Bionic composite nanodrug and preparation method and application thereof

Abstract

The application discloses a kind of bionic composite nanomedicine and its preparation method and application, which is composed of platelet membrane, chemotherapeutic drug DX8951f And topoisomerase II siRNA;The chemotherapeutic drug DX8951f With topoisomerase II siRNA self-assemble to form composite nanoparticles by electrostatic interaction, and the platelet membrane is coated on the surface of the composite nanoparticles.The application successfully constructs a kind of platelet membrane coated DX8951f With topoisomerase II siRNA The carrier-free bionic nanomedicine, overcomes DX8951f Clinical drug resistance problem by chemotherapy-gene synergistic effect, realizes the stable bionic modification of carrier-free nanoparticle by mechanical extrusion coating process, significantly improves drug in-vivo stability, tumor targeting and biological safety.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to a biomimetic composite nanomedicine, its preparation method, and its application. Background Technology

[0002] Triple-negative breast cancer is characterized by a high content of cancer stem cells and low levels of intercellular junction proteins, leading to strong tumor heterogeneity, high malignancy, high recurrence and metastasis rates, and poor patient prognosis. The routine clinical treatment regimen is surgery combined with adjuvant chemotherapy, which easily causes postoperative complications, and the anticancer effect of single chemotherapy drugs is limited, making it difficult to meet clinical treatment needs.

[0003] DX8951f is a highly effective topoisomerase I inhibitor that specifically captures the DNA-topoisomerase I cleavage complex, promoting DNA damage in cancer cells and inducing apoptosis, exhibiting broad-spectrum anticancer activity in various solid tumors, including breast cancer. However, long-term exposure of cancer cells to topoisomerase I inhibitors leads to compensatory increases in the expression and activity of topoisomerase II to compensate for the loss of DNA repair function, significantly reducing the chemotherapeutic effect of DX8951f. Furthermore, DX8951f suffers from poor water solubility, significant off-target toxicity, and easy ring-opening inactivation in the in vivo physiological environment. Significant dose-limiting toxicities were observed in its phase III clinical trials, and the clinical benefit of its combination with gemcitabine was insufficient; therefore, the related study has been terminated.

[0004] Small interfering RNA (siRNA) drugs inhibit target protein expression through RNA interference technology. Specifically, the siRNA's guide strand pairs complementary with the target mRNA, subsequently recruiting various protein components to form an RNA-induced silencing complex, which degrades the target mRNA, thereby reducing target protein expression. However, siRNA is easily degraded by RNase in vivo, and its large molecular weight makes it difficult for cancer cells to actively take up, thus preventing its direct use as a drug for anti-tumor therapy.

[0005] Nanomedicines can achieve passive targeted enrichment of tumor tissues through the EPR effect, increasing drug concentration at the tumor site and providing a solution for the combined delivery of chemotherapeutic drugs and nucleic acid drugs. Existing research has constructed chemotherapeutic drug-nucleic acid composite nanoparticles through electrostatic self-assembly strategies to achieve chemotherapy-gene therapy and improve the treatment efficacy of drug-resistant tumors.

[0006] Cell membrane coating is an emerging biomimetic drug modification technology. Platelet membrane-coated nanomedicines have good immunocompatibility and can achieve active tumor targeting by leveraging the specific binding of P-selectin on the platelet membrane surface to CD44 molecules highly expressed in cancer stem cells. At the same time, it prolongs the drug circulation time in vivo and reduces the risk of immune clearance.

[0007] Traditional cell membrane coating often employs ultrasound technology, suitable for structurally stable nanoparticles with inorganic or polymeric carriers. However, the strong shear force generated by ultrasound can disrupt the structure of carrier-free, self-assembled nanoparticles, making stable coating impossible. Existing research has demonstrated that mechanical extrusion can coat erythrocyte / tumor cell hybrid membranes onto the surface of carrier-free nanoparticles, endowing them with long-term in vivo circulation and tumor-homogeneous targeting capabilities. However, the application of this technology in the field of platelet membrane biomimetic modification still needs further development.

[0008] Therefore, it is necessary to develop a platelet membrane biomimetic composite nanomedicine that can synergistically enhance efficacy, provide stable targeting, has high biosafety, and is applicable to carrier-free nanoparticles, in order to overcome problems such as DX8951f drug resistance, off-target toxicity, in vivo inactivation, and difficulties in siRNA delivery, and meet the needs of clinical cancer combination therapy. Summary of the Invention

[0009] The purpose of this invention is to provide a biomimetic composite nanomedicine, its preparation method, and its application, thereby solving the problems in the prior art such as DX8951f's easy development of drug resistance, poor water solubility, high off-target toxicity, easy in vivo inactivation, siRNA's instability in vivo and difficulty in being taken up by cancer cells, and the inability of carrier-free nanoparticles to achieve cell membrane coating by ultrasound.

[0010] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: According to a first aspect of the present invention, a biomimetic composite nanomedicine is provided, comprising a platelet membrane, a chemotherapeutic drug DX8951f, and a topoisomerase II siRNA; wherein the chemotherapeutic drug DX8951f and the topoisomerase II siRNA self-assemble to form composite nanoparticles through electrostatic interactions, and the platelet membrane is coated on the surface of the composite nanoparticles.

[0011] The sense strand sequence of the topoisomerase II siRNA is 5'-CCGCGUGGUCAAAGAGUCAUU-3', and the antisense strand sequence is 5'-AAUGACUCUUUGACCACGCGG-3'.

[0012] According to a second aspect of the present invention, a method for preparing the biomimetic composite nanomedicine is provided, comprising the following steps: 1) taking peripheral blood from mice, collecting platelets by centrifugation, and preparing a platelet membrane solution by repeated freeze-thaw cycles; 2) preparing DX8951f solution and topoisomerase II siRNA solution separately using DEPC water, mixing them, and then self-assembling to form composite nanoparticles; 3) mixing the composite nanoparticles with the platelet membrane solution, and coating the platelet membrane by mechanical extrusion to obtain the biomimetic composite nanomedicine.

[0013] Preferably, in step 2), the molar ratio of DX8951f to topoisomerase II siRNA is (6~12):1.

[0014] Preferably, in step 3), the mass concentration ratio of the composite nanoparticles to the platelet membrane solution is (3~6):1.

[0015] Preferably, step 1) includes: centrifuging mouse peripheral blood at room temperature, collecting the supernatant, centrifuging again at room temperature to obtain the precipitate, freezing the precipitate at -80 ℃ and thawing it at room temperature, repeating the freeze-thaw cycle 4 to 6 times; washing with pre-cooled phosphate buffer solution, centrifuging at 4 ℃, washing 3 times, resuspending in phosphate buffer solution, sonicating in an ice bath for 5 min, quantifying, and storing at -80 ℃.

[0016] According to a preferred embodiment of the present invention, step 1) includes: centrifuging mouse peripheral blood at 300 g for 5 min at room temperature, collecting the supernatant, centrifuging at 2000 g for 5 min at room temperature to obtain the precipitate, freezing the precipitate at -80 ℃ and thawing it at room temperature, repeating the freeze-thaw cycle 4 to 6 times; washing with pre-cooled phosphate buffer solution, centrifuging at 21000 g for 15 min at 4 ℃, washing 3 times, resuspending in phosphate buffer solution, sonicating in an ice bath for 5 min, quantifying, and storing at -80 ℃.

[0017] Preferably, in step 1), the phosphate buffer solution has the following composition: 2.7 mmol / L KCl, 2.0 mmol / L KH2PO4, 137 mmol / L NaCl, 10 mmol / L Na2HPO4, pH 7.3-7.5, and is sterilized by filtration.

[0018] Preferably, in step 2), the concentration of DX8951f solution is 24-240 μmol / L, and the concentration of topoisomerase II siRNA solution is 4-20 μmol / L; the two solutions are mixed in equal volumes rapidly, vortexed for 5 min, and then shaken overnight at room temperature to form composite nanoparticles.

[0019] Preferably, in step 2), the DEPC water is water that has been treated with 0.1% diethyl pyrocarbonate and sterilized under high temperature and high pressure.

[0020] Preferably, in step 3), the mechanical extrusion is performed using a liposome extruder, which sequentially passes the material through polycarbonate membranes with pore sizes of 400 nm and 200 nm, and is repeatedly extruded 20-40 times to obtain nanoparticles DX / siTOPOⅡα@PMNPs with platelet membranes coated on the surface. After ultrafiltration and centrifugation concentration, the nanoparticles are stored at 4 °C for later use.

[0021] Preferably, the centrifugation conditions in step 7) are 6000 g centrifugation at 4 ℃ for 20 min.

[0022] According to a third aspect of the present invention, the application of the biomimetic composite nanomedicine or the biomimetic composite nanomedicine prepared by the method is provided in the preparation of a cancer treatment drug, wherein the cancer includes gastric cancer, lung adenocarcinoma, triple-negative breast cancer, etc.

[0023] Given the current problems of complex synthesis processes, poor biocompatibility and degradation, and mediocre therapeutic effects of nanomedicines used for tumor treatment, this invention aims to prepare biomimetic composite nanomedicines through simple physical mixing and mechanical extrusion methods. Specifically, DX8951f and topoisomerase II siRNA are self-assembled into nanoparticles through simple physical mixing, and then platelet membranes are coated onto their surface through mechanical extrusion to achieve biomimetic modification. This yields nanomedicines that have both good biocompatibility and precise targeted delivery to tumor tissues. The construction principle is as follows: Figure 1 As shown.

[0024] The biomimetic composite nanomedicine utilizes the natural targeting properties of the platelet membrane to efficiently recognize and target cancer cells, achieving the co-delivery of the chemotherapeutic drug DX8951f and topoisomerase II siRNA. Specifically, the topoisomerase II siRNA effectively inhibits the expression of topoisomerase II within cancer cells, significantly enhancing the sensitivity of cancer cells to the chemotherapeutic drug DX8951f, thereby effectively inhibiting cancer cell proliferation. This biomimetic composite nanomedicine exhibits excellent anti-tumor efficacy, good biosafety, and broad prospects for clinical translation.

[0025] The key inventive point of this invention lies in the first-time combination of the topoisomerase I inhibitor DX8951f and the topoisomerase II siRNA. The siRNA inhibits the compensatory overexpression of topoisomerase II in cancer cells, overcoming DX8951f resistance at its source and achieving synergistic chemotherapy-gene therapy. Secondly, this invention avoids traditional polymer / inorganic carriers, allowing DX8951f and siRNA to directly self-assemble into nanoparticles through electrostatic interactions, thus avoiding the biosafety issues associated with carriers and achieving precise co-delivery of the two drugs. Addressing the issue of carrier-free nanoparticles being intolerant to ultrasound, this invention also employs a mechanical extrusion method to coat the surface of the carrier-free composite nanoparticles with platelet membranes, endowing the nanomedicine with active tumor targeting, long circulation, and immune escape capabilities, while protecting the drug from degradation and reducing off-target toxicity.

[0026] Compared with the prior art, the beneficial effects of the present invention are as follows: 1) The biomimetic composite nanomedicine designed according to the present invention has a simple preparation method and can effectively solve the problems that are difficult to solve with single chemotherapy or single gene therapy. Furthermore, the present invention obtains quasi-spherical composite nanomedicine particles with relatively uniform particle size and distribution by regulating the molar ratio of DX8951f and topoisomerase II siRNA and the self-assembly conditions of their composite. Platelet membranes are then coated onto the surface of the nanoparticles to obtain the biomimetic composite nanomedicine designed according to the present invention. The entire preparation process does not involve any chemical reactions and is simple to operate.

[0027] 2) The biomimetic composite nanomedicine designed in this invention can inhibit the compensatory overexpression of topoisomerase II in cancer cells through topoisomerase II siRNA, thereby increasing the sensitivity of cancer cells to DX8951f treatment; at the same time, it can prevent DX8951f from undergoing ring-opening reaction and becoming ineffective under physiological conditions.

[0028] 3) This invention utilizes a mechanical extrusion process to coat the platelet membrane onto the surface of DX / siTOPOⅡα NPs nanoparticles, achieving biomimetic modification of carrier-free composite nanomedicines. After platelet membrane biomimetic modification, the in vivo circulation time of the composite nanomedicine is prolonged, allowing it to actively target tumor sites and accumulate in tumor tissues, effectively evading immune system clearance, protecting the biological activity of DX8951f and topoisomerase II siRNA, and reducing the off-target toxicity of DX8951f.

[0029] 4) The biomimetic composite nanomedicine designed according to the present invention can simultaneously deliver DX8951f and topoisomerase II siRNA to the tumor site, avoiding the difficulty of simultaneously reaching tumor tissue due to differences in physicochemical properties, in vivo circulation, and metabolism. Furthermore, the present invention avoids the introduction of exogenous carriers, exhibiting high biosafety and promising clinical translation prospects.

[0030] In summary, the present invention provides a biomimetic composite nanomedicine, its preparation method, and its application. For the first time, a carrier-free biomimetic nanomedicine encapsulating platelet membrane-bound DX8951f and topoisomerase II siRNA has been successfully constructed. This overcomes the clinical drug resistance problem of DX8951f through chemotherapy-gene synergy. Stable biomimetic modification of carrier-free nanoparticles is achieved through a mechanical extrusion encapsulation process, significantly improving the drug's in vivo stability, tumor targeting, and biosafety. This effectively solves the shortcomings of existing technologies such as limited efficacy of single chemotherapy, difficulty in siRNA delivery, and the destruction of the carrier-free nanoparticle structure by traditional encapsulation processes. It provides a novel strategy and feasible solution for efficient and safe combination therapy of cancer. Attached Figure Description

[0031] Figure 1 A schematic diagram illustrating the construction principle of a biomimetic composite nanomedicine according to the present invention; Figure 2 TEM images of DX / siTOPOⅡα NPs (A) and biomimetic composite nanomedicine DX / siTOPOⅡα@PM NPs (B) in Example 1 of the present invention; Figure 3 The image shows the DLS diagram of the biomimetic composite nanomedicine in Example 1 of this invention. A is the hydrated particle size distribution diagram of DX / siTOPOⅡα NPs and DX / siTOPOⅡα@PM NPs. B is the particle size change curve of the biomimetic composite nanomedicine dispersed in PBS, C is the particle size dispersed in DMEM and D is the particle size change curve of the biomimetic composite nanomedicine dispersed in 10% FBS liquid environment over time. Figure 4 This is a confocal microscope image of the fluorescently labeled biomimetic composite nanomedicine in Example 1 of this invention; Figure 5 The results of the in vitro cytotoxicity evaluation of the biomimetic composite nanomedicine against various cancer cells in Example 1 of this invention are shown. Figure 6 This is the result of the biomimetic composite nanomedicine being targeted and taken up by MDA-MB-231 cells in Example 1 of the present invention; Figure 7 The results of the tumor inhibition experiment of the biomimetic composite nanomedicine and its control drug in Example 1 of this invention for treating MDA-MB-231 subcutaneous tumor model mice; Figure 8 DLS images of the biomimetic composite nanomedicines prepared in Comparative Examples 1 (A) and 2 (B) of this invention; Figure 9 TEM images of the biomimetic composite nanomedicines prepared in Comparative Examples 3(A) and 4(B) of this invention. Detailed Implementation

[0032] The present invention will be further described below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the techniques used in the embodiments are conventional practices in the art, or experimental methods recommended by the reagent kit and instrument manufacturers. Unless otherwise specified, the reagents and materials used in the embodiments are commercially available.

[0033] Example 1

[0034] 1. Isolation and purification of platelet membrane: First, mouse peripheral blood was centrifuged at 300 g for 5 min at room temperature to collect the supernatant. The supernatant obtained by repeating the centrifugation at 300 g for 5 min was rich in platelets. The supernatant was centrifuged at 2000 g for 5 min at room temperature to obtain a precipitate. The precipitate was then frozen at -80 ℃ for 30 min and then thawed at room temperature for 30 min. The platelet membrane was obtained by repeating the freeze-thaw cycle 5 times. Pre-cooled phosphate buffer solution was added to the precipitate after repeated freeze-thaw cycles. The precipitate was centrifuged at 21000 g for 15 min at 4 ℃, washed 3 times, resuspended in phosphate buffer solution, sonicated in an ice bath for 5 min, and quantified using a BCA kit. The purified platelet membrane solution was then stored at -80 ℃.

[0035] 2. Preparation of DX / siTOPOⅡα NPs: DX8951f (purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number E305074) was dissolved in DEPC water to obtain a DX8951f solution with a concentration of 64 μmol / L; topoisomerase II siRNA (siTOPOⅡα) (purchased from Suzhou Beixin Biomedical Technology Co., Ltd.) was dissolved in DEPC water to obtain a siTOPOⅡα solution with a concentration of 8 μmol / L. Equal volumes of the two drug solutions (DX8951f to siTOPOⅡα molar ratio of 8:1) were then rapidly mixed, vortexed for 5 min, and then transferred to an oscillator and shaken overnight at room temperature to form DX / siTOPOⅡα nanoparticles. TEM results of these nanoparticles are shown below. Figure 2 As shown in Figure A, the particles are quasi-spherical with an average diameter of 99.9 ± 9.4 nm.

[0036] The sense strand sequence of the topoisomerase II siRNA is 5'-CCGCGUGGUCAAAGAGUCAUU-3', and the antisense strand sequence is 5'-AAUGACUCUUUGACCACGCGG-3' (SEQ ID No. 1-2).

[0037] 3. Preparation of DX / siTOPOⅡα@PM NPs: DX / siTOPOⅡα nanoparticles were thoroughly mixed with a platelet membrane solution at a mass concentration ratio of 4.3:1. The mixture was then passed sequentially through a liposome extruder with polycarbonate membranes of 400 nm and 200 nm pore sizes, and extruded repeatedly 20 to 40 times to obtain DX / siTOPOⅡα@PMNPs nanoparticles coated with platelet membranes. After ultrafiltration concentration by centrifugation at 6000 g for 20 min at 4 ℃, the mixture was stored at 4 ℃ for later use. TEM results are shown below. Figure 2In image B, a thin gray layer is clearly visible on the surface of the quasi-spherical particles, indicating that the platelet membrane was successfully coated onto the surface of the nanoparticles and maintained their quasi-spherical structure. TEM results show that the average diameter of DX / siTOPOⅡα@PM NPs is 126.2±11.5 nm, and the platelet membrane layer thickness is approximately 12 nm.

[0038] The dimensional results of the hydrated particle size are shown in Figure 3 In the DLS analysis, the particle sizes of DX / siTOPOⅡα NPs and DX / siTOPOⅡα@PMNPs dispersed in DEPC water were 136.6±1.7 nm and 175.0±13.8 nm, respectively, with corresponding polydispersity indices of 0.177 and 0.179. The stability results of DX / siTOPOⅡα@PMNPs nanoparticles in simulated physiological environments are as follows: Figure 3 As shown in the figure, the particle size did not change significantly within 7 days when dispersed in PBS, DMEM and 10% FBS liquid environments, proving that the nanoparticles have stability.

[0039] The biomimetic modification was verified by colocalization analysis of fluorescence signals using confocal microscopy, such as... Figure 4 The results showed that the DiO-labeled platelet membrane exhibited green fluorescence, while the Cy5.5-labeled DX / siTOPOⅡα NPs showed red fluorescence. The Merge plot showed obvious yellow-orange fluorescence (overlapping area of ​​red and green fluorescence), further proving that the platelet membrane had been successfully coated on the surface of the nanoparticles, resulting in biomimetic modified DX / siTOPOⅡα@PM NPs.

[0040] 4. The toxicity of the nanoparticles to various cancer cells was detected by the MTT assay. Different concentrations of DX / siTOPOⅡα@PM NPs were co-incubated with three cancer cell lines—NCI-N87 (human gastric cancer cells), NCI-H292 (human lung adenocarcinoma cells), and MDA-MB-231 (human breast cancer cells)—for 24, 48, and 72 hours, respectively. The results are shown in the table below. Figure 5 Among them, DX / siTOPOⅡα@PM NPs showed the strongest cytotoxicity against MDA-MB-231 cells, and this cytotoxicity was dose- and time-dependent.

[0041] 5. Results of MDA-MB-231 cell uptake of the nanoparticles are shown in [the table below]. Figure 6 After co-incubating MDA-MB-231 cells with (1) DX / siTOPOⅡα NPs and (2) DX / siTOPOⅡα@PM NPs labeled with cy5.5 for 6 hours, the fluorescence intensity of intracellular cy5.5 was detected by flow cytometry and confocal microscopy. Figure 6The results showed that the fluorescence intensity of cy5.5 in MDA-MB-231 cells treated with (2) DX / siTOPOⅡα@PM NPs was stronger, confirming that the nanoparticles had a significant targeted delivery effect after platelet membrane modification.

[0042] 6. Taking the MDA-MB-231 tumor model mouse as an example, the in vivo antitumor effects of different drug treatment groups (G1: saline, G2: DX8951f, G3: DX / siScrNPs, G4: DX / siTOPOⅡα NPs, and G5: DX / siTOPOⅡα@PM NPs) are as follows: Figure 7 As shown. Figure 7 Figure A shows the tumor volume change curves of mice in each group. It can be seen that the tumor in group G5 shrank significantly, indicating that the nanoparticles have the best therapeutic effect. Figure 7 Figure B shows the weight change curves of mice in each group during the 17-day treatment period. It can be seen that the weight of mice in group G5 did not change significantly, indicating that the nanoparticles have the best biosafety. Figure 7 The image in C is a photograph of the tumor tissue obtained from an autopsy after the 17th day of treatment. It can be seen that the tumor in group G5 is relatively the smallest.

[0043] Comparative Example 1 1. The separation and purification of platelet membranes are the same as in Example 1.

[0044] 2. Preparation of DX / siTOPOⅡα NPs: DX8951f was dissolved in DEPC water to obtain a DX8951f solution with a concentration of 16 μmol / L; topoisomerase II siRNA (siTOPOⅡα) was dissolved in DEPC water to obtain a siTOPOⅡα solution with a concentration of 4 μmol / L. Equal volumes of the two drug solutions (DX8951f to siTOPOⅡα molar ratio of 4:1) were then rapidly mixed, vortexed for 5 min, and then transferred to an oscillator and shaken overnight at room temperature to form DX / siTOPOⅡα nanoparticles.

[0045] 3. Preparation of DX / siTOPOⅡα@PM NPs: DX / siTOPOⅡα nanoparticles were thoroughly mixed with a platelet membrane solution at a mass concentration ratio of 2:1. The mixture was then passed sequentially through a liposome extruder with polycarbonate membranes of 400 nm and 200 nm pore sizes, and extruded repeatedly 20 to 40 times to obtain DX / siTOPOⅡα@PM NPs with a platelet membrane-coated surface. After ultrafiltration concentration by centrifugation at 6000 g for 20 min at 4 ℃, the mixture was stored at 4 ℃ for later use. DLS test results are shown below. Figure 8As shown in Figure A, the hydrated particle sizes of DX / siTOPOⅡα NPs (4:1) and DX / siTOPOⅡα@PM NPs (4:1) are 116.6±39.2 nm and 139.0±10.3 nm, respectively, with corresponding polydispersity indices of 0.640 and 0.383, respectively, which fail to meet the requirement of polydispersity index less than 0.3 for nanomedicines.

[0046] Comparative Example 2 1. The separation and purification of platelet membranes are the same as in Example 1.

[0047] 2. Preparation of DX / siTOPOⅡα NPs: DX8951f was dissolved in DEPC water to obtain a DX8951f solution with a concentration of 160 μmol / L; topoisomerase II siRNA (siTOPOⅡα) was dissolved in DEPC water to obtain a siTOPOⅡα solution with a concentration of 10 μmol / L. Equal volumes of the two drug solutions (molar ratio of DX8951f to siTOPOⅡα 16:1) were then rapidly mixed, vortexed for 5 min, and then transferred to an oscillator and shaken overnight at room temperature to form DX / siTOPOⅡα nanoparticles.

[0048] 3. Preparation of DX / siTOPOⅡα@PM NPs: DX / siTOPOⅡα nanoparticles were thoroughly mixed with a platelet membrane solution at a mass ratio of 10:1. The mixture was then extruded repeatedly 20 to 40 times through a liposome extruder with a 400 nm pore size polycarbonate membrane to obtain DX / siTOPOⅡα@PM NPs with a platelet membrane-coated surface. After ultrafiltration concentration by centrifugation at 6000 g for 20 min at 4 ℃, the mixture was stored at 4 ℃ for later use. DLS test results are shown below. Figure 8 As shown in Figure B, the hydrated particle sizes of DX / siTOPOⅡαNPs (16:1) and DX / siTOPOⅡα@PM NPs (16:1) are 305.2±2.8 nm and 332.3±4.8 nm, respectively, with corresponding polydispersity indices of 0.286 and 0.210. Both types of nanoparticles have particle sizes greater than 300 nm, failing to meet the requirement for nanomedicines with particle sizes less than 200 nm (the particle size range for the EPR effect).

[0049] Comparative Example 3 1. The separation and purification of platelet membranes are the same as in Example 1.

[0050] 2. The preparation of DX / siTOPOⅡα NPs is the same as in Example 1.

[0051] 3. Preparation of DX / siTOPOⅡα@PM NPs: DX / siTOPOⅡα nanoparticles were thoroughly mixed with a platelet membrane solution at a mass concentration ratio of 2:1. The mixture was then passed sequentially through a liposome extruder with polycarbonate membranes of 400 nm and 200 nm pore sizes, and extruded repeatedly 20 to 40 times to obtain DX / siTOPOⅡα@PM NPs with a platelet membrane-coated surface. After ultrafiltration concentration by centrifugation at 6000 g for 20 min at 4 ℃, the mixture was stored at 4 ℃ for later use. TEM test results are shown below. Figure 9 As shown in Figure A, membrane vacuoles without nanomedicine coating are visible (circled in red), indicating that uniformly coated nanomedicine that meets quality requirements could not be obtained.

[0052] Comparative Example 4 1. The separation and purification of platelet membranes are the same as in Example 1.

[0053] 2. The preparation of DX / siTOPOⅡα NPs is the same as in Example 1.

[0054] 3. Preparation of DX / siTOPOⅡα@PM NPs: DX / siTOPOⅡα nanoparticles were thoroughly mixed with a platelet membrane solution at a mass ratio of 7:1. The mixture was then passed sequentially through a liposome extruder with polycarbonate membranes of 400 nm and 200 nm pore sizes, and extruded repeatedly 20 to 40 times to obtain DX / siTOPOⅡα@PM NPs with a platelet membrane-coated surface. After ultrafiltration concentration by centrifugation at 6000 g for 20 min at 4 ℃, the mixture was stored at 4 ℃ for later use. TEM test results are shown below. Figure 9 As shown in Figure B, uncoated nanoparticles are visible (circled in red), indicating that a uniformly coated nanomedicine meeting quality requirements could not be obtained.

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of the invention. Various variations can be made to the above embodiments of the present invention. All simple and equivalent changes and modifications made in accordance with the claims and description of this application fall within the protection scope of the claims of this patent. All aspects not described in detail in this invention are conventional technical content.

Claims

1. A biomimetic composite nanomedicine, characterized in that, It is composed of platelet membrane, chemotherapy drug DX8951f, and topoisomerase II siRNA; the chemotherapy drug DX8951f and topoisomerase II siRNA self-assemble to form composite nanoparticles through electrostatic interaction, and the platelet membrane is coated on the surface of the composite nanoparticles.

2. The biomimetic composite nanomedicine according to claim 1, characterized in that, The sense strand sequence of the topoisomerase II siRNA is 5'-CCGCGUGGUCAAAGAGUCAUU-3', and the antisense strand sequence is 5'-AAUGACUCUUUGACCACGCGG-3'.

3. A method for preparing a biomimetic composite nanomedicine as described in any one of claims 1-2, characterized in that, Includes the following steps: 1) Take peripheral blood from mice, collect platelets by centrifugation, and prepare platelet membrane solution by repeated freeze-thaw cycles; 2) DX8951f solution and topoisomerase II siRNA solution were prepared separately using DEPC water, and then self-assembled to form composite nanoparticles after mixing; 3) The composite nanoparticles are mixed with a platelet membrane solution and coated with a platelet membrane by mechanical extrusion to obtain a biomimetic composite nanomedicine.

4. The preparation method according to claim 3, characterized in that, In step 2), the molar ratio of DX8951f to topoisomerase II siRNA is (6~12):

1.

5. The preparation method according to claim 3, characterized in that, In step 3), the mass concentration ratio of the composite nanoparticles to the platelet membrane solution is (3~6):

1.

6. The preparation method according to claim 3, characterized in that, Step 1) includes: centrifuging mouse peripheral blood at room temperature, collecting the supernatant, centrifuging again at room temperature to obtain the precipitate, freezing the precipitate at -80 ℃ and thawing it at room temperature, repeating the freeze-thaw cycle 4 to 6 times; washing with pre-cooled phosphate buffer solution, centrifuging at 4 ℃, washing 3 times, resuspending in phosphate buffer solution, sonicating in an ice bath for 5 min, quantifying, and storing at -80 ℃.

7. The preparation method according to claim 3, characterized in that, In step 2), the concentration of DX8951f solution is 24-240 μmol / L, and the concentration of topoisomerase II siRNA solution is 4-20 μmol / L. The two solutions are mixed in equal volumes rapidly, vortexed for 4-6 min, and then shaken overnight at room temperature to form composite nanoparticles.

8. The preparation method according to claim 3, characterized in that, In step 3), mechanical extrusion is performed using a liposome extruder, which sequentially passes the nanoparticles through polycarbonate membranes with pore sizes of 400 nm and 200 nm, and extrudes them repeatedly 20-40 times to obtain nanoparticles DX / siTOPOⅡα@PM NPs with platelet membranes coated on the surface. After ultrafiltration and centrifugation concentration, the nanoparticles are stored at 4 °C for later use.

9. The preparation method according to claim 6, characterized in that, In step 1), the phosphate buffer solution has the following composition: 2.7 mmol / L KCl, 2.0 mmol / L KH2PO4, 137 mmol / L NaCl, 10 mmol / L Na2HPO4, pH 7.3-7.5, and is sterilized by filtration.

10. The use of a biomimetic composite nanomedicine as described in any one of claims 1-4, or a biomimetic composite nanomedicine prepared by any one of claims 5-9, in the preparation of a cancer treatment drug, characterized in that, The cancers mentioned include stomach cancer, lung adenocarcinoma, and triple-negative breast cancer.

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