Preparation method of cell membrane camouflage light-driven nano motor

Abstract

The invention belongs to the field of micro-nano motors, and provides a cell membrane camouflage light-driven nano motor and a preparation method and application thereof. The nano-motor Cu < 2-x > Se-PMO is composed of near-infrared light response copper selenide Cu < 2-x > Se nano-particles and periodic mesoporous organosilicon PMO nano-spheres, and the drug-loaded nano-motor and the cell membrane nano-vesicles are mixed and jointly pass through a microporous filter membrane to obtain the cell membrane disguise nano-motor CM (at) Cu < 2-x > Se-PMO. The nano-motor can actively find a target disease site, the cell membrane camouflage light-driven nano-motor can improve the efficiency and targeting property of drug transportation, and the preparation method has the characteristics of high loading rate, high targeting property, low cost and the like.

Description

Technical Field

[0001] This invention belongs to the field of micro-nano motors and relates to a method for preparing micro-nano motors, specifically a method for preparing a cell membrane disguised as a light-driven nanomotor. Background Technology

[0002] The dense extracellular matrix and aberrant vascular distribution of tumors hinder effective drug delivery to their core, causing conventional nanomedicines to typically accumulate at the tumor periphery and fail to reach the inner regions. This uneven drug distribution may result in incomplete tumor eradication, leading to treatment failure and recurrence.

[0003] Several micro-nanomotors have been developed. Patent application CN117653601A discloses a near-infrared II light-driven hydrogel nanomotor. Its preparation method includes: preparing Fe3O4@Cu9S8 nanoparticles, a hydrogel stock solution, and a liposome stock solution; fabricating nanoliposome vesicles encapsulating the hydrogel precursor using a microfluidic chip; Fe3O4@Cu9S8 nanoparticles aggregate at one end of the nanovesicles under the guidance of a magnetic field; and initiating a free radical polymerization reaction between acrylate groups under ultraviolet light to obtain an asymmetric hydrogel nanomotor. This nanomotor can serve as a drug carrier, loading chemotherapeutic drugs to reach tumor sites.

[0004] For example, the invention disclosed in CN118903010A relates to the preparation and application of a dual-drive nanomotor based on polymer vesicles. The nanomotor is a polymer vesicle loaded with cerium dioxide / gold nanoparticles, or a polymer vesicle loaded with cerium dioxide / gold nanoparticles and coated with a cell membrane. The method of this invention is simple to prepare, the product is easy to purify and separate, and it has good transformation prospects. The bowl-shaped polymer vesicles integrating cancer cell membranes, cerium dioxide nanoparticles, and gold nanoparticles can serve as nanomotors, driven by both H2O2 and laser, to achieve motion-enhanced photothermal therapy of tumors and alleviate photothermal damage to the superficial skin. This provides a new approach to promoting the diffusion of nanomedicines in tumors for more efficient tumor treatment.

[0005] Nanomotors, as drug carriers, can move autonomously, freeing drugs from dependence on blood flow and random diffusion. However, traditional nanomotors have certain limitations in performance, such as narrow absorption peaks in the near-infrared region, low drug loading capacity, non-degradability, and low ability to interact with biological interfaces. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a method for preparing a cell membrane disguised as a light-driven nanomotor.

[0007] To overcome the above problems, this invention provides a cell membrane-masked light-driven nanomotor and its preparation method. Specifically, the nanomotor (Cu... 2-x Se-PMO is composed of near-infrared light-responsive copper selenide (Cu). 2-x Composed of Se nanoparticles and periodic mesoporous organosilicon (PMO) nanospheres, the propulsion device Cu 2-x Se offers flexible wavelength selection (e.g., 808, 1064, or 1310 nm) and efficient photothermal conversion capabilities. Furthermore, the mesoporous structure of PMO greatly facilitates drug loading, while cell membrane encapsulation enhances the interaction between nanomotors and biological interfaces. Therefore, Cu... 2-x Se-PMO nanomotors have promising applications in the field of drug delivery.

[0008] In a first aspect, the present invention provides a nanomotor, wherein the nanomotor is Cu 2-x Se-PMO, composed of near-infrared light-responsive copper selenide (Cu). 2-x It consists of Se nanoparticles and periodic mesoporous organosilicon (PMO) nanospheres.

[0009] Janus motors are micro-nano drive technologies based on the Janus structure. They achieve autonomous movement through chemical or external energy sources and are primarily used in the fields of biomedicine and environmental remediation. The Janus structure generates concentration gradients or bubbles through catalytic reactions, which can increase the speed several times at the oil-water interface.

[0010] Periodic mesoporous organosilicon (PMO) is a silica material with a periodic mesoporous structure and containing organic groups. Its core characteristics are the ordered nature of the mesoporous structure and its organic functionalization. In its mesoporous structure, the pore size is between 2 and 50 nanometers, arranged in a periodic manner, and organic groups are introduced through copolymerization or post-grafting methods.

[0011] The nanomotor of this invention can form a cell membrane camouflaged light-driven nanomotor together with cell membrane nanovesicles; it can actively seek target disease sites and optimize delivery targeting and therapeutic effect.

[0012] In a second aspect, the present invention provides a cell membrane camouflaged light-driven nanomotor, wherein the aforementioned nanomotor is loaded with a drug in a cell membrane nanovesicle of a target cell.

[0013] Preferably, the cell membrane-masked light-driven nanomotor can be obtained according to the following preparation method.

[0014] Thirdly, this invention provides a method for preparing a cell membrane-masked light-driven nanomotor. The nanomotor (Cu...) 2-x Se-PMO is composed of near-infrared light-responsive copper selenide (Cu).2-x The Janus motor is composed of Se nanoparticles and periodic mesoporous organosilicon (PMO) nanospheres, and utilizes Cu... 2-x The photothermal effect of Se absorbing light energy creates a temperature gradient on the surface of mesoporous organosilicon spheres, leading to displacement via autothermal migration. A cell membrane-disguised nanomotor (CM@Cu) is obtained by mixing drug-loaded nanomotors and cell membrane nanovesicles and passing them through a microporous filter membrane using a membrane extrusion method. 2-x Se-PMO.

[0015] This invention provides a method for preparing a cell membrane-masked light-driven nanomotor, wherein the nanomotor is made of Cu. 2-x Se-PMO is composed of near-infrared light-responsive copper selenide (Cu). 2-x Composed of Se nanoparticles and periodically mesoporous organosilicon PMO nanospheres, a drug-loaded nanomotor and cell membrane nanovesicles were mixed and passed through a microporous filter membrane to obtain a cell membrane-masked nanomotor CM@Cu. 2- x Se-PMO.

[0016] Preferably, the method for preparing the cell membrane-masked light-driven nanomotor includes the following steps: Step S1, Cu 2-x Preparation of Se nanoparticles: Polyvinylpyrrolidone was dissolved in deionized water, and then selenium dioxide solution and vitamin C solution were added to form mixture I. After stirring, vitamin C solution and copper sulfate pentahydrate were added to mixture I. Finally, the mixture was stirred continuously at room temperature for at least 10 hours, and the stirred product was washed by centrifugation. Step S2, Cu 2-x Preparation of Se-PMO nanomotors: Concentrated ammonia, ethanol, and organosilane were added sequentially to a hexadecyltrimethylammonium bromide aqueous solution to form mixture II. After stirring, mixture II was centrifuged, washed, and the template was removed. Then it was washed with ethanol or water. Step S3, Drug Loading: Dissolve the target drug and add Cu 2-x Se-PMO nanomotors, ultrasonicated and stirred, yield drug-loaded Cu. 2-x Se-PMO nanomotors; Step S4, Preparation and purification of cell membrane nanovesicles: Target cells are lysed with PBS hypotonic buffer, sonicated, and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. Step S5, CM@Cu 2-x Preparation of Se-PMO: The drug-loaded Cu... 2-x Se-PMO was mixed with purified cell membrane nanovesicles and then sequentially passed through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO.

[0017] The steps S1-S5 described above do not necessarily have to be performed in the above order; they only need to conform to the logic of conventional technical means in this field. For example, step S4 can be completed before or after steps S1-S3, but it must be completed before step S5.

[0018] Preferably, in step S1, the amounts of polyvinylpyrrolidone solution, selenium dioxide solution, and vitamin C solution added are 50-100 ml, 0.5-2 ml, and 1-3 ml, respectively. The polyvinylpyrrolidone solution is prepared by adding 20-40 g of polyvinylpyrrolidone per 10 ml of deionized water. The concentration of the selenium dioxide solution is 0.2-0.4 M, and the concentration of the vitamin C solution is 0.2-0.4 M. After stirring, 3-5 ml of vitamin C solution and 0.5-2 ml of copper sulfate pentahydrate are added to mixture I. In a preferred embodiment of the present invention, Cu 2- x The preparation method of Se nanoparticles is as follows: polyvinylpyrrolidone is dissolved in 70 mL of deionized water, followed by the addition of 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M), and stirred for 15 minutes. Then, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) are added to the mixture. Finally, the mixture is stirred continuously at room temperature for 30 hours. The product is then washed twice and centrifuged at 10,000 rpm. It is then stored at 4 °C for later use.

[0019] Preferably, in step S2, 10-30 mg of hexadecyltrimethylammonium bromide is dissolved in 10-30 ml of deionized water to form an aqueous solution of hexadecyltrimethylammonium bromide. The amount of concentrated ammonia added is 200-400 μL, the amount of ethanol added is 50-200 μL, and the amount of organosilane added is 20-40 μL. The temperature at which the ethanol-ammonium nitrate solution is stirred to remove the template is 45-70°C. In a preferred embodiment of the present invention, Cu 2-x The preparation method of Se-PMO nanomotors is as follows: 25 mg of cetyltrimethylammonium bromide in 20 mL of deionized water, then 200-400 μL of concentrated ammonia, 100 μL of ethanol, and 20-40 μL of organosilane are added sequentially. The mixture is stirred at room temperature for 1-3 h, centrifuged, and washed several times with ethanol and water. The template is removed by stirring the ethanol-ammonium nitrate solution at 60℃ for 24 h, and then washed several times with ethanol and water.

[0020] Better results were obtained by purifying cell membrane nanovesicles using iodixanol density gradient centrifugation.

[0021] The method specifically includes the following steps: (1) Cu 2-xPreparation of Se nanoparticles: Polyvinylpyrrolidone was dissolved in 70 mL of deionized water. Then, 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M) were added, and the mixture was stirred for 15 minutes. Next, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) were added to the mixture. After stirring continuously at room temperature for 30 hours, the product was washed twice and centrifuged at 10,000 rpm. It was then stored at 4 °C for later use.

[0022] (2) Cu 2-x Preparation of Se-PMO nanomotors: 25 mg of cetyltrimethylammonium bromide was added to 20 mL of deionized water, followed by the sequential addition of 200-400 μL of concentrated ammonia, 100 μL of ethanol, and 20-40 μL of organosilane. The mixture was stirred at room temperature for 1-3 h, centrifuged, and washed several times with ethanol and water. The template was removed by stirring the ethanol-ammonium nitrate solution at 60 °C for 24 h, followed by washing several times with ethanol and water, and stored at 4 °C for later use.

[0023] (3) Drug loading: Dissolve a specific drug in a solvent and add Cu 2-x Se-PMO nanomotors, ultrasonication and stirring, and washing several times to obtain drug-loaded Cu 2-x Se-PMO nanomotors.

[0024] (4) Preparation and purification of cell membrane nanovesicles: The collected cells were lysed with PBS hypotonic buffer, sonicated, and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. They were purified by density gradient centrifugation with iodixanol for 18 h.

[0025] (5) CM@Cu 2-x Preparation of Se-PMO: The drug-loaded Cu... 2-x Se-PMO was mixed with purified cell membrane nanovesicles at a mass ratio of 1:5 and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO, centrifuged and washed several times, stored at -80℃.

[0026] PBS is phosphate buffer.

[0027] Organosilanes are compounds formed by silicon-hydrogen bonds (Si-H) or silane groups (Si-R) bonded to organic groups, such as silanes (SiH4) and silanes (Si2H6). Their core characteristic is the direct bond between silicon and hydrogen or an organic group; they are chemically reactive and readily undergo hydrogenation or oxidation reactions. Organosilicon precursors refer to organosilicon compounds used in the synthesis of ceramics, semiconductor materials, etc. These substances are typically used as raw materials in ceramic additive manufacturing or chemical vapor deposition (CVD), where they decompose at high temperatures to generate inorganic ceramic materials. Organosilicon precursors possess high chemical stability, controllability, and solubility, thus becoming important raw materials for the preparation of organosilicon materials. There are many types of organosilicon precursors, commonly including siloxanes, silanols, silamides, and silicates.

[0028] In the fabrication of microencapsulated nanomotors, it is typically necessary to remove the template used for molding to form the final cavity structure or pores. The pores or cavities formed after template removal can serve as drug loading areas. Template removal methods include: Physical removal: This involves removing the template material through chemical etching or mechanical stripping. For example, in metal-organic framework (MOF) micro / nano robots, after synthesis using a designable photolithographic template, the template needs to be removed using a specific process to form a customizable cavity structure. Chemical dissolution: This involves dissolving the template using solvents or specific chemical reagents. For example, in the fabrication of light-driven nanomotors, the asymmetric structure of polymer vesicles is transformed into a motor after near-infrared laser irradiation. This may involve removing template materials used in the assembly process. Better, Cu 2-x The particle size of Se nanoparticles is between 30 and 50 nm.

[0029] Ideally, the size of the PMO nanospheres is between 100 and 200 nm.

[0030] The best-quality PMO nanospheres have mesopore sizes between 2 and 10 nm.

[0031] Preferably, the organosilane precursor of PMO is one or more of the following: 1,2-bis(triethoxysilyl)ethane, bis-[3-(triethoxysilyl)propyl]-disulfide, bis-[3-(triethoxysilyl)propyl]-tetrasulfide, and 1,4-bis(triethoxysilyl)benzene.

[0032] Better, Cu 2-x The size of Se-PMO nanomotors is between 150 and 300 nm.

[0033] Better, Cu 2-x The drugs loaded onto the Se-PMO nanomotors are one or more of the following: small molecule chemotherapy drugs, nucleic acid drugs, and protein drugs.

[0034] Better, Cu 2-xThe drug loaded onto the Se-PMO nanomotor is one or more of the following: small molecule chemotherapeutic drugs, nucleic acid drugs, and protein drugs. Examples include various commercially available drugs, siRNA, nucleic acids, peptides, etc. In a preferred embodiment of the invention, the drug is DOX (doxorubicin), a broad-spectrum antitumor drug.

[0035] Ideally, the cell membranes used for camouflaging nanomotors can be derived from biological or plant cells, or simulated cell membranes, exosomes, vesicles, etc.

[0036] A preferred method for preparing cell membrane-masked nanomotors is membrane extrusion.

[0037] This invention provides a cell membrane-camouflaged light-driven nanomotor, its preparation method, and its applications. Nanomotors can actively seek target disease sites, optimizing delivery targeting and therapeutic efficacy. The camouflage of the cell membrane provides a platform for the nanomotor to interact with the biological matrix, enabling it to overcome immune clearance and navigate complex biological environments. This invention loads micro / nanomotor drugs into cell membrane nanovesicles. The drug carrier prepared by this method has the characteristics of high loading rate, high targeting, and low cost. Attached Figure Description

[0038] To more clearly illustrate the technical solutions of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, each drawing described below is for a part of the embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0039] Figure 1 It is Cu 2-x TEM image of Se nanoparticles. TEM (Transmission Electron Microscopy) is a microscope that uses an electron beam to image microscopy. It can observe fine structures smaller than 0.2 micrometers that cannot be seen by optical microscopes; these structures are called submicrostructures or ultrastructures.

[0040] Figure 2 It is Cu 2-x SEM image of Se-PMO nanomotors. SEM (Scanning Electron Microscope) images are high-resolution images generated by scanning electron microscopy, used to observe the microscopic morphology and structure of sample surfaces.

[0041] Figure 3 It is Cu 2-x TEM image of Se-PMO nanomotors. Detailed Implementation

[0042] The technical solution will be clearly and completely described below through embodiments of this application. Obviously, the described embodiments are only some preferred embodiments of this application, and not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the protection scope of this application.

[0043] Example 1 (1) Cu 2-x Preparation of Se: 160 mg of polyvinylpyrrolidone was dissolved in 70 mL of deionized water. Then, 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M) were added, and the mixture was stirred for 15 minutes. Next, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) were added to the mixture. After stirring continuously at room temperature for 30 hours, the product was washed twice and centrifuged at 10,000 rpm to obtain Cu. 2-x Se nanoparticles such as Figure 1 As shown. Store at 4℃ for later use.

[0044] (2) Cu 2-x Preparation of Se-PMO nanomotors: 25 mg of cetyltrimethylammonium bromide was added to 20 mL of deionized water, followed by the sequential addition of 300 μL of concentrated ammonia, 100 μL of ethanol, 20 μL of 1,2-bis(triethoxysilyl)ethane, and 20 μL of bis-[3-(triethoxysilyl)propyl]-disulfide. The mixture was stirred at room temperature for 2 h, centrifuged, and washed several times with ethanol and water. The template was removed by stirring the ethanol-ammonium nitrate solution at 60 °C for 24 h, followed by washing several times with ethanol and water to obtain Cu. 2-x Se-PMO nanomotors, such as Figure 2 and Figure 3 As shown. Store at 4℃ for later use.

[0045] (3) PDL1-siRNA drug loading: PDL1-siRNA was mixed evenly with cationic polymer polyethyleneimine (MW=1800) (PEI-siRNA), and PEI-siRNA was mixed with Cu 2-x Se-PMO nanomotors were mixed at a mass ratio of 1:20, allowed to stand at room temperature for 10 min, and washed several times with enzyme-free water to obtain Cu. 2-x Se-PMO-siRNA.

[0046] (4) Preparation and purification of erythrocyte membrane nanovesicles: Whole blood was collected and centrifuged at 800 g to remove plasma components. Red blood cells were obtained by washing three times with PBS. The red blood cells were resuspended in hypotonic PBS buffer and lysed at 4°C for 2 h. The erythrocyte membrane (RBCM) was collected by centrifugation at 20,000 g, sonicated, and then passed through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. The nanovesicles were washed three times with PBS. The erythrocyte membrane nanovesicles were purified by density gradient centrifugation with iodixanol for 18 h and stored at -80°C for later use.

[0047] (5) RBCM@Cu 2-x Preparation of Se-PMO: Cu 2-x Se-PMO was mixed with purified erythrocyte membrane nanovesicles at a mass ratio of 1:5 and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO, centrifuged and washed three times, yielded RBCM@Cu. 2- x Se-PMO was prepared and stored at -80℃.

[0048] Example 2 (1) Cu 2-x Preparation of Se: 160 mg of polyvinylpyrrolidone was dissolved in 70 mL of deionized water. Then, 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M) were added, and the mixture was stirred for 15 minutes. Next, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) were added to the mixture. After stirring continuously at room temperature for 30 hours, the product was washed twice and centrifuged at 10,000 rpm. It was then stored at 4°C for later use.

[0049] (2) Cu 2-x Preparation of Se-PMO nanomotors: 25 mg of cetyltrimethylammonium bromide was added to 20 mL of deionized water, followed by the sequential addition of 300 μL of concentrated ammonia, 100 μL of ethanol, 20 μL of 1,2-bis(triethoxysilyl)ethane, and 20 μL of bis-[3-(triethoxysilyl)propyl]-disulfide. The mixture was stirred at room temperature for 2 h, centrifuged, and washed several times with ethanol and water. The template was removed by stirring the ethanol-ammonium nitrate solution at 60 °C for 24 h, washed several times with ethanol and water, and stored at 4 °C for later use.

[0050] (3) mRNA drug loading: IL-12 mRNA was mixed evenly with the cationic polymer polyethyleneimine (MW=1800) (PEI-RNA), and PEI-mRNA was then mixed with Cu 2-x Se-PMO nanomotors were mixed at a mass ratio of 1:20, allowed to stand at room temperature for 10 min, and washed several times with enzyme-free water to obtain Cu. 2-xSe-PMO-mRNA.

[0051] (4) Preparation and purification of erythrocyte membrane nanovesicles: Whole blood was collected and centrifuged at 800 g to remove plasma components. Red blood cells were obtained by washing three times with PBS. The red blood cells were resuspended in hypotonic PBS buffer and lysed at 4°C for 2 h. The erythrocyte membrane (RBCM) was collected by centrifugation at 20,000 g, sonicated, and then passed through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. The nanovesicles were washed three times with PBS. The erythrocyte membrane nanovesicles were purified by density gradient centrifugation with iodixanol for 18 h and stored at -80°C for later use.

[0052] (5) RBCM@Cu 2-x Preparation of Se-PMO: Cu 2-x Se-PMO was mixed with purified erythrocyte membrane nanovesicles at a mass ratio of 1:5 and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO, centrifuged and washed three times, yielded RBCM@Cu. 2- x Se-PMO was prepared and stored at -80℃.

[0053] Example 3 (1) Cu 2-x Preparation of Se: 160 mg of polyvinylpyrrolidone was dissolved in 70 mL of deionized water. Then, 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M) were added, and the mixture was stirred for 15 minutes. Next, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) were added to the mixture. After stirring continuously at room temperature for 30 hours, the product was washed twice and centrifuged at 10,000 rpm. It was then stored at 4°C for later use.

[0054] (2) Cu 2-x Preparation of Se-PMO nanomotors: 25 mg of cetyltrimethylammonium bromide was added to 20 mL of deionized water, followed by the sequential addition of 300 μL of concentrated ammonia, 100 μL of ethanol, 20 μL of 1,2-bis(triethoxysilyl)ethane, and 20 μL of bis-[3-(triethoxysilyl)propyl]-disulfide. The mixture was stirred at room temperature for 2 h, centrifuged, and washed several times with ethanol and water. The template was removed by stirring the ethanol-ammonium nitrate solution at 60 °C for 24 h, washed several times with ethanol and water, and stored at 4 °C for later use.

[0055] (3) DOX drug loading: DOX and Cu 2-x Se-PMO nanomotors were mixed and stirred at room temperature in the dark for 24 h, followed by washing several times with deionized water to obtain Cu. 2-x Se-PMO-DOX.

[0056] (4) Preparation and purification of erythrocyte membrane nanovesicles: Whole blood was collected and centrifuged at 800 g to remove plasma components. Red blood cells were obtained by washing three times with PBS. The red blood cells were resuspended in hypotonic PBS buffer and lysed at 4°C for 2 h. The erythrocyte membrane (RBCM) was collected by centrifugation at 20,000 g, sonicated, and then passed through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. The nanovesicles were washed three times with PBS. The erythrocyte membrane nanovesicles were purified by density gradient centrifugation with iodixanol for 18 h and stored at -80°C for later use.

[0057] (5) RBCM@Cu 2-x Preparation of Se-PMO: Cu 2-x Se-PMO was mixed with purified erythrocyte membrane nanovesicles at a mass ratio of 1:5 and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO, centrifuged and washed three times, yielded RBCM@Cu. 2- x Se-PMO was prepared and stored at -80℃.

[0058] Example 4 (1) Cu 2-x Preparation of Se: 160 mg of polyvinylpyrrolidone was dissolved in 70 mL of deionized water. Then, 1 mL of selenium dioxide solution (0.2 M) and 3 mL of vitamin C solution (0.4 M) were added, and the mixture was stirred for 15 minutes. Next, 4 mL of vitamin C solution (0.4 M) and 1 mL of copper sulfate pentahydrate (0.4 M) were added to the mixture. After stirring continuously at room temperature for 30 hours, the product was washed twice and centrifuged at 10,000 rpm. It was then stored at 4°C for later use.

[0059] (2) Cu 2-x Preparation of Se-PMO nanomotors: 25 mg of cetyltrimethylammonium bromide was added to 20 mL of deionized water, followed by the sequential addition of 300 μL of concentrated ammonia, 100 μL of ethanol, 20 μL of 1,2-bis(triethoxysilyl)ethane, and 20 μL of bis-[3-(triethoxysilyl)propyl]-disulfide. The mixture was stirred at room temperature for 2 h, centrifuged, and washed several times with ethanol and water. The template was removed by stirring the ethanol-ammonium nitrate solution at 60 °C for 24 h, washed several times with ethanol and water, and stored at 4 °C for later use.

[0060] (3) DOX drug loading: DOX and Cu 2-x Se-PMO nanomotors were mixed and stirred at room temperature in the dark for 24 h, followed by washing several times with deionized water to obtain Cu. 2-x Se-PMO-DOX.

[0061] (4) Preparation and purification of tumor cell membrane nanovesicles: 4T1 breast cancer cells were cultured to the logarithmic growth phase, digested with trypsin, washed, and then added to HEPES buffer containing 0.25 M sucrose and 1 mM MgCl2. The cell suspension was sonicated on ice for 5 min, and the tumor cell membrane (CCM) was collected by centrifugation at 20,000 g. The cells were washed several times with PBS and then passed through 1 μm and 400 nm nuclear pore membranes to form nanovesicles. The cells were washed three times with PBS. The tumor cell membrane nanovesicles were purified by density gradient centrifugation with iodixanol for 18 h and stored at -80°C for later use.

[0062] (5) CCM@Cu 2-x Preparation of Se-PMO: Cu 2-x Se-PMO was mixed with purified tumor cell membrane nanovesicles at a mass ratio of 1:5 and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to obtain CCM@Cu. 2-x Se-PMO, centrifuged and washed three times, yielded RBCM@Cu. 2-x Se-PMO was prepared and stored at -80℃.

[0063] In the description of this application, the terms "comprising," "including," or any other variations thereof are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising a reference structure" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.

[0064] It should be noted that in this article, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations.

[0065] The embodiments described above are merely specific implementations of this application, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be conceived by those skilled in the art within the scope of the technology disclosed in this application without creative effort should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of protection of the claims in this application.

Claims

1. A method for preparing a cell membrane-masked light-driven nanomotor, characterized in that, Nanomotors Cu 2-x Se-PMO is composed of near-infrared light-responsive copper selenide (Cu). 2-x Composed of Se nanoparticles and periodically mesoporous organosilicon PMO nanospheres, a drug-loaded nanomotor and cell membrane nanovesicles were mixed and passed through a microporous filter membrane to obtain a cell membrane-masked nanomotor CM@Cu. 2-x Se-PMO.

2. The method for preparing a cell membrane-masked light-driven nanomotor according to claim 1, characterized in that, Specifically, it includes the following steps: Step S1, Cu 2-x Preparation of Se nanoparticles: Polyvinylpyrrolidone was dissolved in deionized water, and then selenium dioxide solution and vitamin C solution were added to form mixture I. After stirring, vitamin C solution and copper sulfate pentahydrate were added to mixture I. Finally, the mixture was stirred continuously at room temperature for at least 10 hours, and the stirred product was washed by centrifugation. Step S2, Cu 2-x Preparation of Se-PMO nanomotors: Concentrated ammonia, ethanol, and organosilane were added sequentially to a hexadecyltrimethylammonium bromide aqueous solution to form mixture II. After stirring, mixture II was centrifuged, washed, and the template was removed. Then it was washed with ethanol or water. Step S3, Drug Loading: Dissolve the target drug and add Cu 2-x Se-PMO nanomotors, ultrasonicated and stirred, yield drug-loaded Cu. 2-x Se-PMO nanomotors; Step S4, Preparation and purification of cell membrane nanovesicles: Target cells are lysed with PBS hypotonic buffer, sonicated, and then passed sequentially through 1 μm and 400 nm nuclear pore membranes to form cell membrane nanovesicles. Step S5, CM@Cu 2-x Preparation of Se-PMO: The drug-loaded Cu... 2-x Se-PMO was mixed with purified cell membrane nanovesicles and then sequentially passed through 1 μm and 400 nm nuclear pore membranes to obtain CM@Cu. 2-x Se-PMO.

3. The method for preparing a cell membrane-masked light-driven nanomotor according to claim 1, characterized in that, Cu 2- x The particle size of Se nanoparticles is between 30-50 nm; The size of the PMO nanospheres is between 100 and 200 nm; Cu 2-x The size of Se-PMO nanomotors is between 150 and 300 nm; The organosilane precursor of PMO is one or more of 1,2-bis(triethoxysilyl)ethane, bis-[3-(triethoxysilyl)propyl]-disulfide, bis-[3-(triethoxysilyl)propyl]-tetrasulfide, or 1,4-bis(triethoxysilyl)benzene.

4. The method for preparing a cell membrane-masked light-driven nanomotor according to claim 1, characterized in that, The drug-loaded nanomotors can carry one or more of the following: small molecule chemotherapy drugs, nucleic acid drugs, or protein drugs; the cell membranes used for the nanomotors can be derived from cells of all animals or plants. Cell membrane-masked nanomotors were prepared using a membrane extrusion method.

5. The method for preparing a cell membrane-masked light-driven nanomotor according to claim 2, characterized in that, In step S1, the amounts of polyvinylpyrrolidone solution, selenium dioxide solution, and vitamin C solution added are 50-100 ml, 0.5-2 ml, and 1-3 ml, respectively. The polyvinylpyrrolidone solution is prepared by adding 20-40 g of polyvinylpyrrolidone per 10 ml of deionized water. The concentration of the selenium dioxide solution is 0.2-0.4 M, and the concentration of the vitamin C solution is 0.2-0.4 M. After stirring, 3-5 ml of vitamin C solution and 0.5-2 ml of copper sulfate pentahydrate are added to mixture I. In step S2, 10-30 mg of cetyltrimethylammonium bromide is dissolved in 10-30 ml of deionized water to form an aqueous solution of cetyltrimethylammonium bromide. The amount of concentrated ammonia added is 200-400 μL, the amount of ethanol added is 50-200 μL, and the amount of organosilane added is 20-40 μL. The temperature at which the template is removed by stirring with the ethanol ammonium nitrate solution is 45-70℃. In step S4, cell membrane nanovesicles are purified by density gradient centrifugation with iodixanol.

6. The method for preparing a cell membrane-masked light-driven nanomotor according to claim 5, characterized in that, The mesopore size of PMO nanospheres is between 2 and 10 nm.

7. A nanomotor, characterized in that, The nanomotor mentioned is Cu 2-x Se-PMO, copper selenide Cu that responds to near-infrared light 2-x It consists of Se nanoparticles and periodic mesoporous organosilicon PMO nanospheres.

8. A cell membrane camouflaged light-driven nanomotor, characterized in that, The nanomotor of claim 7 contains a drug-loaded nanovesicle within the cell membrane of the target cell.

9. The cell membrane camouflaged light-driven nanomotor according to claim 8, characterized in that, Obtained according to the preparation method described in claim 1.

10. The application of the nanomotor according to claim 7, characterized in that, Using membrane extrusion, drug-loaded nanomotors are combined with cell membrane nanovesicles to form cell membrane camouflaged light-driven nanomotors; these nanomotors actively seek target disease sites, optimizing delivery targeting and therapeutic efficacy.

Images