Wound-targeted intelligent nano-drug as well as preparation method and application thereof

Abstract

The invention discloses a wound-targeted intelligent nano-drug as well as a preparation method and application thereof. The wound-targeted intelligent nano-drug comprises a platelet membrane and drug-loaded nano-particles, wherein the platelet membrane coats the drug-loaded nano-particles; the drug-loaded nanoparticles contain a tumor angiogenesis inhibitor; the surface of the platelet membrane is connected with an anti-PDL1 antibody. The wound-targeted intelligent nano-drug can significantly improve the curative effect of a tumor angiogenesis inhibitor (sorafenib) and anti-PDL1 combined use, improve the biological distribution of the drug, reduce the toxic and side effects caused by the combined use of the sorafenib and anti-PDL1, greatly reduce the postoperative recurrence rate of early liver cancer, has a strong postoperative recurrence inhibition capability, and has a good application prospect. The biological safety is relatively excellent.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical technology, and in particular to a wound-targeted intelligent nanomedicine for inhibiting postoperative recurrence of early liver cancer, and a preparation method and application thereof. Background Art

[0002] Liver cancer is a common malignant digestive tract tumor, the sixth most common cancer and the third most lethal cancer in the world. Every year, a large number of patients lose their lives to liver cancer, with a total of 830,000 deaths reported in 2020 alone. Liver cancer seriously threatens the lives and health of the people, especially in some developing countries, so the treatment of liver cancer is of great clinical significance.

[0003] At present, 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. Directly removing tumor tissue through surgery can greatly relieve the patient's tumor burden, prolong the patient's survival, and improve the patient's 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. On the other hand, during the operation, tumor cells may enter the blood circulation through damaged blood vessels and metastasize, which will also promote the recurrence of the tumor after surgery. According to statistics, the recurrence rate of early liver cancer 3 years after surgery is as high as 50%.

[0004] Although conventional tumor angiogenesis inhibitors such as sorafenib can be used to prevent postoperative recurrence of early liver cancer, their therapeutic effects are limited, and systemic medication has obvious toxic side effects. A large number of research reports have shown that postoperative recurrence of liver cancer is not only related to obvious angiogenesis, but also closely related to the immunosuppressive tumor microenvironment. Therefore, this provides us with a new therapeutic strategy to inhibit postoperative recurrence. At present, 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, but the response rate and efficacy of the two drugs are limited. In addition, the serious 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 liver cancer, which seriously reduces the overall survival and quality of life of postoperative tumor patients. Summary of the invention

[0005] The purpose of the present invention is to provide an intelligent nano drug which can effectively inhibit the recurrence of liver cancer after surgery.

[0006] In order to achieve the above object, the present invention includes the following technical solutions.

[0007] A wound-targeted smart nanomedicine, comprising a platelet membrane and drug-loaded nanoparticles, wherein the platelet membrane covers the drug-loaded nanoparticles;

[0008] The drug-loaded nanoparticles contain a tumor angiogenesis inhibitor;

[0009] The surface of the platelet membrane is connected with anti-PDL1 antibody.

[0010] Preferably, the tumor angiogenesis inhibitor is sorafenib.

[0011] The present invention also provides the use of the above-mentioned wound-targeted intelligent nanomedicine in the preparation of anti-tumor drugs.

[0012] The present invention also provides the use of the above-mentioned wound-targeted intelligent nanomedicine in the preparation of a drug for preventing and treating postoperative recurrence of liver cancer.

[0013] The present invention also provides a method for preparing the above-mentioned targeted intelligent nanomedicine, comprising the following steps:

[0014] Step 1: preparing the drug-loaded nanoparticles;

[0015] Step 2: obtaining the platelet membrane;

[0016] 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;

[0017] 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.

[0018] The present invention designs and synthesizes a new type of intelligent nano drug 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. The new nano drug delivery system can significantly improve the efficacy of the combined use of tumor angiogenesis inhibitors (sorafenib) and anti-PDL1, improve the biodistribution of the drugs, reduce the toxic side effects caused by the combination of the two, and greatly reduce the postoperative recurrence rate of early liver cancer. The nano platform is based on biologically inert mesoporous silica, and biologically derived platelet membranes are important components. Both have very good biocompatibility, so the intelligent nano drug has a strong ability to inhibit postoperative recurrence while having relatively excellent biosafety.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0020] (1) The wound-targeted nano-drug delivery system (intelligent nano-drug) of the present invention 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 and improve the biodistribution of the drug. It can also 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.

[0021] (2) The present invention utilizes the postoperative wound targeting property and unique tumor tissue targeting property of platelet membrane, so that it can specifically aggregate around residual tumor tissue after surgery, enhance tumor permeability and therapeutic effect, and the introduction of platelet membrane 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

[0022] Figure 1 (a) The preparation flow chart of a-PM-S-MSNP nanomedicine and its transmission electron microscopy characterization (b) are shown. The scale bar is 100 nm.

[0023] Figure 2 This is the SDS-PAGE total protein analysis result of a-PM-S-MSNP nanomedicine.

[0024] Figure 3 Immunocolloidal gold staining was used to verify the presence of key proteins on the platelet membrane surface of a-PM-S-MSNP nanodrugs.

[0025] Figure 4 The release of sorafenib from a-PM-S-MSNP nanomedicine over time.

[0026] Figure 5 In vitro collagen adhesion imaging of a-PM-S-MSNP nanodrugs and its statistical results.

[0027] Figure 6 Establishment of a mouse model of postoperative resection of in situ hepatocellular carcinoma (a) and analysis of the postoperative resection margin targeting ability of a-PM-S-MSNP nanomedicine (b).

[0028] Figure 7 The results of bioluminescence imaging monitoring of tumor recurrence in mice after orthotopic liver cancer surgery.

[0029] Figure 8 Statistics of 70-day survival of mice after orthotopic liver cancer surgery after receiving different groups of nanodrug treatment.

[0030] Fig. 9 This is the weight change of mice during the treatment of mice after orthotopic liver cancer surgery.

[0031] Fig.10 H&E staining analysis results of the main organs of mice at the treatment endpoint.

[0032] Fig.11 Flow cytometry 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

[0033] The technical scheme of the present invention is further illustrated by specific examples below. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0035] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0036] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0037] In order to effectively prevent the postoperative recurrence of liver cancer, the present invention designs a smart nano drug that can actively target the postoperative wound. It uses mesoporous silicon as the main delivery carrier, 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 through a chemical reaction. The smart nano drug can rely on the wound targeting of platelets to deliver tumor angiogenesis inhibitors to the surgical margin position, 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 nano drug prepared by the present invention can significantly inhibit the postoperative recurrence of early liver cancer, providing a new strategy for the prevention and treatment of clinical postoperative recurrence of liver cancer.

[0038] In the first aspect, the present invention provides a wound-targeted nano drug delivery system for the prevention and treatment of postoperative recurrence of early liver cancer. The nano targeted drug delivery system is mainly composed of nano carriers, biofilms and two drugs. The nano carriers can be mesoporous silica nanoparticles, and the biofilm is a platelet membrane that can actively target postoperative wounds. The two drugs are chemotherapy drugs (tumor angiogenesis inhibitors, which can be sorafenib) and antibody drugs PD-L1 antibodies. The tumor angiogenesis inhibitor is encapsulated in the pores of the mesoporous silica, and its outer surface is covered by the platelet membrane. The PD-L1 antibody is further covalently linked to the outside of the platelet membrane through a chemical reaction, thereby forming a nano drug delivery system.

[0039] The preferred nanocarrier of the present invention is mesoporous silica. Mesoporous silica nanoparticles have many advantages: their pore structure is regular and orderly, the pore size distribution is uniform, and the pore diameter can be adjusted according to demand. Mesoporous silica nanoparticles have a high specific surface area and a large pore volume, and they have good biosafety, which lays a foundation for the clinical application of the nanomedicine of the present invention.

[0040] The preferred biological membrane of the present invention is the platelet membrane. The platelet membrane surface has many receptors that can mediate its binding with damaged blood vessels, thereby aggregating to form thrombi and exerting a hemostatic function. The present invention 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 by means 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, and specifically deliver the drug 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 inhibitor or the mesoporous silica nanoparticles loaded with the tumor angiogenesis inhibitor.

[0041] The two preferred drugs of the present invention 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 medication 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, the efficacy is limited, and long-term medication can lead to serious immune side effects. In order to reduce the postoperative recurrence rate of early liver cancer, the present invention preferably uses mesoporous silica as a nano drug delivery platform, encapsulates sorafenib in the internal pores, and coats the outside with platelet membranes. 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.

[0042] Compared with the prior art, the present invention utilizes the cutting edge targeting of platelets for the first time to construct a nanomedicine for the prevention and treatment of postoperative recurrence. In the present invention, 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, and the damaged blood vessels will expose collagen and von Willebrand factor (VWF) and other components, while 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. The present invention utilizes this feature and innovatively wraps the platelet membrane on the outside of the mesoporous silica carrying tumor angiogenesis inhibitors, thereby targeting the drug delivery to the cutting edge position. Furthermore, the present invention also utilizes 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 precisely delivered to the cutting edge position of the tumor after surgery, combining anti-tumor angiogenesis therapy with immunotherapy, and greatly improving the efficacy of its combined medication.

[0043] Specifically, in one embodiment of the present invention, a wound-targeted smart nanomedicine is provided, which comprises a platelet membrane and drug-loaded nanoparticles, wherein the platelet membrane covers the drug-loaded nanoparticles;

[0044] The drug-loaded nanoparticles contain a tumor angiogenesis inhibitor;

[0045] The surface of the platelet membrane is connected with anti-PDL1 antibody.

[0046] In some embodiments, the tumor angiogenesis inhibitor is sorafenib.

[0047] In some embodiments, the drug-loaded nanoparticles are obtained by encapsulating tumor angiogenesis inhibitors in mesoporous silica nanoparticles.

[0048] 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.

[0049] In some of the embodiments, the content of tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 3% to 50%.

[0050] In some of the embodiments, the content of tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 5% to 20%.

[0051] In some of the embodiments, the content of tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 10% to 20%.

[0052] In some of the embodiments, the content of tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 14% to 19%.

[0053] In some of the embodiments, the content of tumor angiogenesis inhibitor in the wound-targeted smart nanomedicine is 16% to 19%.

[0054] 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.

[0055] In some of the embodiments, the mass ratio of the tumor angiogenesis inhibitor to the mesoporous silica nanoparticles is 1:0.2-5, preferably 1:0.5-2.

[0056] In some of the 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.

[0057] In some of the embodiments, the particle size of the wound-targeted smart nanomedicine is 50nm-200nm, for example, it can be 50nm, 100nm, 120nm, 150nm, 180nm, 200nm, etc.

[0058] In one embodiment of the present invention, there is also provided the use of the wound-targeted intelligent nanomedicine in the preparation of a drug for preventing and treating postoperative tumor recurrence.

[0059] In some embodiments, the tumor is liver cancer. The targeted smart nanomedicine of the present invention can significantly inhibit the postoperative recurrence of early liver cancer in mice, thereby significantly prolonging the survival of patients with early liver cancer who have undergone postoperative resection.

[0060] In one embodiment of the present invention, the use of the wound-targeted smart nanomedicine in the preparation of anti-tumor drugs is also provided.

[0061] In one embodiment of the present invention, the use of the wound-targeted smart nanomedicine in the preparation of a drug for improving the immune activation ability of tumor tissue is also provided. The wound-targeted smart nanomedicine of the present invention can significantly increase the infiltration of CD4+T and CD8+T in tumor tissue, stimulate a stronger immune response, thereby significantly improving its killing effect on tumor cells and improving the inhibitory effect on postoperative recurrence of early liver cancer.

[0062] In a second aspect, the present invention provides a method for preparing the wound-targeted nano-targeted delivery system. The present invention coats the platelet membrane on the surface of the mesoporous silica carrying the tumor angiogenesis inhibitor by ultrasound, and further connects the PD-L1 antibody to the platelet membrane surface by chemical connection, thereby preparing an intelligent nano-drug delivery system.

[0063] Specifically, in one embodiment of the present invention, the method for preparing the above-mentioned targeted smart nanomedicine comprises the following steps:

[0064] Step 1: preparing the drug-loaded nanoparticles;

[0065] Step 2: obtaining the platelet membrane;

[0066] 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;

[0067] 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.

[0068] In some of the embodiments, the feed ratio of the platelet membrane and the drug-loaded nanoparticles in step 3 is 1:1.2-2.

[0069] 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.

[0070] 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.

[0071] In some embodiments, the centrifugation conditions in step 3 include: centrifugation at 7000 rpm-9000 rpm for 8 min-15 min.

[0072] 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.

[0073] In some embodiments, the coupling buffer is a PBS-EDTA buffer with a pH of 7.5-8.5.

[0074] 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: the molar ratio of the activator B to the anti-PD-L1 antibody is 4-6:1, the temperature is 0°C-8°C, and the time is 1h-3h.

[0075] In some embodiments, the reaction conditions in step 4 include: temperature of 0°C-8°C, and time of 4h-12h.

[0076] In some embodiments, the centrifugation conditions in step 4 include: centrifugation at 9000 rpm-11000 rpm for 8 min-15 min.

[0077] 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.

[0078] In some embodiments, the feed ratio of the mesoporous silica nanoparticles to sorafenib is 5:1 to 1:5.

[0079] In some embodiments, the feed ratio of the mesoporous silica nanoparticles to sorafenib is 2:1 to 1:2.

[0080] In some of the embodiments, the reaction conditions for preparing the drug-loaded nanoparticles include: reacting at 1500 rpm-2500 rpm for 6 h-10 h.

[0081] The following are specific embodiments.

[0082] Example 1a - Synthesis and Characterization of PM-S-MSNPs

[0083] (I) Preparation of S-MSNPs

[0084] Mesoporous silica nanoparticles (MSNPs) with a negative surface charge (charge of -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 ratio shown in Table 1, and reacted at 2000 rpm for 8 h. After the reaction, centrifugation was performed at room temperature for 15 min to remove the free drug not encapsulated in MSN in the supernatant, and the precipitate was the nanoparticles loaded with sorafenib, namely S-MSNPs.

[0085] Table 1

[0086]

[0087] (II) Preparation of platelet membrane

[0088] First, whole blood was collected from the retro-orbital venous plexus of BALB / c mice, and ACD solution (39mM citric acid, 75mM sodium citrate, 135mM glucose, pH=7.4) was added. After the centrifuge tube was moistened with ACD in advance, ACD was added according to blood: ACD=9:1. The anticoagulated whole blood was gently and thoroughly mixed, and then centrifuged at 100g for 20min at room temperature to obtain platelet-rich plasma. Platelet-rich plasma was further centrifuged at 800g for 20min to obtain platelets. The precipitate was resuspended and then dispersed in Tyrode buffer. At the same time, 1μM PGE1 and 1mM PMSF (dilution ratio=1:100) were added to the solution to prevent platelet activation and membrane protein degradation. The above solution was placed in a -80℃ refrigerator and repeatedly frozen and thawed for 5 cycles to fully release the contents of the platelets to obtain platelet membranes. Centrifugation was continued at 4℃ and 12000rpm for 30min to obtain platelet membrane components.

[0089] (III) Preparation of PM-S-MSNPs

[0090] In order to coat the platelet membrane on the surface of S-MSNP, the platelet membrane was first ultrasonically treated for 10 minutes (100W, 42kHz) and fully dispersed in deionized water. S-MSNP was added according to the mass ratio of platelet membrane: MSN-S = 1:1.5, and the ultrasonic treatment was continued for 20 minutes under the same conditions as above, so that the platelet membrane was fully coated on the surface of the nanoparticles loaded with sorafenib. Finally, centrifugation was performed at 8000rpm for 10 minutes, and the resulting precipitate was the nanoparticles loaded with sorafenib (PM-S-MSNP) coated with platelet membrane.

[0091] (IV) Synthesis and characterization of a-PM-S-MSNP

[0092] In order to efficiently connect the anti-PDL1 antibody to the surface of PM-S-MSNP, the nanoparticles (the volume mass ratio of Traut's solution to nanoparticles was 15mL:1mg) were first treated with Traut's solution (Traut's reagent dissolved in coupling buffer (PBS-EDTA, pH8.0) at a concentration of 2mg / mL) for 45min to fully activate the thiol groups on the surface of the platelet membrane 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 at a ratio of 1:5 (molar ratio) and reacted at 4°C for 2h. The product was dialysis bag with a molecular weight cutoff of 10KD to remove unreacted Sulfo-SMCC to obtain aPDL1-Sulfo-SMCC. Next, PM-S-MSNP was mixed with excess aPD-L1-sulfo-SMCC and reacted for 12 hours at 4°C. The reaction product was centrifuged at 10,000 g for 10 minutes at room temperature to remove excess anti-PD-L1 antibodies in the supernatant, and the lower precipitate was the final a-PM-S-MSNP nanomedicine.

[0093] The supernatant of step (I) was collected, and the concentration of free sorafenib in the supernatant that was not encapsulated into MSN was determined by high performance liquid chromatography, thereby indirectly determining the content of sorafenib in the nanoparticles. Since the anti-PD-L1 antibody is covalently linked to the surface of the platelet membrane by coupling with the thiol groups on the surface of the platelet membrane, the thiol content on the surface of the platelet membrane before and after the reaction was determined by a non-protein thiol content detection kit (https: / / www.solarbio.com / goods-6214.html), so that the total amount of thiol groups consumed during the reaction can be obtained. The mass of the coupled anti-PD-L1 can be calculated by multiplying the total amount of thiol groups consumed by the molecular weight of anti-PD-L1 (40KD). At the same time, a non-protein thiol content detection kit was used to detect the thiol content in the supernatant, and platelet membranes of different concentrations were prepared and the corresponding thiol content was measured to draw a standard curve, so that the platelet content in the supernatant was calculated based on the standard curve, and the platelet content on the surface of the nanoparticles was indirectly determined, and finally the total mass of the mesoporous silica was determined. Taking the mass ratio of MSNP / sorafenib 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.

[0094] 10 μL of a-PM-S-MSNP solution was absorbed on the surface of the copper mesh. After 10 minutes of deposition, the solution was dried with filter paper, and 10 μL of uranyl acetate was added to stain for 10 minutes in the dark. After the filter paper was dried, the morphology was observed using a biological transmission electron microscope (Hitachi, HT7700, 100 kV). Figure 1 As shown in b, a-PM-S-MSNP is a relatively regular spherical shape with an obvious core-shell structure and a particle size of about 100 nm.

[0095] Example 2. SDS-Page total protein analysis

[0096] Platelets were extracted and the platelet membranes were lysed and denatured, and then the treated materials of each group were added to the polyacrylamide gel and electrophoresis was started. After the electrophoresis, the protein gel was stained with Coomassie brilliant blue, and after washing with decolorizing solution, the protein bands were analyzed by an imaging device.

[0097] The specific steps of extracting platelets and lysing and denaturing the platelet membrane are as follows: prepare ACD solution (39mM citric acid, 75mM sodium citrate, 135mM glucose, pH = 7.4), and mix the whole blood of BALB / c mice with ACD in a ratio of 9:1. Centrifuge at 100g for 20min at room temperature to obtain platelet-rich plasma. Centrifuge again at 800g for 20min to obtain platelet precipitate and resuspend in Tyrode buffer. Add 1μM PGE1 and 1mM PMSF (dilution ratio = 1:100) to the solution to prevent platelet activation or membrane protein degradation. Further, place the above platelet-rich solution in a -80℃ refrigerator and repeat freezing and thawing for 5 cycles to fully release the contents of the platelets. Finally, centrifuge at 4℃ and 12000rpm for 30min, and the resulting precipitate is 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.

[0098] The analysis results are as follows Figure 2 As shown: a-PM-S-MSNP nanodrug contains platelet surface membrane protein and aPDL1 protein, indicating that the surface of a-PM-S-MSNP nanodrug is successfully coated with platelet membrane connected to aPDL1 protein.

[0099] Example 3. Immunocolloidal gold staining experiment

[0100] 10μL a-PM-S-MSNP solution was added dropwise to the copper mesh, incubated for 30 minutes and dried with filter paper. Next, the copper mesh was washed twice with PBS (1% BSA, 50nM glycine) solution containing glycine and BSA. Then 10μL of CD41, CD61, and CD62P antibodies were added dropwise to the copper mesh and incubated at room temperature for 30 minutes. After blocking with BSA (1%) for 20 minutes, 10μL 5nM IgG colloidal gold was added dropwise and incubated for 1 hour. Fix with 1% glutaraldehyde solution for 10 minutes, add 10μL of 1% uranyl acetate for staining for 10 minutes, and observe with transmission electron microscopy.

[0101] The results are as follows Figure 3 As shown: The gold particles on the surface of a-PM-S-MSNP indicate the presence of CD41, CD61 and CD62p on its surface, indicating that the platelet membrane is successfully coated on the surface of a-PM-S-MSNP.

[0102] Example 4. Determination of in vitro drug release

[0103] First, 2 mg of a-PM-S-MSNP was placed in a dialysis bag (molecular weight cutoff of 2000 Da), and the dialysis bag was placed in 20 ml of PBS solution and oscillated at a constant temperature (37 ° C, 100 rpm). At the corresponding time point, 1 ml of dialysate was taken out and supplemented with 1 ml of fresh PBS solution. The released sorafenib content was measured by high performance liquid chromatography (HPLC), and the cumulative release dose of the drug at different time points was calculated.

[0104] The results are as follows Figure 4 As shown: Sorafenib in a-PM-S-MSNP exhibits a time-dependent release behavior.

[0105] Example 5. In vitro collagen binding assay

[0106] 100 μL of 0.5 mg / mL type IV collagen solution was added to each well of the 96-well plate, and the plates were blocked with 2% BSA after overnight coating at 4°C, and washed twice with PBS solution. 100 μL of each group of Cy5.5-labeled nanoparticles (1 mg / mL) was added to each well and incubated for 2 min 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 in the nanomedicine, and analyzed by a fluorescent imaging instrument.

[0107] The results are as follows Figure 5 As shown: 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 targeting adhesion ability.

[0108] Example 6. In vivo therapeutic effect of nanomedicine

[0109] 1. Construction of orthotopic hepatocellular carcinoma resection model

[0110] After the mice were anesthetized, the liver tissue was fully exposed by dissection, and the suspension of Hep 1-6 tumor cells (Hep1-6-luc) labeled with luciferase 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. The tumor volume was approximately 200mm 3 When the tumor tissue was removed, the abdomen was opened again, leaving about 5% of residual tumor tissue to simulate the clinical postoperative tumor resection model (such as Figure 6 (as shown in Figure a).

[0111] 2. Testing the targeting ability of nanomedicines at the in vivo resection margin

[0112] After the in situ liver cancer resection model was successfully established, each group of nanodrugs labeled with Cy5.5 (10 mg / kg) were intraperitoneally injected, and the Cy5.5 fluorescence signal was observed by in vivo fluorescence imaging after a period of administration. The co-localization of Cy5.5 and tumor tissue was observed to evaluate its postoperative resection margin targeting ability.

[0113] The results are as follows Figure 6 As shown in Figure b: 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 edge.

[0114] 3. Testing of the Anti-tumor Efficacy of Nanomedicine

[0115] After the in situ postoperative resection model was successfully established, the mice were randomly divided into 7 groups (n=6). On the second day after surgery, the mice were given normal saline, PM-MSNP, free drug combination (sorafenib + anti-PDL1 monoclonal antibody), S-PM-MSNP, a-PM-MSNP, a-RM-S-MSNP (RM is erythrocyte membrane, and its preparation method is the same as that of a-PM-S-MSNP, except that the platelet membrane is replaced by erythrocyte membrane), and a-PM-S-MSNP treatment (sorafenib: 30 mg / kg; antibody: 10 mg / kg) by intraperitoneal injection. The drug was given once every 3 days for a total of 4 treatments. During this period, the postoperative recurrence of liver cancer at different time points was observed using a small animal in vivo imaging system. The weight and survival period of the mice were recorded. At the end of the treatment, the liver and other organs of the mice were dissected and separated, fixed with 4% paraformaldehyde, and analyzed by H&E staining sections.

[0116] Bioluminescence imaging monitoring results of tumor recurrence in mice after orthotopic liver cancer surgery Figure 7 As shown, the intensity of bioluminescence can reflect the size of the tumor. The results showed that a-PM-S-MSNP can significantly inhibit the postoperative recurrence of early liver cancer in mice, while other drug treatment groups cannot completely inhibit the postoperative recurrence of liver cancer.

[0117] The 70-day survival statistics of mice after receiving different groups of nanomedicine treatment are as follows Figure 8 As shown: 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 is significantly longer than that in the free drug group of sorafenib + anti-PDL1 antibody, indicating that the a-PM-S-MSNP nanomedicine prepared by the present invention can significantly improve the therapeutic effect of the combined use of sorafenib and anti-PDL1 antibody. In addition, the survival of mice in the a-PM-S-MSNP nanomedicine group is longer than that of the a-RM-S-MSNP nanomedicine, indicating that the present invention selects platelet membranes to prepare nanomedicines loaded with sorafenib + anti-PDL1 antibodies, and its synergistic effect on the combined inhibition of the recurrence of early liver cancer after surgery by sorafenib and anti-PDL1 antibodies is significantly better than that of red blood cell membranes.

[0118] The weight changes of mice during treatment were as follows Fig. 9 As shown: Treatment of mice with orthotopic hepatocellular carcinoma resection model with a-PM-S-MSNP did not cause weight loss in mice.

[0119] The results of H&E staining analysis of the main organs of mice at the end of treatment are as follows Fig.10 As shown: Treatment of orthotopic hepatocellular carcinoma resection model mice with a-PM-S-MSNP did not cause damage to the main organs of the mice, indicating that it has good biosafety.

[0120] Example 7 Test of the immune activation ability of nanomedicine

[0121] An in situ hepatocellular carcinoma resection mouse model was constructed according to the method of Example 6. In order to ensure that liver cancer tissue of sufficient size can be obtained for flow analysis, 50% of the tumor tissue was retained during in situ surgical resection of tumor-bearing mice in the immune activation capacity analysis experiment. On the second day after resection, the mice were randomly divided into 7 groups and given intraperitoneal injection of normal saline, PM-MSNP, free drug combination (sorafenib + anti-PDL1 monoclonal antibody), S-PM-MSNP, a-PM-MSNP, a-RM-S-MSNP, a-PM-S-MSNP treatment (sorafenib: 30 mg / kg; antibody: 10 mg / kg) once every 3 days for a total of 2 times. 12 hours after the second administration, the mice were anesthetized and killed, and the tumor tissue was removed and homogenized to prepare a single cell suspension. The corresponding flow antibodies were added for cell staining, and the CD4 + T cells and CD8 + T cell infiltration.

[0122] The results are as follows Fig.11 As shown: a-PM-S-MSNP nanodrug can significantly increase the number of CD4 in tumor tissues compared with sorafenib + anti-PDL1 antibody free drug combination. + T and CD8 + The infiltration of T cells stimulates a stronger immune response, thereby significantly improving the therapeutic effect on tumors and improving the inhibitory effect on the postoperative recurrence of early liver cancer.

[0123] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described 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.

[0124] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

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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