration, oral administration, rectal administration, vaginal administration, transdermal administration, pulmonary administration, nasal administration, oral administration or ophthalmic administration. The route of administration is obvious to those skilled in the art and depends on various factors, including the type and severity of the disease being treated, the type and age of the animal or human patient being treated, etc. The parenteral administration includes any route of administration characterized by physically breaking open the patient's tissue and administering through a tissue rupture. Therefore, parenteral administration includes but is not limited to administration by injection, application of the drug to or through surgical incision, application of the drug to or through tissue penetrating non-surgical wounds, etc. Specifically, parenteral administration includes but is not limited to skin administration, subcutaneous administration, intraperitoneal administration, intramuscular injection, intrasternal administration, intravenous injection and intra-arterial administration.

[0067] It is understood that the above description of the PLGA-PSP nanoparticles of the first aspect of the present invention is also applicable to this aspect of the present invention. Therefore, for the sake of brevity, no further details will be given here.

[0068] Example Experimental materials used in the examples: PLGA 10K-PEG 3400-NHS was purchased from Xi'an Ruixi Biotechnology Co., Ltd.; Platelet-specific peptides TRYLRIHPQSWVHQI (VBP), [GPO]7 (CBP), and HHLGGAKQAGDV (FMP) were custom synthesized by Jiangsu Jitai Peptide Technology Co., Ltd.; N,N-Dimethylformamide (DMF), triethylamine, and dialysis tubing were purchased from Sigma-Aldrich, Germany; Dimethyl sulfoxide (DMSO) was purchased from Beijing Solaibao Technology Co., Ltd.; Platelet-rich plasma (PRP) preparation: Whole blood was drawn from healthy volunteers who had not taken any medication within two weeks and centrifuged at 1500 g for 15 minutes at room temperature. The supernatant was retained as PRP. Platelet count in PRP was monitored using an automated blood and fluid analyzer (model XN-350, purchased from Sysmex, Japan) and maintained at 150,000 / μL (the lower limit of normal platelet count). This study was approved by the Second Affiliated Hospital of the Fourth Military Medical University and adhered to the principles of the Declaration of Helsinki. All volunteers provided signed informed consent.

[0069] Experimental mice: Male Kunming (KM) mice, 6-8 weeks old, matched for age and weight, were purchased from the Animal Center of the Fourth Military Medical University in Xi'an. All mice were housed in a specific pathogen-free environment with ample water and food. Purchased mice were acclimated to their diet for one week before the experiment.

[0070] To establish a thrombocytopenic mouse model, normal mice were intraperitoneally injected with busulfan (Cat. No. 55-98-1, purchased from MedChemexpress Biotech, USA) at a dose of 2 mg / kg once daily for two weeks. The model was successfully established when the platelet count decreased by 50-60% compared to normal mice.

[0071] Example 1. Preparation of PLGA-PSP nanoparticles The PLGA-PSP nanoparticles of the present invention are prepared according to the following method: 100 mg of PLGA 10k-PEG 3400-NHS was dissolved in 3 ml of DMF. VBP (1.1 eq.) and triethylamine (3.0 eq.) were then added to the DMF solution and stirred to dissolve completely. After 12 hours of reaction at room temperature, the linear peptide conjugate, PLGA 10k-PEG 3400-VBP, was obtained. The reaction solution was dialyzed against double-distilled water (molecular weight cutoff, 2000 Da) for 24 hours. The dialyzate was collected and freeze-dried to obtain the lyophilized linear peptide conjugate, PLGA 10k-PEG 3400-VBP.

[0072] Lyophilized linear conjugated peptide PLGA 10k-PEG 3400-CBP was prepared in the same manner except that CBP (1.1 eq.) and triethylamine (3.0 eq.) were added to the DMF solution.

[0073] Lyophilized linear conjugated peptide PLGA 10k-PEG 3400-FMP was prepared in the same manner except that FMP (1.1 eq.) and triethylamine (3.0 eq.) were added to the DMF solution.

[0074] 60 mg of each of the three lyophilized linear peptide conjugates (PLGA 10k-PEG 3400-VBP, PLGA 10k-PEG 3400-CBP, and PLGA 10k-PEG 3400-FMP) was dissolved in 500 μL of DMSO. 25 μL of each dissolved linear peptide conjugate was thoroughly mixed with 0.5 mL of DMSO and then added dropwise to 4.5 mL of distilled water. The mixture was stirred at 500 rpm for 2 hours at room temperature and then dialyzed against doubly distilled water (molecular weight cutoff, 3500 Da) for 2 days to obtain PLGA-PSP nanoparticles.

[0075] Example 2, Characterization and Detection of PLGA-PSP Nanoparticles The PLGA-PSP nanoparticles prepared in Example 1 were subjected to the following characterization tests: 1) Ultraviolet absorption spectrum The ultraviolet absorption of PLGA-PEG, PLGA-PEG-FMP, PLGA-PEG-VBP, PLGA-PEG-CBP and PLGA-PSP nanoparticles was detected using an enzyme-labeled detector (model Epoch, purchased from BioTeK Co., Ltd., USA).

[0076] The results are as follows Figure 1As shown, the UV absorption spectrum of PLGA-PSP shows a significant absorption enhancement in the 201-250nm region compared to PLGA-PEG, which is consistent with the strong absorption characteristics of the three peptides PLGA-PEG-FMP, PLGA-PEG-VBP, and PLGA-PEG-CBP in this wavelength range. In particular, at approximately 204nm (red arrow) and approximately 230nm (blue arrow), the PLGA-PSP absorption spectrum shows two absorption peaks, corresponding to the characteristic absorption peaks of the three peptides PLGA-PEG-FMP, PLGA-PEG-VBP, and PLGA-PEG-CBP in the corresponding wavelength bands (the areas indicated by the red and blue circles). The above UV absorption spectrum shows that the active peptide is successfully coupled to the PLGA-PEG structure.

[0077] 2) Nanoparticle tracking analysis to determine the particle size distribution of PLGA-PSP nanoparticles The particle size distribution and average particle size of PLGA-PSP nanoparticles were determined using a nanoparticle tracking analyzer (model Zetaview, purchased from Particle Metrix, Germany).

[0078] The results are as follows Figure 2 As shown in the figure (the horizontal axis is particle size, the vertical axis is number), the average particle size of the synthesized PLGA-PSP nanoparticles is 120 nm, and the particle size distribution is relatively uniform.

[0079] 3) Transmission electron microscopy observation of PLGA-PSP nanoparticles The morphology of PLGA-PSP nanoparticles was observed using a transmission electron microscope (HT7800 / HT7700, purchased from HITACHI, Japan).

[0080] The results are as follows Figure 3 As shown in Figure A, the freshly prepared PLGA-PSP nanoparticles are spherical particles that are evenly distributed and have no agglomeration, proving that PLGA-PSP nanoparticles with a morphology similar to that of natural platelets have been successfully synthesized; Figure 3 As shown in Figure B, after conventional storage at 4°C for 6 months, the PLGA-PSP nanoparticles still have a relatively complete vesicle-like structure, and their morphology and particle size have not changed significantly.

[0081] Example 3: Detection of platelet adhesion induced by PLGA-PSP nanoparticles PRP was centrifuged at 13,000 g for 5 minutes to obtain the platelet pellet, which was then washed three times with PBS. Platelets were then stained using the Calcein AM Cell Viability Assay Kit (Cat. No. C2013FT, purchased from Beyotime Biotech) at room temperature in the dark for 30 minutes. PLGA-PSP nanoparticles and PLGA-PEG, along with PBS, were mixed with a cell membrane red fluorescent probe (Cat. No. C1036, purchased from Beyotime Biotech) and added to a collagen-coated 96-well plate (Cat. No. WHB-96-CC, purchased from WHB). The platelets were incubated at room temperature in the dark for 20 minutes, followed by two washes. Fluorescently labeled platelets were then added to the 96-well plate and incubated with the nanoparticles for 5–10 minutes at room temperature in the dark. After two washes, the platelets were observed using an inverted fluorescence imaging microscope (Model Eclipse Ti2-A, purchased from Nikon, Japan).

[0082] The results are as follows Figure 4 As shown, compared with PBS, PLGA-PEG nanoparticles have no effect on platelet adhesion, while the PLGA-PSP nanoparticles synthesized in Example 1 can significantly induce platelet adhesion.

[0083] Example 4: Detection of the ability of PLGA-PSP nanoparticles to induce platelet aggregation 50 µL of PRP suspension, 10 µL of PLGA-PSP nanoparticles, PLGA-PEG, and PBS were added to a 96-well plate and incubated at room temperature for 5 minutes. ADP (a platelet stimulator, Catalog No. A2754, purchased from Sigma-Aldrich, Germany) was added to a final concentration of 20 µM and mixed thoroughly. Platelets were then poured onto microscope slides, and platelet aggregation was observed under an upright microscope (Eclipse CI, purchased from Nikon, Japan).

[0084] The results are as follows Figure 5 As shown, compared with PBS, in the absence of platelet stimulants, neither PLGA-PEG nor PLGA-PSP nanoparticles induced spontaneous platelet aggregation with “resting” platelets ( Figure 5 A in the figure), and when the stimulator ADP acts on platelets, platelets will aggregate, but the aggregation degree of platelets after PLGA-PSP nanoparticles treatment is much higher than that of PBS and PLGA-PEG nanoparticles ( Figure 5The above results indicate that the PLGA-PSP nanoparticles synthesized in Example 1 do not induce spontaneous platelet aggregation, but significantly promote platelet aggregation only in the presence of a platelet stimulant.

[0085] Example 5: Detection of the hemostatic ability of PLGA-PSP nanoparticles on venous bleeding in traumatic mice PLGA-PSP nanoparticles and PLGA-PEG, along with 100µL of PBS, were injected into mice via the tail vein. A 5mm section of the mouse tail was amputated with a scalpel blade. Five minutes after nanoparticle injection, the tail was then immersed in saline containing an anticoagulant. A stopwatch was used to record the time it took for bleeding to stop. Blood was collected and analyzed using an automated blood and fluid analyzer (Model XN-350, purchased from Sysmex, Japan). The red blood cells (RBCs) in the collected blood represent the amount of blood lost by the trauma.

[0086] The results are as follows Figure 6 As shown in Figure 3, compared with PBS, PLGA-PEG nanoparticles had no effect on tail vein bleeding in normal mice, while PLGA-PSP nanoparticles treated mice significantly reduced the bleeding time (P < 0.01, Figure 6 A in the figure) and the amount of bleeding (P<0.01, Figure 6 In the thrombocytopenia mouse model, the tail vein bleeding time of mice treated with PLGA-PSP nanoparticles was shortened by 40% compared with that of mice treated with PBS (P<0.001, Figure 6 C in), bleeding volume (P<0.01, Figure 6 The above results show that the PLGA-PSP nanoparticles synthesized in Example 1 can achieve rapid hemostasis of venous bleeding in traumatic bleeding under the conditions of normal or reduced platelet count.

[0087] Example 6: Detection of the hemostatic ability of PLGA-PSP nanoparticles on visceral bleeding in traumatized mice PLGA-PSP nanoparticles, PLGA-PEG, and 100 µL of PBS were injected into mice via the tail vein. Twenty minutes later, the mice were anesthetized with isoflurane. A scalpel was used to open the thoracic and abdominal regions along the rostral and caudal axis to expose the liver. A 3 mm long incision was made at the outer edge of the left lobe of the liver using scissors. A pre-weighed absorbent gauze was placed inside the mouse cavity. Hemostasis time was recorded when bleeding from the injured liver site completely ceased.

[0088] The results are as follows Figure 7 As shown in Figure 2, compared with PBS, PLGA-PSP nanoparticles significantly improved the bleeding time of liver injury in normal mice (P<0.001, Figure 7 A in the figure) and the amount of bleeding (P<0.01, Figure 7B); In addition, PLGA-PSP nanoparticles have an effect on the liver bleeding time of thrombocytopenic mice (P<0.0001, Figure 7 C in) and bleeding volume (P<0.01, Figure 7 The above results show that the PLGA-PSP nanoparticles synthesized in Example 1 can achieve rapid hemostasis of visceral hemorrhage in traumatic bleeding under conditions of normal or reduced platelet count.

[0089] Example 7: Detection of the hemostatic ability of PLGA-PSP nanoparticles on arterial bleeding in traumatic mice PLGA-PSP nanoparticles, PLGA-PEG, and 100 µL of PBS were injected into the tail vein catheter of mice 20 minutes later. The mice were anesthetized with isoflurane, and the skin and muscle were excised with a scalpel to expose the femoral artery. A 26-gauge needle was used to puncture the mid-segment of the femoral artery to induce bleeding. A pre-weighed absorbent gauze was placed in the femoral artery to record the bleeding time and amount.

[0090] The results are as follows Figure 8 As shown in Figure 2, compared with PBS (control), PLGA-PSP nanoparticles significantly improved the femoral artery bleeding time of normal mice (P<0.01, Figure 8 A in the figure) and bleeding volume (P<0.05, Figure 8 Even in thrombocytopenic mice, PLGA-PSP nanoparticles significantly reduced the bleeding time of the femoral artery compared with PBS (P < 0.05, Figure 8 C in) and bleeding volume (P<0.05, Figure 8 The above results show that the PLGA-PSP nanoparticles synthesized in Example 1 can achieve rapid hemostasis in traumatic bleeding arteries under conditions of normal or reduced platelet count.

[0091] Example 8: Detection of microthrombosis and inflammation levels in vivo using PLGA-PSP nanoparticles PLGA-PSP nanoparticles, PLGA-PEG, and 100 μL of PBS were injected into Kunming mice via the tail vein. Five minutes later, the mice were euthanized, and major tissues and organs (liver, lungs, kidneys, and spleen) were removed and fixed in 2 mL of 4% paraformaldehyde for 48 hours. The fully fixed tissues and organs were then washed with 1× PBS, dehydrated, cleared, waxed, embedded, sectioned, and dried before HE staining.

[0092] from Figure 9It can be seen that the histopathological staining results of mice treated with PLGA-PSP nanoparticles are the same as those of mice treated with PBS and PLGA-PEG nanoparticles, which indicates that PLGA-PSP nanoparticles do not cause inflammation and microthrombosis in tissues and organs and are safe and applicable to the human body.

Claims

1. A PLGA-PSP nanoparticle comprising a plurality of PSP conjugates, each PSP conjugate being represented by the following formula: PLGA-PEG-PSP, in: The PLGA represents poly(lactic-co-glycolic acid), which constitutes the core of the nanoparticles. The PEG represents polyethylene glycol, which is used to connect the PLGA and the PSP. The PSP represents a platelet-specific polypeptide, located on the surface of the nanoparticle, and includes one or more, for example, two or three, of collagen-binding peptide (CBP), vWF-binding peptide (VBP) and fibrinogen mimetic peptide (FMP).

2. PLGA-PSP nanoparticles according to claim 1, characterized in that, The PLGA-PSP nanoparticles comprise one or more, for example, two or three, of PLGA-PEG-CBP, PLGA-PEG-VBP, and PLGA-PEG-FMP; Preferably, the PLGA-PSP nanoparticles comprise PLGA-PEG-CBP, PLGA-PEG-VBP and PLGA-PEG-FMP in a molar ratio of 1:1:

1.

3. The PLGA-PSP nanoparticle according to any one of claims 1-2, characterized in that The VBP comprises the amino acid sequence TRYLRIHPQSWVHQI.

4. The PLGA-PSP nanoparticle according to any one of claims 1 to 3, characterized in that The CBP comprises the amino acid sequence [GPO]7.

5. The PLGA-PSP nanoparticle according to any one of claims 1 to 4, characterized in that The FMP comprises the amino acid sequence HHLGGAKQAGDV.

6. The PLGA-PSP nanoparticles according to any one of claims 1 to 5, characterized in that In the PLGA, the degree of polymerization of polyglycolic acid is 30-90, preferably 30-70, and the degree of polymerization of polylactic acid is 50-100, preferably 50-90; Preferably, the degree of polymerization of the PEG is 40-130, preferably 50-80.

7. The PLGA-PSP nanoparticles according to any one of claims 1 to 6, characterized in that The PLGA-PSP nanoparticles are vesicle-like structures.

8. The PLGA-PSP nanoparticles according to any one of claims 1 to 7, characterized in that The particle size distribution of the PLGA-PSP nanoparticles is 50-200 nm.

9. The PLGA-PSP nanoparticle according to any one of claims 1 to 8, characterized in that The PLGA-PSP nanoparticles still maintain a substantially complete vesicle-like structure after being stored at 4° C. for at least 6 months, and their particle size distribution remains between 50 and 200 nm.

10. Use of the PLGA-PSP nanoparticles according to any one of claims 1 to 9 in the preparation of a medicament for preventing or treating: a) bleeding, such as arterial bleeding, venous bleeding and / or capillary bleeding; b) Vascular damage, such as vascular damage caused by trauma or surgery; c) Bleeding disorders caused by thrombocytopenia or platelet dysfunction.

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