Platelet exosome-based composite nanodelivery particles, preparation and application thereof

Abstract

This invention discloses a composite drug delivery nanoparticle based on platelet exosomes, its preparation, and its application. The composite drug delivery nanoparticle comprises platelet exosomes, DPPS liposomes, and a multifunctional molecule; both the DPPS liposomes and the multifunctional molecule are embedded on the outer surface of the outer layer of the platelet exosome. The structural formula of the multifunctional molecule is: R1-R2-R3-R4. The composite drug delivery nanoparticle of this invention can increase the loading capacity of the hydrophobic chemotherapeutic drug PTX and the photothermal agent ICG without damaging the exosome particle size and membrane protein characteristics. It achieves controlled release of immune checkpoint inhibitors in a tumor-associated enzyme environment. This system exhibits good drug enrichment capacity, photothermal heating performance, and can effectively block PD-1 / PD-L1 interaction. This invention provides a feasible nanodelivery scheme for the synergistic application of chemotherapy, photothermal therapy, and immunotherapy.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterial preparation technology, specifically relating to a composite nanoparticle for drug delivery based on platelet exosomes, its preparation and application. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is a highly challenging malignant tumor to treat. Traditional chemotherapy, radiotherapy, and their combination therapies all have limitations in terms of efficacy and safety. In recent years, immune checkpoint inhibitors have shown promising applications in tumor treatment, but their effectiveness in ESCC treatment remains limited by factors such as low drug delivery efficiency and immunosuppression in the tumor microenvironment. Existing immune checkpoint inhibitors are prone to non-specific distribution and rapid clearance in vivo, making it difficult to maintain effective concentrations at the tumor site. To improve the stability and delivery efficiency of immunotherapies, researchers have attempted to introduce nanocarriers for delivery, with exosomes attracting attention due to their good biocompatibility and natural origin. However, existing exosome drug delivery technologies still have shortcomings in the synergistic loading of multiple therapeutic factors, particularly lacking effective control methods for the synergistic loading efficiency and stability of hydrophobic chemotherapeutic drugs and photothermal agents. Furthermore, current technologies often introduce immune checkpoint inhibitors into nanocarriers via covalent coupling or physical adsorption, lacking effective regulation of the release behavior of immune checkpoint inhibitors, making it difficult to achieve specific activation or release in the tumor microenvironment, thus affecting their immune blocking effect. Current technology lacks a composite nanoparticle that can achieve synergistic loading of multiple therapeutic factors and controllable release of immune checkpoint inhibitors in the tumor microenvironment while ensuring the stability of the exosome structure. Summary of the Invention

[0003] The purpose of this invention is to provide a composite nanoparticle for drug delivery based on platelet exosomes, as well as its preparation and application, to solve the problems in the prior art such as the difficulty in stably and synergistically loading multiple therapeutic factors in the same nanocarrier, the low delivery efficiency of immune checkpoint inhibitors, and the lack of tumor microenvironment response release mechanisms.

[0004] To achieve the above objectives, the present invention provides a composite nanoparticle for drug delivery based on platelet exosomes, which includes platelet exosomes, DPPS liposomes and multifunctional molecules. The DPPS liposomes and multifunctional molecules are embedded on the outer surface of the platelet exosome outer layer.

[0005] Furthermore, the mass ratio of platelet exosomes to DPPS liposomes is 10:1.

[0006] Furthermore, the multifunctional molecule includes a targeting segment, a retention segment, a response segment, and an anchoring segment, and the targeting segment, retention segment, response segment, and anchoring segment are connected in sequence.

[0007] Furthermore, the structural formula of the multifunctional molecule is as follows: R1-R2-R3-R4. Among them, R1 is an immune checkpoint inhibitor; R2 is an enzyme-responsive polymorphic peptide sequence that can be recognized and cleaved by tumor-associated proteases; R3 is a polypeptide sequence that can self-assemble and form a fibrotic structure; R4 is a lipid anchoring group; and the linkage is amide bond.

[0008] Furthermore, the R1 structure is as follows: ; The R2 structure is as follows: The R3 structure is as follows: The R4 structure is as follows: Another object of the present invention is to provide a method for preparing the above-mentioned composite nanoparticles for drug delivery, the method specifically comprising the following steps: S1) Obtaining and pretreatment of platelet exosome carriers: Platelet exosomes with a particle size of 30-200 nm were obtained by differential centrifugation, and the concentration was adjusted to 1.2 × 10⁻⁶. 10 Platelet exosomes were obtained at a density of 1 / mL to produce platelet exosome vectors; S2) Place 0.5 mM DPPS liposomes and the platelet exosome carrier prepared in S1) in the same aqueous system and incubate them together at 37°C for at least 2 h to allow the hydrophobic alkyl groups of DPPS liposomes to insert into the outer layer of the exosome lipid bilayer, thereby obtaining a DPPS liposome modified platelet exosome carrier. S3) Add the multifunctional molecule to the DPPS liposome-modified platelet exosome carrier system obtained in S2), and incubate in a co-bath at 37°C for at least 2 hours to allow one end of the multifunctional molecule to insert into the outer layer of the DPPS liposome-modified platelet exosome carrier (inserted into the outer layer of the DPPS liposome-modified exosome), thus obtaining composite nanoparticles for drug delivery.

[0009] Furthermore, the multifunctional molecule is synthesized using a polypeptide solid-phase synthesis method.

[0010] Furthermore, the solid-phase synthesis method for polypeptides specifically includes the following steps: S1) Resin swelling: Fmoc-Gly-Wang's resin was swollen with N,N-dimethylformamide for 4-6 hours; S2) Deprotection: After repeatedly rinsing with dichloromethane and N,N-dimethylformamide solvent alternately, add 7-8 mL of deprotecting agent and shake on a shaker for 15-20 min to completely remove the Fmoc group of the amino acid; S3) Detection: Rinse repeatedly with dichloromethane and N,N-dimethylformamide solvent. Add Kasserian reagent and a small amount of Fmoc-Gly-Wang resin to a 1.5 mL centrifuge tube and heat in boiling water for 1-2 min. If the Fmoc-Gly-Wang resin particles turn purple, it indicates that the Fmoc protecting group has been removed. If the purple color is very light or the resin does not turn purple, repeat step S2) until Fmoc is completely removed. S4) Coupling: Weigh out 10 equivalents of Fmoc protected amino acid and coupling agent relative to the loading of Fmoc-Gly-Wang's resin, pre-react in N,N-dimethylformamide for 10-15 min, then add to the peptide synthesis tube and react on a shaker for 1-2 h. S5) Detection: Use the Kassel test reagent to detect the amino acid coupling status. If the coupling is incomplete, repeat step S4). S6) Circular coupling: Repeat steps S2) to S5) to complete the solid-phase synthesis of the complete amino acid sequence of the enzyme-responsive peptide PVGLIG and the fibrinogenic peptide FFVDF in sequence; after the last amino acid is completely coupled, remove the Fmoc protecting group with a deprotecting agent. S7) Coupling of lipid anchoring groups: The lipid anchoring group DPPS or its derivatives are linked to the peptide chain under the action of a coupling agent to form a target segment-retention segment-response segment-anchoring segment structure; S8) Pyrolysis and purification: The resin is pyrolyzed using a pyrolysis buffer, and after precipitation, washing and drying, multifunctional molecules are obtained.

[0011] This invention uses platelet exosomes as nanocarriers. Through the design of platelet exosome separation, drug loading and functionalization modification processes, the platelet exosomes can specifically target tumor cells by retaining the characteristic proteins on the platelet membrane, thereby achieving the synergistic delivery of chemotherapeutic drugs, photothermal agents and immune checkpoint inhibitors in the same nanosystem.

[0012] Specifically, this invention first separates and purifies platelet exosomes from purchased platelet supernatant to obtain exosome nanocarriers with a lipid bilayer structure. Subsequently, the exosome nanocarriers are co-treated with DPPS liposomes, resulting in an exosome concentration of approximately 1.2 × 10⁻⁶. 10The concentration of DPPS was 0.5 mM, with DPPS molecules embedded in the exosome lipid bilayer via hydrophobic interactions, thereby regulating the lipid composition of the exosome surface. Subsequently, a hydrophobic chemotherapeutic drug was introduced into the DPPS-modified exosome dispersion system under mild conditions, allowing it to embed into the exosome lipid bilayer through hydrophobic interactions, forming an initial intercalation loading structure. Based on this, the local polarity state of the exosome lipid bilayer was altered by controlling the solvent environment in the system, with an ethanol / water volume ratio of 20%. A photothermal agent was introduced to achieve synergistic distribution between the photothermal agent and the intercalated chemotherapeutic drug within the lipid bilayer region, thereby improving the loading efficiency and stability of both within the exosomes.

[0013] Simultaneously, this invention constructs a multifunctional molecule for the controlled delivery and release of immune checkpoint inhibitors. The multifunctional molecule is characterized by comprising ICI1 (targeting segment)-FFVDF (retention segment)-PVGLIG (response segment)-DPPS (anchoring segment), and its molecular structure has the structure shown in Formula I: Formula I R1 is an immune checkpoint inhibitor; R2 is an enzyme-responsive polymorphic peptide sequence that can be recognized and cleaved by tumor-associated proteases; R3 is a polypeptide sequence capable of self-assembly and forming a fibrotic structure; R4 is a lipid anchoring group; and each unit is connected in sequence by an amide bond.

[0014] In the structure described, PVGLIG is a tumor-associated enzyme response linker, FFVDF is a polypeptide sequence capable of self-assembly and forming a fibrotic structure, and DPPS is a lipid anchoring group. Each functional unit is sequentially linked by an amide bond. The immunomodulatory functional unit is prepared using a polypeptide solid-phase synthesis method, sequentially completing the stepwise solid-phase synthesis of the peptide chain. Under solid-phase conditions, an immune checkpoint inhibitor is coupled to the linker peptide, followed by the introduction of a lipid anchoring group, resulting in a functional unit with a targeting unit-linker unit-self-assembly unit-lipid structure.

[0015] Subsequently, the multifunctional molecule was introduced into the exosome system that had already undergone drug co-loading, allowing the lipid anchoring groups to embed into the exosome lipid bilayer through hydrophobic interactions, thereby obtaining composite nanoparticles that simultaneously load chemotherapeutic drugs, photothermal agents, and carry enzyme-responsive immune checkpoint blocking units, such as... Figure 1 As shown, when the composite nanoparticles enter the tumor microenvironment, the linker peptides break down under the action of specific enzymes, releasing the immune checkpoint inhibitor from its lipid-anchored state and exposing its immune-active sites. This effectively regulates the immunosuppressive state of the tumor microenvironment, achieving a synergistic therapeutic effect of chemotherapy, photothermal therapy, and immunotherapy.

[0016] The beneficial effects of the present invention are as follows: due to the adoption of the above technical solution, the composite nanoparticles of the present invention can significantly improve the enrichment efficiency of drugs in esophageal squamous cell carcinoma lesions and exhibit excellent tumor-targeted delivery ability. Compared with free drugs, the enrichment amount in the tumor area is increased by about 142% 4 hours after administration, showing excellent tumor-targeted delivery ability.

[0017] Furthermore, this invention enables the immune checkpoint inhibitor to be specifically released and exert a blocking effect in the tumor microenvironment by synergistically loading chemotherapeutic drugs and photothermal agents and introducing an enzyme-responsive immune checkpoint inhibitor structure. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the composite nanoparticles used in Example 1; Figure 2 This is a transmission electron microscope image of the pre-DPPS exosomes loaded in Example 1; Figure 3 This is a transmission electron microscope image of the exosomes before PTX / ICG loading in Example 1; Figure 4 This is a statistical diagram of the particle size distribution of exosomes after DPPS loading in Example 1; Figure 5 This is a statistical diagram of the particle size distribution of exosomes after PTX / ICG loading in Example 1; Figure 6 This is a protein imprinting detection image of exosome characteristic proteins before and after loading DPPS in Example 1; Figure 7 This is a quantitative statistical chart of the loading amounts of PTX and ICG in exosomes in Example 1; Figure 8 This is a comparison chart of PTX / ICG synergistic loading efficiency under different solvent volume fractions in Example 2; Figure 9 This is a quantitative statistical chart of the surface loading amount of ICI1 in exosomes in Example 3; Figure 10 This is a statistical chart of the amount of drugs in tumors from different treatment groups in Example 4; Figure 11 This is a quantitative statistical chart of in vitro fluorescence intensity of different treatment groups in Example 4, where the control group is a free drug system and the experimental group is a composite nanoparticle system; Figure 12 This is the HPLC analysis chromatogram of the composite molecule and MMP2 enzyme before co-bathing in Example 4; Figure 13 This is the HPLC analysis chromatogram of the composite molecule and MMP2 enzyme after co-bathing in Example 4. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the technical features or combinations of technical features described in the following embodiments should not be considered in isolation, but can be combined with each other to achieve better technical effects.

[0020] This invention relates to a composite nanoparticle for drug delivery based on platelet exosomes, which includes platelet exosomes, DPPS liposomes, and multifunctional molecules. The DPPS liposomes and multifunctional molecules are embedded on the outer surface of the platelet exosome outer layer.

[0021] Furthermore, the mass ratio of platelet exosomes to DPPS liposomes is 10:1.

[0022] Furthermore, the multifunctional molecule includes a targeting segment, a retention segment, a response segment, and an anchoring segment, and the targeting segment, retention segment, response segment, and anchoring segment are connected in sequence.

[0023] Furthermore, the structural formula of the multifunctional molecule is as follows: R1-R2-R3-R4. Among them, R1 is an immune checkpoint inhibitor; R2 is an enzyme-responsive polymorphic peptide sequence that can be recognized and cleaved by tumor-associated proteases; R3 is a polypeptide sequence that can self-assemble and form a fibrotic structure; R4 is a lipid anchoring group; and the linkage is amide bond.

[0024] Example 1 Exosomes were isolated from platelet-derived cell culture supernatant using a combination of differential and ultracentrifugation to obtain exosome nanocarriers. These exosomes were then placed in the same aqueous system with DPPS liposomes (dipalmitoylphosphatidylserine), where the concentration of exosomes was approximately 1.2 × 10⁻⁶. 10 The exosomes were incubated at 37°C with 0.5 mM DPPS per mL to allow the hydrophobic alkyl groups of DPPS to insert into the outer layer of the exosome lipid bilayer, resulting in a DPPS-modified exosome system. The morphology of the DPPS-loaded exosomes was observed using transmission electron microscopy, and the results are as follows: Figure 2 As shown in the figure, the exosomes retain their typical vesicle-like structure after DPPS insertion, with intact morphology and clear boundaries, without obvious membrane collapse or aggregation. Further, after loading PTX and ICG into the DPPS-modified exosomes, their morphology was observed using transmission electron microscopy, and the results are shown in the figure. Figure 3 As shown, the results indicate that the drug loading process did not cause significant damage to the exosome structure.

[0025] Further statistical analysis was performed on the particle size distribution of exosomes loaded with DPPS and those loaded with PTX / ICG. The results are as follows: Figure 4 and Figure 5 As shown in the figure. The results showed that the particle size of exosomes before and after DPPS modification and drug loading was mainly distributed in the 30-200 nm range, with only slight changes, and the system maintained good dispersibility and particle size stability. To verify whether the DPPS insertion and drug loading process affected the membrane protein characteristics of exosomes, protein imprinting was performed on the characteristic proteins of exosomes before and after DPPS modification, and the results are shown in the figure. Figure 6 As shown in the figure. The results showed that typical exosome characteristic protein bands could be detected in exosomes before and after DPPS insertion, and the position and intensity of the bands did not change significantly, indicating that DPPS insertion did not disrupt the membrane protein composition and biological characteristics of exosomes. Based on the confirmation of exosome structural integrity and stable membrane protein characteristics, the loading of PTX and ICG in exosomes was quantitatively analyzed, and the results are shown in the figure. Figure 7 As shown in the figure. The results showed that the loading capacity of exosomes with DPPS insertion was significantly higher than that of unmodified exosomes (ICG loading increased from 4.53% to 6.2%, and PTX loading increased from 3.89% to 5.13%), indicating that DPPS insertion effectively improved the overall drug loading capacity of the exosome system without damaging the exosome structure and membrane protein characteristics.

[0026] Example 2 In the DPPS-modified exosome system obtained in Example 1, the effect of the volume fraction of ethanol in the solvent environment on the loading behavior of the hydrophobic chemotherapeutic drug PTX and the photothermal agent ICG inside the exosomes was further investigated. Specifically, the DPPS-modified exosome system with multifunctional molecular surface modification was placed in solvent environments with different volume fractions of ethanol, namely 0%, 10%, 20%, 30%, 50%, and 100%. Under each solvent condition, PTX and ICG were simultaneously added to the system at the concentrations of Example 1, and incubated together at 37°C for 4 hours to allow PTX and ICG to be loaded inside the exosomes. After incubation, unbound drugs were removed by centrifugation and dialysis, and the loading amounts of PTX and ICG in the exosomes under different ethanol volume fractions were quantitatively analyzed.

[0027] The results showed that the volume fraction of ethanol had a significant impact on the internal loading behavior of PTX and ICG. Figure 8As shown, the loading of PTX and ICG in exosomes showed a significant trend with increasing ethanol volume fraction; the loading of PTX and ICG reached its peak at 20% ethanol volume fraction (5.9% and 8.7%, respectively); when the ethanol volume fraction was further increased to 40%, the loading behavior of PTX and ICG changed significantly, and their loading was negatively correlated with the ethanol volume fraction.

[0028] Example 3 Multifunctional molecules with the defined structure ICI1-FFVDF-PVGLIG-DPPS were placed in the same aqueous system with DPPS-modified exosome nanoparticles and incubated at 37°C to investigate the loading behavior of the multifunctional molecules on the exosome surface under different loading ratios. The mass ratio of the multifunctional molecules to the exosome characteristic protein CD9 was set at 5:1, 10:1, 20:1, and 30:1, respectively. After incubation, unbound molecules were removed and the multifunctional molecules loaded on the exosome surface were quantitatively analyzed to obtain the surface loading rates under different mixing ratios (4.1%, 6.8%, 6.9%, and 6.7%, respectively). Figure 9 As shown.

[0029] The results showed that as the proportion of multifunctional molecules added increased, their loading on the surface of exosomes gradually increased; when the mass ratio of multifunctional molecules to exosome characteristic proteins reached 10:1, the surface loading of the multifunctional molecules tended to saturate; further increasing the proportion of multifunctional molecules added no longer significantly increased their loading on the surface of exosomes.

[0030] Example 4 The platelet exosome-based drug delivery systems prepared in the three aforementioned cases were added to the in vitro tumor cell culture system, with the free drug system serving as a control group. The drug delivery and enrichment capabilities of the different treatment groups were compared and analyzed. Platelet exosomes retain the characteristics of platelet membrane-associated proteins and possess a natural affinity for tumor blood vessels and tumor tissues, providing a basis for targeted enrichment of drugs. The results of fluorescence imaging and quantitative analysis of the in vitro fluorescence intensity of different treatment groups are as follows: Figure 10 As shown in the figure, the control group consisted of the free drug system, while the experimental group consisted of the drug delivery system. The results showed that the fluorescence signal intensity of the drug delivery system group was significantly higher than that of the control group, indicating that it could effectively improve the in vitro delivery and intracellular accumulation of the drug (the drug accumulation was increased by 142%). Further ultraviolet absorption spectroscopy was performed on the different treatment groups, and the results are shown below. Figure 11 As shown, the absorption intensity of the experimental group at the characteristic absorption band of the drug was significantly enhanced, which is consistent with the results of quantitative fluorescence, further verifying the delivery advantages of the drug delivery system.

[0031] To verify the responsive release behavior of the surface-modified enzyme-responsive multifunctional molecule ICI1-PVGLIG-FFVDF-DPPS in the tumor microenvironment, the multifunctional molecule was incubated in a buffer system containing the tumor-associated protease MMP2, and its structural changes were analyzed by high-performance liquid chromatography (HPLC). The HPLC analysis results of the multifunctional molecule before incubation with MMP2 are as follows: Figure 12 As shown, it exhibits a single and stable characteristic peak; after co-bathing with MMP2 enzyme, its HPLC analysis results are as follows. Figure 13 As shown, the original main peak significantly weakened and was accompanied by the appearance of a new separation peak, indicating that the enzyme-responsive linker PVGLIG underwent selective cleavage under the action of MMP2, causing the immune checkpoint inhibitor ICI1 to dissociate from its lipid-anchored state. These results demonstrate that the drug delivery system constructed in this embodiment possesses both excellent drug delivery and enrichment capabilities and tumor microenvironment-responsive release characteristics.

[0032] The foregoing has provided a detailed description of a platelet exosome-based composite nanoparticle for drug delivery, its preparation, and its application, as provided in the embodiments of this application. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this application; furthermore, those skilled in the art will recognize that, based on the ideas of this application, there will be changes in the specific implementation methods and application scope. Therefore, the content of this specification should not be construed as a limitation of this application.

[0033] Certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that hardware manufacturers may use different names to refer to the same component. This specification and claims do not distinguish components based on differences in name, but rather on differences in function. The terms "comprising" and "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising / including but not limited to". "Approximately" means that within an acceptable margin of error, those skilled in the art can solve the technical problem and substantially achieve the technical effect within a certain margin of error. The following descriptions in the specification are preferred embodiments for carrying out this application; however, these descriptions are for the purpose of illustrating the general principles of this application and are not intended to limit the scope of this application. The scope of protection of this application shall be determined by the appended claims.

[0034] It should also be noted that the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a product or system comprising a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a product or system. Without further limitation, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the product or system that includes said element.

[0035] It should be understood that the term "and / or" used in this article is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this article generally indicates that the preceding and following related objects have an "or" relationship.

[0036] The foregoing description illustrates and describes several preferred embodiments of this application. However, as previously stated, it should be understood that this application is not limited to the forms disclosed herein and should not be construed as excluding other embodiments. It can be used in various other combinations, modifications, and environments, and can be altered within the scope of the application concept described herein through the foregoing teachings or techniques or knowledge in related fields. Any modifications and variations made by those skilled in the art that do not depart from the spirit and scope of this application should be within the protection scope of the appended claims.

Claims

1. A composite nanoparticle for drug delivery based on platelet exosomes, characterized in that, The composite nanoparticles for drug delivery include platelet exosomes, DPPS liposomes, and multifunctional molecules. The DPPS liposomes and multifunctional molecules are embedded on the outer surface of the platelet exosome outer layer.

2. The composite nanoparticles for drug delivery according to claim 1, characterized in that, The mass ratio of platelet exosomes to DPPS liposomes is 10:

1.

3. The composite nanoparticles for drug delivery according to claim 1, characterized in that, The multifunctional molecule includes a targeting segment, a retention segment, a response segment, and an anchoring segment, which are connected sequentially.

4. The composite nanoparticles for drug delivery according to claim 3, characterized in that, The structural formula of the multifunctional molecule is: R1-R2-R3-R4. Among them, R1 is an immune checkpoint inhibitor; R2 is an enzyme-responsive polymorphic peptide sequence that can be recognized and cleaved by tumor-associated proteases; R3 is a polypeptide sequence that can self-assemble and form a fibrotic structure; R4 is a lipid anchoring group; and the linkage is amide bond.

5. The composite nanoparticles for drug delivery according to claim 4, characterized in that, The R1 structure is as follows: ； The R2 structure is as follows: The R3 structure is as follows: The R4 structure is as follows: 。 6. A method for preparing composite nanoparticles for drug delivery as described in any one of claims 1-5, characterized in that, The method specifically includes the following steps: S1) Obtaining and pretreatment of exosome vectors: Platelet exosomes with a particle size of 30-200 nm were obtained by differential centrifugation, and the concentration was adjusted to 1.2 × 10⁻⁶. 10 Platelet exosomes were obtained at a density of 1 / mL to produce platelet exosome vectors; S2) Place 0.5 mM DPPS liposomes and the platelet exosome carrier prepared in S1) in the same aqueous system and incubate them together at 37°C for at least 2 h to allow the hydrophobic alkyl groups of the DPPS liposomes to insert into the outer layer of the exosome lipid bilayer, thereby obtaining the DPPS liposome modified platelet exosome carrier. S3) Add the multifunctional molecule to the DPPS liposome-modified platelet exosome carrier system obtained in S2), and incubate in a co-bath at 37°C for at least 2 hours to allow one end of the multifunctional molecule to be inserted into the outer layer of the DPPS liposome-modified platelet exosome carrier, thereby obtaining composite nanoparticles for drug delivery.

7. The method according to claim 6, characterized in that, The multifunctional molecule was synthesized using a polypeptide solid-phase synthesis method.

8. The method according to claim 7, characterized in that, The solid-phase synthesis method for polypeptides specifically includes the following steps: S1) Resin swelling: Fmoc-Gly-Wang's resin was swollen with N,N-dimethylformamide for 4-6 hours; S2) Deprotection: After repeatedly rinsing with dichloromethane and N,N-dimethylformamide solvents, add 7-8 mL of deprotecting agent and shake on a shaker for 15-20 min to completely remove the Fmoc group of the amino acid; S3) Detection: Rinse repeatedly with dichloromethane and N,N-dimethylformamide solvent. Add Kasserian reagent and a small amount of Fmoc-Gly-Wang resin to a 1.5 mL centrifuge tube and heat in boiling water for 1-2 min. If the Fmoc-Gly-Wang resin particles turn purple, it indicates that the Fmoc protecting group has been removed. If the purple color is very light or the resin does not turn purple, repeat step S2) until Fmoc is completely removed. S4) Coupling: Weigh out 10 equivalents of Fmoc protected amino acid and coupling agent relative to the loading of Fmoc-Gly-Wang's resin, pre-react in N,N-dimethylformamide for 10-15 min, then add to the peptide synthesis tube and react on a shaker for 1-2 h. S5) Detection: Use the Kassel test reagent to detect the amino acid coupling status. If the coupling is incomplete, repeat step S4). S6) Circular coupling: Repeat steps S2) to S5) to complete the solid-phase synthesis of the complete amino acid sequence of the enzyme-responsive peptide PVGLIG and the fibrinogenic peptide FFVDF in sequence; after the last amino acid is completely coupled, remove the Fmoc protecting group with a deprotecting agent. S7) Coupling of lipid anchoring groups: The lipid anchoring group DPPS or its derivatives are linked to the peptide chain under the action of a coupling agent to form a target segment-retention segment-response segment-anchoring segment structure; S8) Pyrolysis and purification: The resin is pyrolyzed using a pyrolysis buffer, and after precipitation, washing and drying, multifunctional molecules are obtained.

9. The application of a composite nanoparticle for drug delivery as described in any one of claims 1-5 in the treatment of tumors.

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