Intelligent wound-targeting nano-drug and preparation method therefor and use thereof

Abstract

An intelligent wound-targeting nano-drug and a preparation method therefor and the use thereof. The intelligent wound-targeting nano-drug contains a platelet membrane and drug-loading nanoparticles, wherein the drug-loading nanoparticles are coated with the platelet membrane, the drug-loading nanoparticles contain a tumor angiogenesis inhibitor, and the surface of the platelet membrane is linked with an anti-PDL1 antibody. The intelligent wound-targeting nano-drug can significantly improve the therapeutic effect of the tumor angiogenesis inhibitor (sorafenib) in combination with the anti-PDL1 antibody, can improve the biodistribution of the drugs, can reduce toxic side effects caused by the combined use of the two, and can greatly reduce the postoperative recurrence rate of early liver cancer. The intelligent wound-targeting nano-drug has a strong capability for inhibiting postoperative recurrence and a relatively good biological safety.

Description

Wound-targeted smart nanomedicine and its preparation method and application

[0001] Related cross-references

[0002] This application claims priority to the Chinese patent application filed with the China Patent Office on June 26, 2023, with application number "202310759353.9" and public name "Wound-targeted intelligent nanomedicines and their preparation methods and applications", the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present application relates to the field of pharmaceutical technology, and in particular to a wound-targeted intelligent nanomedicine for inhibiting postoperative recurrence of early-stage liver cancer, and a preparation method and application thereof. Background Art

[0004] Liver cancer, a common malignant digestive tract tumor, is the sixth most common cancer and the third leading cause of cancer death worldwide. Every year, a large number of patients lose their lives to liver cancer, with 830,000 deaths reported in 2020 alone. Liver cancer poses a serious threat to the lives and health of the general public, especially in developing countries. Therefore, its treatment is of vital clinical importance.

[0005] Currently, the treatment of liver cancer is still mainly based on traditional chemotherapy and surgery. Surgery is one of the most effective treatments for early- and mid-stage liver cancer. Direct surgical removal of tumor tissue can greatly reduce the patient's tumor burden, prolong their survival, and improve their quality of life. However, surgery is often unable to completely remove liver cancer tissue from the liver, resulting in the recurrence of residual tumor cells at the surgical margin. Furthermore, during surgery, tumor cells may enter the bloodstream through damaged blood vessels, leading to metastasis and promoting postoperative recurrence. According to statistics, the recurrence rate of early-stage liver cancer is as high as 50% three years after surgery.

[0006] Although conventional tumor angiogenesis inhibitors such as sorafenib can be used to prevent postoperative recurrence of early-stage liver cancer, their therapeutic efficacy is limited, and systemic administration has significant toxic side effects. Numerous studies have shown that postoperative recurrence of liver cancer is not only associated with significant angiogenesis but also closely related to an immunosuppressive tumor microenvironment. This provides us with a new therapeutic strategy to inhibit postoperative recurrence. Currently, the combined use of tumor angiogenesis inhibitors and immune checkpoint antibodies has been reported to inhibit postoperative recurrence of liver cancer to a certain extent. However, the response rate and efficacy of both drugs are limited, and the severe toxic side effects induced by long-term treatment have greatly limited the further development of this strategy. Therefore, there is no effective treatment for the prevention and treatment of postoperative recurrence of early-stage liver cancer, which seriously reduces the overall survival and quality of life of postoperative tumor patients.

[0007] Summary of the Invention

[0008] The present application discloses a wound-targeted intelligent nanomedicine and its preparation method and application, which can effectively inhibit the recurrence of liver cancer after surgery.

[0009] In order to achieve the above-mentioned objectives, the present application discloses, in a first aspect, a wound-targeted smart nanomedicine, which comprises a platelet membrane and drug-loaded nanoparticles, wherein the platelet membrane coats the drug-loaded nanoparticles;

[0010] The drug-loaded nanoparticles contain a tumor angiogenesis inhibitor;

[0011] The surface of the platelet membrane is connected with anti-PDL1 antibody.

[0012] Preferably, the tumor angiogenesis inhibitor is sorafenib.

[0013] In a second aspect, the present application also provides the use of the above-mentioned wound-targeted smart nanomedicine in the preparation of anti-tumor drugs.

[0014] In a third aspect, the present application also provides the use of the above-mentioned wound-targeted smart nanomedicine in the preparation of drugs for preventing and treating postoperative recurrence of liver cancer.

[0015] In a fourth aspect, the present application also provides a method for preparing the above-mentioned targeted smart nanomedicine, comprising the following steps:

[0016] Step 1: preparing the drug-loaded nanoparticles;

[0017] Step 2: obtaining the platelet membrane;

[0018] Step 3: dispersing the platelet membrane in water, adding the drug-loaded nanoparticles, sonicating to allow the platelet membrane to coat the surface of the drug-loaded nanoparticles, centrifuging, and collecting the precipitate;

[0019] Step 4: The precipitate obtained in step 3 is treated with activator A, and then mixed with an excess of anti-PD-L1 antibody treated with activator B, reacted, centrifuged, and the precipitate is collected to obtain the targeted smart nanomedicine.

[0020] The present application designs and synthesizes a new type of intelligent nanomedicine with tumor postoperative margin targeting. The platelet membrane on its surface can target the wound formed by tumor resection, and after the wound heals, it can continue to be enriched in the tumor tissue by virtue of the tumor targeting of the platelet membrane and the anti-PDL1 antibody, while inhibiting tumor angiogenesis, activating the immune system and killing residual tumors. This new nano drug delivery system can significantly improve the efficacy of the combination of tumor angiogenesis inhibitor (sorafenib) and anti-PDL1, improve the biodistribution of the drug, reduce the toxic side effects caused by the combination of the two, and greatly reduce the recurrence rate of early liver cancer after surgery. The nano platform is based on bioinert mesoporous silica and biologically derived platelet membrane as an important component. Both have very good biocompatibility, so the intelligent nano drug has a strong ability to inhibit postoperative recurrence while having relatively excellent biosafety.

[0021] Compared with the prior art, this application has the following beneficial effects:

[0022] (1) The wound-targeted nano-drug delivery system (smart nano-drug) of the present application improves the bioavailability by increasing the water solubility of the drug and the enrichment effect of residual tumor tissue after surgery. Compared with traditional drug administration strategies, it can reduce its toxic side effects, improve the biodistribution of the drug, and significantly improve the efficacy of the combined use of tumor angiogenesis inhibitors (sorafenib) and anti-PDL1, reduce the postoperative recurrence rate of early liver cancer, and prolong the survival of patients.

[0023] (2) The present application utilizes the postoperative wound targeting and unique tumor tissue targeting of platelet membranes, so that they can specifically aggregate around residual tumor tissues after surgery, enhance tumor permeability and therapeutic effects, and the introduction of platelet membranes can effectively prevent the leakage of sorafenib from the inside of mesoporous silica in the blood circulation, thereby reducing the toxic side effects of the drug. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 shows the preparation process of a-PM-S-MSNP nanomedicine (a) and transmission electron microscopy characterization (b), with the scale bar being 100 nm;

[0026] Figure 2 shows the SDS-PAGE total protein analysis results of a-PM-S-MSNP nanomedicine;

[0027] Figure 3 shows the presence of key proteins on the platelet membrane surface verified by immunocolloidal gold staining of a-PM-S-MSNP nanomedicine;

[0028] Figure 4 shows the release of sorafenib from a-PM-S-MSNP nanomedicine over time;

[0029] Figure 5 shows the in vitro collagen adhesion imaging and statistical results of a-PM-S-MSNP nanomedicine;

[0030] Figure 6 shows the establishment of a mouse model of orthotopic hepatocellular carcinoma after surgery (a) and the analysis of the postoperative resection margin targeting ability of a-PM-S-MSNP nanomedicine (b);

[0031] FIG7 shows the results of bioluminescence imaging monitoring of tumor recurrence in mice after orthotopic liver cancer surgery;

[0032] Figure 8 shows the 70-day survival statistics of mice after orthotopic liver cancer surgery and treatment with different groups of nanomedicines;

[0033] Figure 9 shows the weight changes of mice during the treatment of orthotopic liver cancer surgery;

[0034] Figure 10 shows the results of H&E staining analysis of the main organs of mice at the end of treatment;

[0035] Figure 11 Flow cytometric analysis of the abundance of CD4+T cells (a, b) and CD8+T cells (c, d) in tumor tissues after treatment with a-PM-S-MSNP and control group nanodrugs. DETAILED DESCRIPTION

[0036] The technical solution of the present application is further illustrated below by specific examples. Those skilled in the art should understand that the examples are only for facilitating understanding of the present application and should not be regarded as specific limitations of the present application.

[0037] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by those skilled in the art to which this application belongs. The terms used in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0038] As used herein, the terms "comprise," "comprising," and "having," and any variations thereof, are intended to cover non-exclusive inclusions. For example, a process, method, apparatus, product, or device comprising a series of steps is not limited to the listed steps or modules but may optionally include steps not listed, or other steps inherent to the process, method, product, or device.

[0039] In this application, "plurality" refers to two or more. "And / or" describes the relationship between related objects, indicating that three possible relationships exist. For example, "A and / or B" can mean: A exists alone, A and B exist simultaneously, or B exists alone. The character " / " generally indicates that the related objects are in an "or" relationship.

[0040] In order to effectively prevent the postoperative recurrence of liver cancer, the present application has designed a smart nanomedicine that can actively target the postoperative wound. It is mainly delivered by mesoporous silicon as a carrier for delivering tumor angiogenesis inhibitors (sorafenib), and at the same time, a platelet membrane with wound targeting is coated on the surface of the mesoporous silicon, and then the anti-PDL1 antibody is covalently modified on the outer platelet membrane surface by chemical reaction. This smart nanomedicine can deliver the tumor angiogenesis inhibitor to the surgical margin position by virtue of the wound targeting of platelets, inhibit the formation of new blood vessels, destroy the "soil" of tumor recurrence, and thus inhibit the growth and recurrence of residual tumors. In addition, the introduction of anti-PDL1 can relieve the immunosuppressive state of the tumor microenvironment, induce the body's T cells to directly kill residual tumor cells, and destroy the "seeds" of tumor recurrence. Through a two-pronged strategy, the nanomedicine prepared by the present application can significantly inhibit the postoperative recurrence of early-stage liver cancer, providing a new strategy for the prevention and treatment of clinical postoperative recurrence of liver cancer.

[0041] In the first aspect, the present application provides a wound-targeted nano-drug delivery system for the prevention and treatment of postoperative recurrence of early-stage liver cancer. The nano-targeted drug delivery system is mainly composed of a nano-carrier, which can be mesoporous silica nanoparticles, a bio-membrane, and two drugs. The nano-carrier can be mesoporous silica nanoparticles, and the bio-membrane is a platelet membrane that can actively target postoperative wounds. The two drugs are a chemotherapy drug (a tumor angiogenesis inhibitor, which can be sorafenib) and an antibody drug PD-L1 antibody. The tumor angiogenesis inhibitor is encapsulated in the pores of the mesoporous silica, the outer surface of which is covered by the platelet membrane. The PD-L1 antibody is further covalently linked to the outer side of the platelet membrane through a chemical reaction, thereby forming a nano-drug delivery system.

[0042] Mesoporous silica is the preferred nanocarrier for this application. Mesoporous silica nanoparticles offer numerous advantages: a well-organized pore structure, uniform pore size distribution, and adjustable pore diameter. Mesoporous silica nanoparticles also possess a high specific surface area and large pore volume, along with excellent biosafety, laying the foundation for the clinical application of the nanomedicines described in this application.

[0043] The preferred biological membrane of the present application is the platelet membrane. There are many receptors on the surface of the platelet membrane that can mediate its binding to damaged blood vessels, thereby aggregating to form thrombi and exerting a hemostatic function. The present application coats the platelet membrane on the outside of the mesoporous silica loaded with tumor angiogenesis inhibitors, which can prevent the premature leakage of drugs during the blood circulation process, and with the help of the receptors on the surface of the platelet membrane, the nanocarrier is given the ability to actively target the collagen exposed by the blood vessels at the surgical margin, specifically targeting the delivery of drugs to the damaged position of the blood vessels at the surgical margin. The platelet membrane is a natural component in the blood circulation, so it has good biocompatibility, and can also prolong the blood half-life of the angiogenesis inhibitors or the mesoporous silica nanoparticles loaded with tumor angiogenesis inhibitors.

[0044] The two drugs preferred in this application are sorafenib, a tumor angiogenesis inhibitor, and PD-L1 antibody, an immune checkpoint inhibitor. Sorafenib is used in the treatment of tumors with relatively rich blood supply such as liver cancer and lung cancer. Clinical results show that sorafenib can significantly improve the survival of tumor patients, but its therapeutic effect is limited, and systemic administration has obvious toxic side effects. In addition, immune checkpoint inhibitors anti-PD1 or anti-PDL1 are also widely used in the prevention and treatment of postoperative recurrence of liver cancer, but their response rate is not high, their efficacy is limited, and long-term use can lead to serious immune side effects. In order to reduce the postoperative recurrence rate of early liver cancer, this application prefers mesoporous silica as a nano drug delivery platform, encapsulating sorafenib in the internal pores and coating it with platelet membranes on the outside. At the same time, the immune checkpoint inhibitor drug anti-PDL1 is covalently linked to the surface of the platelet membrane for the prevention of postoperative recurrence of liver cancer, which can significantly improve its effect in preventing and treating postoperative recurrence of liver cancer.

[0045] Compared with the prior art, this application uses the cutting edge targeting of platelets for the first time to construct a nanomedicine for the prevention and treatment of postoperative recurrence. In this application, according to the fact that early liver cancer resection surgery will lead to the production of cutting edges, there will be a large number of damaged blood vessels in the cutting edge part. After the blood vessels are damaged, collagen and von Willebrand factor (VWF) and other components will be exposed, and platelets can specifically bind to damaged blood vessels through membrane receptors such as GPIb, GPIa-IIa, CD41 and CD61, thereby inducing coagulation and preventing a large amount of blood loss. This application uses this feature to innovatively wrap the platelet membrane on the outside of mesoporous silica loaded with tumor angiogenesis inhibitors, thereby targeting the drug delivery to the cutting edge position. Furthermore, this application also uses the thiol group on the surface of the platelet membrane to covalently couple the PD-L1 antibody to the surface of the platelet membrane, so that the immune checkpoint inhibitor and the angiogenesis inhibitor can be accurately delivered to the cutting edge position after tumor surgery, combining anti-tumor angiogenesis treatment with immunotherapy, and greatly improving the efficacy of its combination therapy.

[0046] Specifically, in one embodiment of the present application, a wound-targeted smart nanomedicine is provided, which comprises a platelet membrane and drug-loaded nanoparticles, wherein the platelet membrane coats the drug-loaded nanoparticles;

[0047] The drug-loaded nanoparticles contain a tumor angiogenesis inhibitor;

[0048] The surface of the platelet membrane is connected with anti-PDL1 antibody.

[0049] In some embodiments, the tumor angiogenesis inhibitor is sorafenib.

[0050] In some embodiments, the drug-loaded nanoparticles are obtained by encapsulating tumor angiogenesis inhibitors in mesoporous silica nanoparticles.

[0051] In some embodiments, the particle size of the mesoporous silica nanoparticles is 50nm-200nm, for example, it can be 50nm, 100nm, 120nm, 150nm, 180nm, 200nm, etc.; the pore size of the mesoporous silica nanoparticles is 4nm-10nm, for example, it can be 4nm, 5nm, 6nm, 7nm, 8nm, 9nm, 10nm; the charge of the mesoporous silica nanoparticles is -15mV to -25mV, for example, it can be: -15mV, -18mV, -20mV, -22mV, -23mV, -25mV.

[0052] In some embodiments, the content of the tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 3% to 50%.

[0053] In some embodiments, the content of the tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 5% to 20%.

[0054] In some embodiments, the content of the tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 10% to 20%.

[0055] In some embodiments, the content of the tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 14% to 19%.

[0056] In some embodiments, the content of the tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 16% to 19%.

[0057] In some embodiments, the mass ratio of the anti-PDL1 antibody to the tumor angiogenesis inhibitor is 1:0.05-20, preferably 1:1-10, preferably 1:2-6, preferably 1:2-4, and more preferably 1:3.

[0058] In some embodiments, the mass ratio of the tumor angiogenesis inhibitor to the mesoporous silica nanoparticles is 1:0.2-5, preferably 1:0.5-2.

[0059] In some embodiments, in the wound-targeted smart nanomedicine, the mass proportions of anti-PD-L1, sorafenib, mesoporous silica, and platelet membrane are 6%-7%, 18%-21%, 40%-45%, and 30%-35%, respectively.

[0060] In some embodiments, the particle size of the wound-targeted smart nanomedicine is 50 nm-200 nm, for example, it can be 50 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, etc.

[0061] In one embodiment of the present application, there is also provided the use of the wound-targeted smart nanomedicine in the preparation of a drug for preventing and treating postoperative tumor recurrence.

[0062] In some embodiments, the tumor is liver cancer. The targeted smart nanomedicine of the present application can significantly inhibit the postoperative recurrence of early-stage liver cancer in mice, thereby significantly prolonging the survival of patients who have undergone postoperative resection of early-stage liver cancer.

[0063] In one embodiment of the present application, there is also provided the use of the wound-targeted smart nanomedicine in the preparation of anti-tumor drugs.

[0064] In one embodiment of the present application, the use of the wound-targeted smart nanomedicine in the preparation of a drug for enhancing the immune activation ability of tumor tissue is also provided. The wound-targeted smart nanomedicine of the present application can significantly increase the infiltration of CD4+ and CD8+ T cells in tumor tissue, stimulating a more powerful immune response, thereby significantly improving its tumor cell-killing effect and improving the inhibitory effect on postoperative recurrence of early-stage liver cancer.

[0065] Secondly, the present application provides a method for preparing the aforementioned wound-targeted nano-targeted delivery system. The present application uses ultrasound to coat platelet membranes on a mesoporous silica surface loaded with a tumor angiogenesis inhibitor, and further chemically connects PD-L1 antibodies to the platelet membrane surface, thereby preparing an intelligent nano-drug delivery system.

[0066] Specifically, in one embodiment of the present application, the method for preparing the above-mentioned targeted smart nanomedicine comprises the following steps:

[0067] Step 1: preparing the drug-loaded nanoparticles;

[0068] Step 2: obtaining the platelet membrane;

[0069] Step 3: dispersing the platelet membrane in water, adding the drug-loaded nanoparticles, sonicating to allow the platelet membrane to coat the surface of the drug-loaded nanoparticles, centrifuging, and collecting the precipitate;

[0070] Step 4: The precipitate obtained in step 3 is treated with activator A, and then mixed with an excess of anti-PD-L1 antibody treated with activator B, reacted, centrifuged, and the precipitate is collected to obtain the targeted smart nanomedicine.

[0071] In some embodiments, the feed ratio of the platelet membrane and the drug-loaded nanoparticles in step 3 is 1:1.2-2.

[0072] In some embodiments, step 3 of dispersing the platelet membrane in water is performed by ultrasonic dispersion, and the ultrasonic conditions include: power of 80W-120W, frequency of 35kHz-50kHz, and time of 8min-15min.

[0073] In some embodiments, the ultrasonic conditions after adding the drug-loaded nanoparticles in step 3 include: power of 80W-120W, frequency of 35kHz-50kHz, and time of 15min-25min.

[0074] In some embodiments, the centrifugation conditions in step 3 include: centrifugation at 7000 rpm-9000 rpm for 8 min-15 min.

[0075] In some embodiments, the activator A is Traut's reagent, and the step of treating the precipitate with the activator A includes: dissolving the Traut's reagent in a coupling buffer to obtain a Traut's solution with a concentration of 1.5 mg / mL to 2.5 mg / mL, treating the precipitate with the Traut's solution, the mass volume ratio of the Traut's solution to the precipitate is 20 mL to 10 mL: 1 mg, and the treatment time is 40 min to 50 min.

[0076] In some embodiments, the coupling buffer is a PBS-EDTA buffer with a pH of 7.5-8.5.

[0077] In some embodiments, the activator B is (n-maleimidomethyl)-sulfosuccinimidyl)-cyclohexanecarboxylate, and the reaction conditions for treating the anti-PD-L1 antibody with the activator B include: a molar ratio of the activator B to the anti-PD-L1 antibody of 4-6:1, a temperature of 0°C-8°C, and a time of 1h-3h.

[0078] In some embodiments, the reaction conditions in step 4 include: temperature of 0°C-8°C, and time of 4h-12h.

[0079] In some embodiments, the centrifugation conditions in step 4 include: centrifugation at 9000 rpm-11000 rpm for 8 min-15 min.

[0080] In some embodiments, the step of preparing the drug-loaded nanoparticles includes: mixing mesoporous silica nanoparticles with sorafenib, reacting, and centrifuging to obtain the drug-loaded nanoparticles.

[0081] In some embodiments, the feeding ratio of the mesoporous silica nanoparticles to sorafenib is 5:1 to 1:5.

[0082] In some embodiments, the feeding ratio of the mesoporous silica nanoparticles to sorafenib is 2:1 to 1:2.

[0083] In some embodiments, the reaction conditions for preparing drug-loaded nanoparticles include: reacting at 1500 rpm-2500 rpm for 6 h-10 h.

[0084] The following are specific examples.

[0085] Example 1. Synthesis and characterization of a-PM-S-MSNP

[0086] (1) Preparation of S-MSNPs

[0087] Mesoporous silica nanoparticles (MSNPs) with a negative surface charge (-20 mV), a particle size of 100 nm, and a pore size of 6 nm were mixed with sorafenib in methanol according to the mass ratios shown in Table 1 and reacted at 2000 rpm for 8 hours. After the reaction, the supernatant was centrifuged at 10,000 rpm for 15 minutes at room temperature to remove free drug not entrapped in the MSNPs. The resulting precipitate was the sorafenib-loaded nanoparticles, or S-MSNPs.

[0088] Table 1

[0089] (2) Preparation of platelet membrane

[0090] First, whole blood was collected from the retro-orbital venous plexus of BALB / c mice and added to an ACD solution (39 mM citric acid, 75 mM sodium citrate, 135 mM glucose, pH 7.4). After pre-wetting a centrifuge tube with ACD, ACD was added at a ratio of 9:1 blood:ACD. The anticoagulated whole blood was gently mixed thoroughly and centrifuged at 100 g for 20 minutes at room temperature to obtain platelet-rich plasma. The platelet-rich plasma was then centrifuged at 800 g for 20 minutes to obtain platelets. The pellet was resuspended and dispersed in Tyrode's buffer. 1 μM PGE1 and 1 mM PMSF (dilution ratio = 1:100) were added to the solution to prevent platelet activation and membrane protein degradation. The solution was frozen at -80°C and repeatedly freeze-thawed for five cycles to fully release the platelet contents and obtain platelet membranes. The platelet membrane fraction was then obtained by further centrifugation at 12,000 rpm at 4°C for 30 minutes.

[0091] (III) Preparation of PM-S-MSNPs

[0092] To coat the platelet membranes on the surface of the S-MSNPs, the platelet membranes were first sonicated for 10 minutes (100W, 42kHz) and thoroughly dispersed in deionized water. S-MSNPs were then added at a mass ratio of 1:1.5 for platelet membranes:MSN-S. Ultrasonication was continued for 20 minutes under the same conditions as above to ensure that the platelet membranes fully coated the surface of the sorafenib-loaded nanoparticles. Finally, the platelet membranes were centrifuged at 8000 rpm for 10 minutes. The resulting precipitate was the sorafenib-loaded nanoparticles coated with platelet membranes (PM-S-MSNPs).

[0093] (IV) Synthesis and characterization of a-PM-S-MSNP

[0094] In order to efficiently connect the anti-PDL1 antibody to the surface of PM-S-MSNP, the nanoparticles were first treated with Traut's solution (Traut's reagent dissolved in coupling buffer (PBS-EDTA, pH 8.0) at a concentration of 2 mg / mL) (the volume mass ratio of Traut's solution to nanoparticles was 15 mL: 1 mg) for 45 minutes to fully activate the sulfhydryl groups on the platelet membrane surface for subsequent connection reactions. After repeated washing with Tyrode's buffer three times, the anti-PD-L1 antibody (aPDL1) and 4-(n-maleimidomethyl)-sulfosuccinimidyl)-cyclohexanecarboxylate (Sulfo-SMCC) were mixed in a ratio of 1:5 (molar ratio) and reacted at 4°C for 2 hours. The product was dialyzed using a 10KD molecular weight cutoff bag to remove unreacted Sulfo-SMCC to obtain aPDL1-Sulfo-SMCC. Next, PM-S-MSNPs were mixed with an excess of aPD-L1-sulfo-SMCC and reacted at 4°C for 12 hours. The reaction product was centrifuged at 10,000 g for 10 minutes at room temperature to remove excess anti-PD-L1 antibodies from the supernatant. The precipitate at the bottom was the final a-PM-S-MSNP nanomedicine.

[0095] The supernatant from step (1) was collected and the concentration of free sorafenib in the supernatant that was not encapsulated into the MSN was determined by HPLC, thereby indirectly determining the sorafenib content in the nanoparticles. Since the anti-PD-L1 antibody is covalently attached to the platelet membrane surface by coupling with the sulfhydryl groups on the platelet membrane surface, the sulfhydryl content of the platelet membrane surface before and after the reaction was measured using a non-protein sulfhydryl content detection kit (https: / / www.solarbio.com / goods-6214.html). This provides the total amount of sulfhydryl groups consumed during the reaction. The mass of the coupled anti-PD-L1 can be calculated by multiplying the total amount of sulfhydryl groups consumed by the molecular weight of anti-PD-L1 (40KD). The thiol content in the supernatant was also measured using a non-protein thiol content detection kit. Platelet membranes of varying concentrations were prepared and the corresponding thiol content was measured to plot a standard curve. The platelet content in the supernatant was then inferred from the standard curve, indirectly determining the platelet content on the nanoparticle surface and ultimately the total mass of the mesoporous silica. Using a MSNP / sorafenib mass ratio of 2:1 as an example, the mass proportions of anti-PD-L1, sorafenib, silica, and platelet membrane in the prepared a-PM-S-MSNP nanomedicine were measured to be 6.27%, 18.84%, 41.27%, and 33.62%, respectively.

[0096] 10 μL of the a-PM-S-MSNP solution was pipetted onto a copper mesh. After 10 minutes of deposition, the solution was blotted dry with filter paper and 10 μL of uranyl acetate was added dropwise for staining in the dark for 10 minutes. After drying with the filter paper, the morphology was observed using a biological transmission electron microscope (Hitachi, HT7700, 100 kV). As shown in Figure 1(b), the a-PM-S-MSNPs exhibited a relatively regular spherical shape with a distinct core-shell structure and a particle size of approximately 100 nm.

[0097] Example 2. SDS-Page total protein analysis

[0098] Platelets were extracted and their membranes lysed and denatured. Each treated group of materials was then loaded onto a polyacrylamide gel and electrophoresis was initiated. Following electrophoresis, the gel was stained with Coomassie Brilliant Blue, washed with destaining solution, and analyzed for protein bands using an imaging device.

[0099] The specific steps for platelet extraction and platelet membrane lysis and denaturation are as follows: ACD solution (39 mM citric acid, 75 mM sodium citrate, 135 mM glucose, pH 7.4) was prepared and mixed with whole blood from BALB / c mice at a ratio of 9:1. Platelet-rich plasma was obtained by centrifugation at 100 g for 20 minutes at room temperature. The platelet pellet was centrifuged again at 800 g for 20 minutes to obtain a platelet pellet, which was resuspended in Tyrode's buffer. 1 μM PGE1 and 1 mM PMSF (dilution ratio = 1:100) were added to the solution to prevent platelet activation and membrane protein degradation. The platelet-rich solution was then placed in a -80°C freezer and subjected to five freeze-thaw cycles to fully release the platelet contents. Finally, the platelet pellet was centrifuged at 12,000 rpm at 4°C for 30 minutes to obtain the platelet membrane component. The platelet membrane was boiled in SDS-PAGE loading buffer for 10 min. After the sample was cooled to room temperature, the denatured protein sample was subjected to SDS-PAGE electrophoresis analysis.

[0100] The analysis results are shown in Figure 2: a-PM-S-MSNP nanodrugs contain platelet surface membrane proteins and aPDL1 proteins, indicating that the surface of a-PM-S-MSNP nanodrugs is successfully coated with platelet membranes connected to aPDL1 proteins.

[0101] Example 3. Immunocolloidal gold staining experiment

[0102] 10 μL of a-PM-S-MSNP solution was added dropwise to the copper mesh, incubated for 30 minutes, and blotted dry with filter paper. The copper mesh was then washed twice with a PBS solution containing glycine and BSA (1% BSA, 50 nM glycine). 10 μL of antibodies against CD41, CD61, and CD62P were then added dropwise to the copper mesh and incubated at room temperature for 30 minutes. After blocking with BSA (1%) for 20 minutes, 10 μL of 5 nM IgG colloidal gold was added dropwise and incubated for 1 hour. The mesh was fixed with 1% glutaraldehyde solution for 10 minutes, stained with 10 μL of 1% uranyl acetate for 10 minutes, and observed using a transmission electron microscope.

[0103] The results are shown in Figure 3: the gold particles on the surface of a-PM-S-MSNP showed the presence of CD41, CD61 and CD62p on its surface, indicating that the platelet membrane was successfully coated on the surface of a-PM-S-MSNP.

[0104] Example 4. Determination of in vitro drug release

[0105] First, 2 mg of a-PM-S-MSNP was placed in a dialysis bag (molecular weight cut-off 2000 Da). The bag was then placed in 20 ml of PBS solution and shaken at a constant temperature (37°C, 100 rpm). At the corresponding time point, 1 ml of the dialysate was removed and replaced with 1 ml of fresh PBS solution. The released sorafenib content was measured using high-performance liquid chromatography (HPLC), and the cumulative drug release dose at different time points was calculated.

[0106] The results are shown in Figure 4: Sorafenib in a-PM-S-MSNP showed a time-dependent release behavior.

[0107] Example 5. In vitro collagen binding assay

[0108] 100 μL of 0.5 mg / mL type IV collagen solution was added to each well of a 96-well plate. After coating overnight at 4°C, the plates were blocked with 2% BSA and washed twice with PBS. 100 μL of each Cy5.5-labeled nanoparticle group (1 mg / mL) was added to each well and incubated for 2 minutes at room temperature. After washing the 96-well plate three times with PBS, 100 μL of dimethyl sulfoxide was added to completely dissolve the Cy5.5 fluorescent dye from the nanomedicines. The plates were then analyzed using a fluorescence imaging instrument.

[0109] The results are shown in Figure 5: there is obvious fluorescence in the collagen-coated wells, while no obvious fluorescence is observed in the blank wells, indicating that a-PM-S-MSNP has good collagen-targeted adhesion ability.

[0110] Example 6. In vivo therapeutic effects of nanomedicine

[0111] 1. Construction of an orthotopic hepatocellular carcinoma resection model

[0112] After the mice were anesthetized, the liver tissue was fully exposed by dissection, and a suspension of luciferase-labeled Hep 1-6 tumor cells (Hep 1-6-luc) was gently injected into the edge of the outer lobe of the mouse liver (500,000 tumor cells / mouse). After the liver was repositioned, it was sutured to construct an orthotopic liver cancer model. Three days after the model was established, the growth of the orthotopic Hep 1-6 tumor in the mouse liver was monitored using a small animal in vivo imaging system. When the tumor volume was approximately 200 mm3, the tumor tissue was removed again by laparotomy, leaving about 5% of residual tumor tissue to simulate the clinical postoperative tumor resection model (as shown in Figure 6a).

[0113] 2. Testing the targeting ability of nanomedicines at the in vivo resection margin

[0114] After the successful establishment of an orthotopic liver cancer resection model, each group of Cy5.5-labeled nanodrugs (10 mg / kg) were intraperitoneally injected. After a period of administration, in vivo fluorescence imaging was performed to observe the Cy5.5 fluorescence signal. The colocalization of Cy5.5 with tumor tissue was observed to assess its ability to target the resection margin after surgery.

[0115] The results are shown in Figure 6b: the bioluminescence of tumor cells and Cy5.5 fluorescence have a good co-localization effect, indicating that a-PM-S-MSNP can actively target the postoperative resection margin.

[0116] 3. Testing the Anti-tumor Efficacy of Nanomedicines

[0117] After successful establishment of an orthotopic postoperative resection model, mice were randomly divided into seven groups (n=6). On the second day after surgery, mice were intraperitoneally injected with saline, PM-MSNPs, a combination of free drugs (sorafenib and anti-PDL1 monoclonal antibody), S-PM-MSNPs, a-PM-MSNPs, a-RM-S-MSNPs (RM represents erythrocyte membranes, prepared similarly to a-PM-S-MSNPs, except that platelet membranes are replaced with erythrocyte membranes), and a-PM-S-MSNPs (sorafenib: 30 mg / kg; antibody: 10 mg / kg). Dosing was repeated every three days for a total of four treatments. Postoperative recurrence of HCC was observed at different time points using a small animal in vivo imaging system. Mouse body weight and survival were recorded. At the end of treatment, the livers and other organs were dissected, fixed with 4% paraformaldehyde, and analyzed by H&E staining.

[0118] The results of bioluminescence imaging monitoring of tumor recurrence in mice after orthotopic liver cancer surgery are shown in Figure 7. The intensity of bioluminescence can reflect the size of the tumor. The results show that a-PM-S-MSNP can significantly inhibit the postoperative recurrence of early-stage liver cancer in mice, while other drug treatment groups cannot completely inhibit the postoperative recurrence of liver cancer.

[0119] The 70-day survival statistics of mice after receiving treatment with different groups of nanomedicines are shown in Figure 8: a-PM-S-MSNP nanomedicine can significantly prolong the survival of mice in the early liver cancer postoperative resection model. In addition, the survival of mice in the a-PM-S-MSNP nanomedicine group was significantly longer than that in the sorafenib + anti-PDL1 antibody free drug group, indicating that the a-PM-S-MSNP nanomedicine prepared in this application can significantly improve the therapeutic effect of the combination of sorafenib and anti-PDL1 antibodies. In addition, the survival of mice in the a-PM-S-MSNP nanomedicine group was longer than that of the a-RM-S-MSNP nanomedicine, indicating that the platelet membrane was selected in this application to prepare the nanomedicine loaded with sorafenib + anti-PDL1 antibody, and its synergistic effect on the combined inhibition of the recurrence of early liver cancer after surgery by sorafenib and anti-PDL1 antibody was significantly better than that of red blood cell membrane.

[0120] The weight changes of mice during the treatment are shown in Figure 9: Treatment of orthotopic hepatocellular carcinoma resection model mice with a-PM-S-MSNP did not cause weight loss in the mice.

[0121] The results of H&E staining analysis of the main organs of mice at the treatment endpoint are shown in Figure 10: Treatment of mice with orthotopic hepatocellular carcinoma resection model with a-PM-S-MSNP did not cause damage to the main organs of the mice, indicating that it has good biosafety.

[0122] Example 7. Test of the immune activation ability of nanomedicine

[0123] An orthotopic hepatocellular carcinoma resection mouse model was constructed according to the method of Example 6. To ensure that liver cancer tissue of sufficient size could be obtained for flow cytometric analysis, 50% of the tumor tissue was retained during orthotopic surgical resection of tumor-bearing mice in the immune activation capacity analysis experiment. Two days after resection, the mice were randomly divided into seven groups and treated intraperitoneally with saline, PM-MSNPs, a combination of free drugs (sorafenib + anti-PDL1 monoclonal antibody), S-PM-MSNPs, a-PM-MSNPs, a-RM-S-MSNPs, and a-PM-S-MSNPs (sorafenib: 30 mg / kg; antibody: 10 mg / kg), once every three days for a total of two doses. 12 hours after the second dose, the mice were anesthetized and sacrificed, and the tumor tissue was removed and homogenized to prepare a single-cell suspension. Cells were stained with the corresponding flow cytometric antibodies, and the infiltration of CD4+ and CD8+ T cells in the tumor tissue was analyzed by flow cytometry.

[0124] The results are shown in Figure 11: Compared with the free drug combination of sorafenib + anti-PDL1 antibody, the a-PM-S-MSNP nanodrug can significantly increase the infiltration of CD4+T and CD8+T in tumor tissues, stimulate a stronger immune response, thereby significantly improving the therapeutic effect on tumors and improving the inhibitory effect on postoperative recurrence of early liver cancer.

[0125] The technical features of the above-mentioned embodiments can be combined arbitrarily. In order to make the description concise, not all possible combinations of the technical features in the above-mentioned embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0126] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present application.

Claims

1. A wound-targeted smart nanomedicine, characterized in that: It comprises platelet membrane and drug-loaded nanoparticles, wherein the platelet membrane covers the drug-loaded nanoparticles; The drug-loaded nanoparticles contain a tumor angiogenesis inhibitor; The surface of the platelet membrane is connected with anti-PDL1 antibody.

2. The wound-targeted smart nanomedicine according to claim 1, characterized in that: The tumor angiogenesis inhibitor is sorafenib.

3. The wound-targeted smart nanomedicine according to claim 1, characterized in that: The drug-loaded nanoparticles are obtained by encapsulating tumor angiogenesis inhibitors in mesoporous silica nanoparticles.

4. The wound-targeted smart nanomedicine according to claim 3, characterized in that: The mesoporous silica nanoparticles have a particle size of 50 nm to 200 nm, a pore size of 4 nm to 10 nm, and a charge of -15 mV to -25 mV; and / or, The particle size of the wound-targeted intelligent nanomedicine is 50nm to 200nm.

5. The wound-targeted smart nanomedicine according to any one of claims 1 to 4, characterized in that: The content of the tumor angiogenesis inhibitor in the wound-targeted intelligent nanomedicine is 5% to 20%; and / or, The mass ratio of the anti-PDL1 antibody to the tumor angiogenesis inhibitor is 1:20 to 20:

1.

6. Use of the wound-targeted smart nanomedicine according to any one of claims 1 to 5 in the preparation of anti-tumor drugs or drugs for preventing and treating postoperative tumor recurrence; preferably, the tumor is liver cancer.

7. A method for preparing the targeted smart nanomedicine according to any one of claims 1 to 5, characterized in that: The steps include: Step 1: preparing the drug-loaded nanoparticles; Step 2: obtaining the platelet membrane; Step 3: dispersing the platelet membrane in water, adding the drug-loaded nanoparticles, ultrasonicating to allow the platelet membrane to coat the surface of the drug-loaded nanoparticles, centrifuging, and collecting the precipitate; Step 4: The precipitate obtained in step 3 is treated with activator A, and then mixed with an excess of anti-PD-L1 antibody treated with activator B, reacted, centrifuged, and the precipitate is obtained to obtain the targeted smart nanomedicine.

8. The method for preparing the targeted smart nanomedicine according to claim 7, characterized in that: In step 3, the feed ratio of the platelet membrane to the drug-loaded nanoparticles is 1:1.2-2; and / or, Step 3: Dispersing the platelet membrane in water by ultrasonic dispersion, wherein the ultrasonic conditions include: power of 80W-120W, frequency of 35kHz-50kHz, and time of 8min-15min; and / or, The ultrasonic conditions after adding the drug-loaded nanoparticles in step 3 include: power of 80W-120W, frequency of 35kHz-50kHz, and time of 15min-25min; and / or, The centrifugal conditions in step 3 include: centrifugation at 7000 rpm-9000 rpm for 8 min-15 min; and / or, The activator A is Traut's reagent, and the step of treating the precipitate with the activator A comprises: dissolving the Traut's reagent in a coupling buffer to obtain a Traut's solution with a concentration of 1.5 mg / mL-2.5 mg / mL, treating the precipitate with the Traut's solution, the mass volume ratio of the Traut's solution to the precipitate is 20 mL-10 mL: 1 mg, and the treatment time is 40 min-50 min; and / or, The activator B is (n-maleimidomethyl)-sulfosuccinimidyl)-cyclohexanecarboxylate, and the reaction conditions for treating the anti-PD-L1 antibody with the activator B include: a molar ratio of the activator B to the anti-PD-L1 antibody of 4-6:1, a temperature of 0°C-8°C, and a time of 1h-3h; and / or, The reaction conditions in step 4 include: temperature of 0°C-8°C, time of 4h-12h; and / or, The centrifugal conditions in step 4 include: centrifugation at 9000 rpm-11000 rpm for 8 min-15 min.

9. The method for preparing the targeted smart nanomedicine according to claim 7 or 8, characterized in that: The steps of preparing the drug-loaded nanoparticles include: mixing the mesoporous silica nanoparticles with sorafenib, reacting, and centrifuging to obtain the drug-loaded nanoparticles.

10. The method for preparing the targeted smart nanomedicine according to claim 9, characterized in that: The feeding ratio of the mesoporous silica nanoparticles to sorafenib is 5:1 to 1:5; and / or, The reaction conditions for preparing the drug-loaded nanoparticles include: reacting at 1500rpm-2500rpm for 6h-10h.

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