pH / redox dual-responsive nanocarrier for ovarian cancer and preparation method thereof

Abstract

The application belongs to the technical field of nano-carrier, and particularly relates to a pH / oxidation-reduction dual-response nano-carrier for ovarian cancer and a preparation method thereof; the pH / oxidation-reduction dual-response nano-carrier for ovarian cancer comprises the following raw materials in parts by weight: 5-15 parts of functionalized modified mesoporous silica nanoparticles, 5-15 parts of functionalized biomimetic cell membrane vesicles, and 2-8 parts of doxorubicin hydrochloride; the application constructs a reduction-response drug release site by grafting a disulfide bond in the mesoporous silica as a skeleton, forms an intelligent response network on the surface by cross-linking polyhistidine through a disulfide bond, releases the drug only when a weak acid and a high reduction environment coexist in the tumor, and remains stable under single stimulation; meanwhile, the ovarian cancer cell membrane is modified by anchoring cholesterol to GALA peptide to prepare biomimetic vesicles and coat the drug-loaded particles on the outer layer, the circulation time is prolonged and the tumor enrichment is enhanced by utilizing homologous targeting and immune escape characteristics, and deep matrix penetration is realized by means of the GALA peptide.

Description

Technical Field

[0001] This invention belongs to the field of nanocarrier technology, specifically relating to a pH / redox dual-responsive nanocarrier for ovarian cancer and its preparation method. Background Technology

[0002] Currently, the mainstream clinical treatment for ovarian cancer is surgery combined with chemotherapy, with chemotherapy playing a central role in suppressing both primary and metastatic lesions. However, traditional free chemotherapy drugs suffer from poor in vivo circulation stability, weak tumor-targeting enrichment ability, and severe non-specific damage to normal tissues, easily leading to significant systemic toxic side effects. Furthermore, ovarian cancer cells readily initiate drug efflux and damage repair mechanisms, resulting in frequent chemotherapy resistance, significantly reducing clinical treatment efficacy, and are a core contributing factor to recurrence and poor prognosis after ovarian cancer treatment.

[0003] Among them, pH / redox dual-response nanocarriers constructed based on the weakly acidic tumor microenvironment and the highly reducing environment within tumor cells can achieve targeted recognition of tumor sites and controlled intracellular drug release, effectively improving the tumor selectivity and intracellular accumulation of chemotherapy drugs, and showing significant application advantages compared to traditional nanocarriers. However, existing conventional pH / redox dual-response nanodelivery systems still have many inherent technical defects, making it difficult to meet the clinical needs of precise and efficient treatment of ovarian cancer. First, existing dual-response carriers mostly adopt single-condition triggering or logical response mechanisms for drug release. Physiological blood circulation or weakly abnormal microenvironments in normal tissues can trigger premature drug leakage, which not only reduces the effective utilization rate of drugs but also has certain systemic toxic side effects, and cannot achieve precise targeting and site-specific release of drugs. Second, conventional dual-response carriers only have simple drug release regulation functions and lack stable physical barrier structures, making them prone to structural perturbations and premature activation of responding groups in the complex in vivo environment. Therefore, developing an integrated biomimetic intelligent nanodelivery system that simultaneously possesses homologous targeting and deep penetration capabilities is a technical problem that urgently needs to be solved in the field of precision targeted chemotherapy for ovarian cancer. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a pH / redox dual-responsive nanocarrier for ovarian cancer and its preparation method. Using mesoporous silica as the carrier framework, redox-responsive drug release sites are constructed sequentially by grafting disulfide bonds onto the inner walls of the pores. A three-dimensional dense network is formed by cross-linking polyhistidines on the surface, achieving stable drug locking in the normal physiological environment and precise drug release specific to the tumor microenvironment. Furthermore, GALA penetrating peptides are stably embedded into the ovarian cancer cell membrane via cholesterol anchoring, preparing functionalized biomimetic cell membrane vesicles that are then coated onto the surface of drug-loaded nanoparticles. This constructs an integrated intelligent drug delivery system with homologous targeting, immune escape, long circulation, and deep tumor penetration characteristics. This effectively solves the technical defects of traditional drug delivery systems, such as low drug release accuracy, poor stability, insufficient targeted penetration, and significant off-target toxicity. It can significantly increase drug accumulation in ovarian cancer cells, reverse chemotherapy resistance, and efficiently inhibit tumor proliferation and metastasis, while also possessing excellent biosafety and clinical translational application value.

[0005] To address the shortcomings of existing technologies, the present invention adopts the following technical solution: This invention provides a pH / redox dual-responsive nanocarrier for ovarian cancer, wherein the pH / redox dual-responsive nanocarrier for ovarian cancer comprises the following raw materials in parts by weight: 5-15 parts of functionalized modified mesoporous silica nanoparticles, 5-15 parts of functionalized biomimetic cell membrane vesicles, and 2-8 parts of doxorubicin hydrochloride. The functionalized biomimetic cell membrane vesicles comprise the following raw materials in the following mass ratio: SKOV-3 human ovarian cancer cell line: GALA peptide: cholesterol hemisuccinate = 1:0.05-0.2:0.02; The method for preparing the functionalized biomimetic cell membrane vesicles includes the following steps: (1) Culture of SKOV-3 ovarian cancer cells: SKOV-3 human ovarian cancer cell line was cultured in McCoy's 5A complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics and cultured in a 37℃, 5% CO2 incubator until the cell confluence reached 80-90% for cell membrane extraction. (2) Synthesis of cholesterol-anchored GALA peptide: Pure GALA peptide was prepared by solid-phase synthesis. Cholesterol hemisuccinate was weighed and dissolved in anhydrous DMF solvent. 0.3-0.5 mL of anhydrous DMF was added to the EDC / NHS activation system for every 1 μmol of GALA peptide. The carboxyl group was activated by stirring at room temperature for 30 minutes. Then, GALA peptide was added and the reaction was carried out by stirring at room temperature for 12 h for amidation reaction. After the reaction was completed, the high-purity cholesterol-anchored GALA peptide was obtained by high-performance liquid chromatography. After freeze-drying, it was sealed and stored at -20℃ for later use. (3) SKOV-3 cell membrane extraction: Collect SKOV-3 cells cultured to the logarithmic growth phase, with a total cell count of 5 × 10⁻⁶. 7 Each cell was washed twice with pre-cooled PBS, and then lysed on ice for 30 minutes with membrane protein extraction buffer containing protease inhibitor (PMSF). After homogenization using a Dunns homogenizer, the cells were centrifuged at 4°C and 1000×g for 10 minutes to remove the nuclei and unbroken cells. The supernatant was then centrifuged at 4°C and 10000×g for 30 minutes. The precipitate was cell membrane fragments. The membrane precipitate was resuspended in pre-cooled PBS to obtain a cell membrane suspension. (4) GALA peptide embedding into the cell membrane: The cell membrane suspension was mixed with cholesterol-anchored GALA peptide (the mass ratio of the GALA peptide to the membrane protein was 1:10) and incubated on ice for 30 minutes to allow the GALA peptide to insert into the cell membrane through the cholesterol anchor. After incubation, the mixture was centrifuged at 4°C and 10000×g for 10 minutes to remove the un-embedded free cholesterol-anchored GALA peptide. The mixture was then resuspended in PBS and washed twice to obtain functionalized biomimetic cell membrane vesicles.

[0006] The functionalized modified mesoporous silica nanoparticles comprise the following raw materials in parts by weight: 0.1-0.3 parts hexadecyltrimethylammonium bromide (CTAB), 0.8-1.5 parts tetraethyl orthosilicate (TEOS), 0.2-0.6 parts 3-aminopropyltriethoxysilane, 0.3-0.9 parts 3,3'-dithiodipropionic acid, 0.2-0.7 parts 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), 0.1-0.4 parts N-hydroxysuccinimide (NHS), and 0.5-1.2 parts polyhistidine; The method for preparing the functionalized modified mesoporous silica nanoparticles includes the following steps: i. Dissolve CTAB in ultrapure water at a ratio of 1g:450-500mL. Adjust the pH to 11.0 with 2M NaOH solution. Heat and stir in an 80℃ water bath. After the temperature stabilizes, slowly add TEOS dropwise while stirring continuously for 2 hours. After the reaction is complete, collect the white precipitate by centrifugation, wash, and disperse the obtained product in an ethanol solution containing 6 g / L NH4NO3. Reflux and extract at 60℃ for 12 hours to remove the CTAB template. Collect the product by centrifugation, wash, and vacuum dry at 40℃ to obtain MSN. ii. Weigh MSN and disperse it in anhydrous ethanol at a ratio of 10-15 mg: 1 mL. Add 3-aminopropyltriethoxysilane and reflux at 80 °C for 24 hours under nitrogen protection. After the reaction is complete, collect the product by centrifugation, wash, and dry under vacuum at 40 °C to obtain aminated MSN (MSN-NH2). iii. Weigh MSN-NH2 and disperse it in 20 mL of anhydrous ethanol. Sonicate the dispersion for 10 minutes, add 3,3'-dithiodipropionic acid, and simultaneously add EDC·HCl and NHS as an activation system. Stir magnetically for 12 hours at room temperature in the dark. After the reaction is complete, collect the product by centrifugation, wash, and dry under vacuum at 40 °C to obtain disulfide bond-functionalized MSN. iv. Polyhistidine (PLH) was dissolved in anhydrous dimethyl sulfoxide (DMSO), and 2-iminothione hydrochloride (Traut's reagent, molar ratio of PLH 20:1) was added. The mixture was stirred at room temperature under nitrogen protection for 2 hours to introduce thiol groups. After the reaction was completed, the mixture was purified by dialyzing for 24 hours and then freeze-dried to obtain thiolized polyhistidine. v. Disulfide-functionalized MSN was dispersed in phosphate-buffered saline (PBS) at pH 7.4 at a concentration of 2 mg / mL. Thiolized polyhistidine (at a mass ratio of 1.5:1 to MSN-SS) was added, and the mixture was stirred at room temperature for 4 hours to allow PLH-SH to bind to the surface of MSN-SS via electrostatic adsorption. Subsequently, aqueous H2O2 solution was slowly added until the final concentration of H2O2 in the reaction solution was 0.01%. The mixture was stirred at room temperature in the dark for 2 hours. After the reaction was completed, the mixture was purified by centrifugation, washed three times with PBS, and freeze-dried to obtain functionalized modified mesoporous silica nanoparticles.

[0007] This invention also provides a method for preparing pH / redox dual-responsive nanocarriers for ovarian cancer, specifically including the following steps: S1, functionalized modified mesoporous silica nanoparticles were mixed with doxorubicin hydrochloride at a mass ratio of 2:1 and magnetically stirred at room temperature for 24 hours under light-protected conditions. The drug was loaded by the dual effects of physical adsorption through mesoporous channels and chemical anchoring by disulfide bonds. After the drug loading was completed, the product was collected by centrifugation, the unloaded free drug on the surface was gently washed with deionized water, and then freeze-dried to obtain the drug-loaded nanocore. S2, the drug-loaded nanocores were dispersed in PBS and mixed with functionalized biomimetic cell membrane vesicles at a mass ratio of 1:1. The mixture was repeatedly extruded 15-21 times using a polycarbonate membrane extruder to ensure that the cell membrane was uniformly coated on the surface of the nanoparticles. After extrusion, the product was centrifuged for 10 minutes and resuspended in PBS and washed twice to obtain the pH / redox dual-responsive nanocarrier for ovarian cancer.

[0008] Compared with the prior art, the present invention achieves the following beneficial effects: This invention utilizes a sol-gel method combined with template extraction to prepare a mesoporous silica framework, resulting in a nanocarrier substrate with a regular structure, uniform pore size, and excellent specific surface area. This provides ample loading space for doxorubicin, ensuring efficient drug loading and stable anchoring. The carrier is modified by amination, introducing active amino functional groups onto the particle surface and pore walls, providing stable reaction sites for the covalent grafting of disulfide functional groups. This invention also employs amidation to covalently graft disulfide redox responsive groups onto the pore walls, endowing the carrier pores with a specific response capability to the high-reduction environment within tumor cells. In a normal physiological environment, the disulfide bond structure is stable, firmly anchoring doxorubicin; upon entering ovarian cancer cells, the high-glutathione environment within the cells precisely triggers disulfide bond breakage, achieving controlled drug release. Simultaneously, the disulfide bonds on the pore walls and the surface cross-linking network form a hierarchical response structure in space, with the inner layer providing the basis for the reduction response and the outer layer providing a pH-responsive barrier; these two layers synergistically constitute a dual protection.

[0009] This invention innovatively modifies polyhistidine by thiolization and constructs a three-dimensional polyhistidine network with disulfide bonds on the carrier surface through oxidative crosslinking, overcoming the limitations of traditional single pH response technology. Under physiological neutral conditions, the crosslinked network tightly seals the pores, synergistically locking the drug in conjunction with the inner disulfide bonds, effectively preventing drug leakage in the bloodstream. Under the weakly acidic environment of tumors, the protonation of polyhistidine causes the network to expand moderately, but the crosslinked disulfide bonds remain intact, preventing non-specific drug release. Only when the carrier enters ovarian cancer cells, simultaneously meeting the dual conditions of weak acidity and high reduction, does the network disintegrate and the disulfide bonds in the pores break, achieving precise drug release.

[0010] This invention employs a cholesterol-anchored approach to stably embed GALA-penetrating peptides into the membrane of ovarian cancer cells. The natural ovarian cancer cell membrane endows the carrier with excellent biocompatibility, immune escape capability, and homologous targeting properties, prolonging in vivo circulation time and increasing the enrichment efficiency of ovarian cancer lesions in situ and metastases. The embedded GALA peptides endow the carrier with pH-responsive membrane fusion capability, promoting deep tumor penetration and overcoming the dense matrix barrier of ovarian cancer. Simultaneously, the outer biomimetic cell membrane forms a protective shell, shielding the inner pH-sensitive network and disulfide bond-responsive structure, preventing premature activation caused by acidic or redox substances in the blood environment, and ensuring the functional integrity of the hierarchical response structure. Attached Figure Description

[0011] Figure 1 Zeta plot of the pH / redox dual-responsive nanocarrier for ovarian cancer prepared in this invention; Figure 2 Confocal images of the uptake by ID8 and B16F10 of the pH / redox dual-responsive nanocarrier for ovarian cancer prepared in Example 2 of the present invention. Detailed Implementation

[0012] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0013] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those familiar to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein may be applied to this invention. The preferred embodiments and materials described herein are for illustrative purposes only and do not limit the scope of this application.

[0014] Unless otherwise specified, all methods used in the following examples are conventional. Unless otherwise specified, all materials used in the following examples are new materials purchased from the market. The GALA peptide sequence is: WEAALAEALAEALAEHLAEALAEALEALAA (SEQ ID NO:1); the molecular weight of the polyhistidine is 10-20 kDa.

[0015] Example 1: This example provides a pH / redox dual-responsive nanocarrier for ovarian cancer, comprising the following raw materials in parts by weight: 5 parts functionalized modified mesoporous silica nanoparticles, 5 parts functionalized biomimetic cell membrane vesicles, and 2 parts doxorubicin hydrochloride. The functionalized biomimetic cell membrane vesicles comprise the following raw materials in the following mass ratio: SKOV-3 human ovarian cancer cell line: GALA peptide: cholesterol hemisuccinate = 1:0.05:0.02; The method for preparing the functionalized biomimetic cell membrane vesicles includes the following steps: (1) Culture of SKOV-3 ovarian cancer cells: SKOV-3 human ovarian cancer cell line was cultured in McCoy's 5A complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics and cultured at 37℃ in a 5% CO2 incubator until the cell confluence reached 80% for cell membrane extraction. (2) Synthesis of cholesterol-anchored GALA peptide: Pure GALA peptide was prepared by solid-phase synthesis. Cholesterol hemisuccinate was weighed and dissolved in anhydrous DMF solvent. 0.3 mL of anhydrous DMF was added to each 1 μmol GALA peptide and the EDC / NHS activation system was added. The carboxyl group was activated by stirring at room temperature for 30 minutes. Then, GALA peptide was added and the reaction was carried out by stirring at room temperature for 12 h for amidation reaction. After the reaction was completed, the high-purity cholesterol-anchored GALA peptide was obtained by high-performance liquid chromatography. After freeze-drying, it was sealed and stored at -20℃ for later use. (3) SKOV-3 cell membrane extraction: Collect SKOV-3 cells cultured to the logarithmic growth phase, with a total cell count of 5 × 10⁻⁶. 7 Each cell was washed twice with pre-cooled PBS, and then lysed on ice for 30 minutes with membrane protein extraction buffer containing protease inhibitor (PMSF). After homogenization using a Dunns homogenizer, the cells were centrifuged at 4°C and 1000×g for 10 minutes to remove the nuclei and unbroken cells. The supernatant was then centrifuged at 4°C and 10000×g for 30 minutes. The precipitate was cell membrane fragments. The membrane precipitate was resuspended in pre-cooled PBS to obtain a cell membrane suspension. (4) GALA peptide insertion into the cell membrane: The cell membrane suspension and cholesterol-anchored GALA peptide were mixed at a mass ratio of membrane protein to cholesterol-anchored GALA peptide of 10:1 and incubated on ice for 30 minutes to allow the GALA peptide to insert into the cell membrane through the cholesterol anchor. After incubation, the mixture was centrifuged at 4°C and 10000×g for 10 minutes to remove the un-intercalated free cholesterol-anchored GALA peptide. The mixture was then resuspended and washed twice with PBS to obtain functionalized biomimetic cell membrane vesicles.

[0016] The functionalized modified mesoporous silica nanoparticles comprise the following raw materials in parts by weight: 0.1 parts CTAB, 0.8 parts TEOS, 0.2 parts 3-aminopropyltriethoxysilane, 0.3 parts 3,3'-dithiodipropionic acid, 0.2 parts EDC·HCl, 0.1 parts NHS, and 0.5 parts polyhistidine. The method for preparing the functionalized modified mesoporous silica nanoparticles includes the following steps: i. Dissolve CTAB in ultrapure water at a ratio of 1 g:450 mL. Adjust the pH to 11.0 with 2 M NaOH solution. Heat and stir in an 80°C water bath. After the temperature stabilizes, slowly add TEOS dropwise while stirring continuously for 2 hours. After the reaction is complete, collect the white precipitate by centrifugation. Wash the precipitate three times with anhydrous ethanol and ultrapure water, respectively. Disperse the obtained product in an ethanol solution containing 6 g / L NH4NO3 and reflux at 60°C for 12 hours to remove the CTAB template. Repeat the operation twice. Collect the product by centrifugation and wash it with ethanol and ultrapure water, respectively. Dry it under vacuum at 40°C to obtain MSN. ii. Weigh MSN and disperse it in anhydrous ethanol at a ratio of 10 mg:1 mL. Add 3-aminopropyltriethoxysilane and reflux at 80 °C for 24 hours under nitrogen protection. After the reaction is complete, centrifuge to collect the product, wash it three times with anhydrous ethanol to remove unreacted APTES, and dry it under vacuum at 40 °C to obtain amino-modified MSN (MSN-NH2). iii. Weigh MSN-NH2 and disperse it in 20 mL of anhydrous ethanol. Sonicate the dispersion for 10 minutes, add 3,3'-dithiodipropionic acid, and simultaneously add EDC·HCl and NHS as an activation system. Stir magnetically for 12 hours at room temperature in the dark. After the reaction is complete, collect the product by centrifugation, wash it three times alternately with deionized water and anhydrous ethanol to remove ungrafted free small molecules, and dry it under vacuum at 40 °C to obtain disulfide bond-functionalized MSN. iv. Polyhistidine (PLH) was dissolved in anhydrous dimethyl sulfoxide (DMSO), and 2-iminothione hydrochloride (20:1 molar ratio to PLH) was added. The mixture was stirred at room temperature under nitrogen protection for 2 hours to introduce thiol groups. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against deionized water for 24 hours, with the water changed 6 times during the process. The dialysate was freeze-dried to obtain thiolized polyhistidine. v. Disulfide-functionalized MSN was dispersed in phosphate-buffered saline (PBS) at pH 7.4 at a concentration of 2 mg / mL. Thiolized polyhistidine (mass ratio of 1.5:1 to disulfide-functionalized MSN) was added, and the mixture was stirred at room temperature for 4 hours to allow the thiolized polyhistidine to bind to the surface of the disulfide-functionalized MSN via electrostatic adsorption. Subsequently, H2O2 aqueous solution was slowly added until the final concentration of H2O2 in the reaction solution was 0.01%. The mixture was stirred at room temperature in the dark for 2 hours to promote the formation of disulfide bond crosslinks between thiolized polyhistidine molecules through thiol oxidation, thus constructing a three-dimensional crosslinked PLH network on the MSN surface. After the reaction was completed, the mixture was purified by centrifugation, washed three times with PBS, and freeze-dried to obtain functionalized modified mesoporous silica nanoparticles.

[0017] This embodiment also provides a method for preparing pH / redox dual-responsive nanocarriers for ovarian cancer, specifically including the following steps: S1, functionalized modified mesoporous silica nanoparticles were mixed with doxorubicin hydrochloride at a mass ratio of 2:1 and magnetically stirred at room temperature for 24 hours under light-protected conditions. The drug was loaded by the dual effects of physical adsorption through mesoporous channels and chemical anchoring by disulfide bonds. After the drug loading was completed, the product was collected by centrifugation, the unloaded free drug on the surface was gently washed with deionized water, and then freeze-dried to obtain the drug-loaded nanocore. S2, the drug-loaded nanocores were dispersed in PBS at a concentration of 2 mg / mL, then mixed with functionalized biomimetic cell membrane vesicles, and repeatedly extruded 15 times using a polycarbonate membrane extruder (pore size 200 nm) to uniformly coat the surface of the nanoparticles with cell membrane. After extrusion, the product was centrifuged at 4°C and 10000×g for 10 minutes to remove uncoated free cell membrane fragments, and then resuspended and washed twice with PBS to obtain the pH / redox dual-responsive nanocarrier for ovarian cancer.

[0018] Example 2: This example provides a pH / redox dual-responsive nanocarrier for ovarian cancer, comprising the following raw materials in parts by weight: 10 parts functionalized modified mesoporous silica nanoparticles, 10 parts functionalized biomimetic cell membrane vesicles, and 5 parts doxorubicin hydrochloride. The functionalized biomimetic cell membrane vesicles comprise raw materials in the following mass ratio: SKOV-3 human ovarian cancer cell line: GALA peptide: cholesterol hemisuccinate = 1:0.1:0.02; The method for preparing the functionalized biomimetic cell membrane vesicles includes the following steps: (1) Culture of SKOV-3 ovarian cancer cells: SKOV-3 human ovarian cancer cell line was cultured in McCoy's 5A complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics and cultured in a 37℃, 5% CO2 incubator until the cell confluence reached 85% for cell membrane extraction. (2) Synthesis of cholesterol-anchored GALA peptide: Pure GALA peptide was prepared by solid-phase synthesis. Cholesterol hemisuccinate was weighed and dissolved in anhydrous DMF solvent. 0.4 mL of anhydrous DMF was added to each 1 μmol GALA peptide and the EDC / NHS activation system was added. The carboxyl group was activated by stirring at room temperature for 30 minutes. Then, GALA peptide was added and the reaction was carried out by stirring at room temperature for 12 h for amidation reaction. After the reaction was completed, the high-purity cholesterol-anchored GALA peptide was obtained by high-performance liquid chromatography. After freeze-drying, it was sealed and stored at -20℃ for later use. (3) SKOV-3 cell membrane extraction: Collect SKOV-3 cells cultured to the logarithmic growth phase, with a total cell count of 5 × 10⁻⁶. 7 Each cell was washed twice with pre-cooled PBS, and then lysed on ice for 30 minutes with membrane protein extraction buffer containing protease inhibitor (PMSF). After homogenization using a Dunns homogenizer, the cells were centrifuged at 4°C and 1000×g for 10 minutes to remove the nuclei and unbroken cells. The supernatant was then centrifuged at 4°C and 10000×g for 30 minutes. The precipitate was cell membrane fragments. The membrane precipitate was resuspended in pre-cooled PBS to obtain a cell membrane suspension. (4) GALA peptide insertion into the cell membrane: The cell membrane suspension and cholesterol-anchored GALA peptide were mixed at a mass ratio of membrane protein to cholesterol-anchored GALA peptide of 10:1 and incubated on ice for 30 minutes to allow the GALA peptide to insert into the cell membrane through the cholesterol anchor. After incubation, the mixture was centrifuged at 4°C and 10000×g for 10 minutes to remove the un-intercalated free cholesterol-anchored GALA peptide. The mixture was then resuspended and washed twice with PBS to obtain functionalized biomimetic cell membrane vesicles.

[0019] The functionalized modified mesoporous silica nanoparticles comprise the following raw materials in parts by weight: 0.2 parts CTAB, 1.0 parts TEOS, 0.3 parts 3-aminopropyltriethoxysilane, 0.6 parts 3,3'-dithiodipropionic acid, 0.5 parts EDC·HCl, 0.2 parts NHS, and 0.9 parts polyhistidine. The method for preparing the functionalized modified mesoporous silica nanoparticles includes the following steps: i. Dissolve CTAB in ultrapure water at a ratio of 1 g:450 mL. Adjust the pH to 11.0 with 2 M NaOH solution. Heat and stir in an 80°C water bath. After the temperature stabilizes, slowly add TEOS dropwise while stirring continuously for 2 hours. After the reaction is complete, collect the white precipitate by centrifugation. Wash the precipitate three times with anhydrous ethanol and ultrapure water, respectively. Disperse the obtained product in an ethanol solution containing 6 g / L NH4NO3 and reflux at 60°C for 12 hours to remove the CTAB template. Repeat the operation twice. Collect the product by centrifugation and wash it with ethanol and ultrapure water, respectively. Dry it under vacuum at 40°C to obtain MSN. ii. Weigh MSN and disperse it in anhydrous ethanol at a ratio of 12 mg to 1 mL. Add 3-aminopropyltriethoxysilane and reflux at 80 °C for 24 hours under nitrogen protection. After the reaction is complete, centrifuge to collect the product, wash it three times with anhydrous ethanol to remove unreacted APTES, and dry it under vacuum at 40 °C to obtain amino-modified MSN (MSN-NH2). iii. Weigh 200 mg MSN-NH2 and disperse it in 20 mL of anhydrous ethanol. Sonicate the mixture for 10 minutes, add 3,3'-dithiodipropionic acid, and simultaneously add EDC·HCl and NHS as an activation system. Stir magnetically for 12 hours at room temperature in the dark. After the reaction is complete, collect the product by centrifugation, wash it three times alternately with deionized water and anhydrous ethanol to remove ungrafted free small molecules, and dry it under vacuum at 40 °C to obtain disulfide bond-functionalized MSN. iv. Polyhistidine (PLH) was dissolved in anhydrous dimethyl sulfoxide (DMSO), and 2-iminothione hydrochloride (20:1 molar ratio to PLH) was added. The mixture was stirred at room temperature under nitrogen protection for 2 hours to introduce thiol groups. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against deionized water for 24 hours, with the water changed 6 times during the process. The dialysate was freeze-dried to obtain thiolized polyhistidine. v. Disulfide-functionalized MSN was dispersed in phosphate-buffered saline (PBS) at pH 7.4 at a concentration of 2 mg / mL. Thiolized polyhistidine (mass ratio of 1.5:1 to disulfide-functionalized MSN) was added, and the mixture was stirred at room temperature for 4 hours to allow the thiolized polyhistidine to bind to the surface of the disulfide-functionalized MSN via electrostatic adsorption. Subsequently, H2O2 aqueous solution was slowly added until the final concentration of H2O2 in the reaction solution was 0.01%. The mixture was stirred at room temperature in the dark for 2 hours to promote the formation of disulfide bond crosslinks between thiolized polyhistidine molecules through thiol oxidation, thus constructing a three-dimensional crosslinked PLH network on the MSN surface. After the reaction was completed, the mixture was purified by centrifugation, washed three times with PBS, and freeze-dried to obtain functionalized modified mesoporous silica nanoparticles.

[0020] This embodiment also provides a method for preparing pH / redox dual-responsive nanocarriers for ovarian cancer, specifically including the following steps: S1, functionalized modified mesoporous silica nanoparticles were mixed with doxorubicin hydrochloride at a mass ratio of 2:1 and magnetically stirred at room temperature for 24 hours under light-protected conditions. The drug was loaded by the dual effects of physical adsorption through mesoporous channels and chemical anchoring by disulfide bonds. After the drug loading was completed, the product was collected by centrifugation, the unloaded free drug on the surface was gently washed with deionized water, and then freeze-dried to obtain the drug-loaded nanocore. S2, the drug-loaded nanocores were dispersed in PBS at a concentration of 2 mg / mL, then mixed with functionalized biomimetic cell membrane vesicles, and repeatedly extruded 18 times using a polycarbonate membrane extruder (pore size 200 nm) to uniformly coat the surface of the nanoparticles with cell membrane. After extrusion, the product was centrifuged at 6°C and 10000×g for 10 minutes to remove uncoated free cell membrane fragments, and then resuspended and washed twice with PBS to obtain the pH / redox dual-responsive nanocarrier for ovarian cancer.

[0021] Example 3: This example provides a pH / redox dual-responsive nanocarrier for ovarian cancer, comprising the following raw materials in parts by weight: 15 parts functionalized modified mesoporous silica nanoparticles, 15 parts functionalized biomimetic cell membrane vesicles, and 8 parts doxorubicin hydrochloride. The functionalized biomimetic cell membrane vesicles comprise the following raw materials in the following mass ratio: SKOV-3 human ovarian cancer cell line: GALA peptide: cholesterol hemisuccinate = 1:0.2:0.02; The method for preparing the functionalized biomimetic cell membrane vesicles includes the following steps: (1) Culture of SKOV-3 ovarian cancer cells: SKOV-3 human ovarian cancer cell line was cultured in McCoy's 5A complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin double antibiotics and cultured at 37℃ in a 5% CO2 incubator until the cell confluence reached 90% for cell membrane extraction. (2) Synthesis of cholesterol-anchored GALA peptide: Pure GALA peptide was prepared by solid-phase synthesis. Cholesterol hemisuccinate was weighed and dissolved in anhydrous DMF solvent. 0.5 mL of anhydrous DMF was added to each 1 μmol GALA peptide and the EDC / NHS activation system was added. The carboxyl group was activated by stirring at room temperature for 30 minutes. Then, GALA peptide was added and the reaction was carried out by stirring at room temperature for 12 h for amidation reaction. After the reaction was completed, the high-purity cholesterol-anchored GALA peptide was obtained by high-performance liquid chromatography. After freeze-drying, it was sealed and stored at -20℃ for later use. (3) SKOV-3 cell membrane extraction: Collect SKOV-3 cells cultured to the logarithmic growth phase, with a total cell count of 5 × 10⁻⁶. 7 Each cell was washed twice with pre-cooled PBS, and then lysed on ice for 30 minutes with membrane protein extraction buffer containing protease inhibitor (PMSF). After homogenization using a Dunns homogenizer, the cells were centrifuged at 4°C and 1000×g for 10 minutes to remove the nuclei and unbroken cells. The supernatant was then centrifuged at 4°C and 10000×g for 30 minutes. The precipitate was cell membrane fragments. The membrane precipitate was resuspended in pre-cooled PBS to obtain a cell membrane suspension. (4) GALA peptide insertion into the cell membrane: The cell membrane suspension and cholesterol-anchored GALA peptide were mixed at a mass ratio of membrane protein to cholesterol-anchored GALA peptide of 10:1 and incubated on ice for 30 minutes to allow the GALA peptide to insert into the cell membrane through the cholesterol anchor. After incubation, the mixture was centrifuged at 4°C and 10000×g for 10 minutes to remove the un-intercalated free cholesterol-anchored GALA peptide. The mixture was then resuspended and washed twice with PBS to obtain functionalized biomimetic cell membrane vesicles.

[0022] The functionalized modified mesoporous silica nanoparticles comprise the following raw materials in parts by weight: 0.3 parts CTAB, 1.5 parts TEOS, 0.6 parts 3-aminopropyltriethoxysilane, 0.9 parts 3,3'-dithiodipropionic acid, 0.7 parts EDC·HCl, 0.4 parts NHS, and 1.2 parts polyhistidine. The method for preparing the functionalized modified mesoporous silica nanoparticles includes the following steps: i. Dissolve CTAB in ultrapure water at a ratio of 1 g to 500 mL. Adjust the pH to 11.0 with 2 M NaOH solution. Heat and stir in an 80°C water bath. After the temperature stabilizes, slowly add TEOS dropwise while stirring continuously for 2 hours. After the reaction is complete, collect the white precipitate by centrifugation. Wash the precipitate three times with anhydrous ethanol and ultrapure water, respectively. Disperse the obtained product in an ethanol solution containing 6 g / L NH4NO3 and reflux at 60°C for 12 hours to remove the CTAB template. Repeat the operation twice. Collect the product by centrifugation and wash it with ethanol and ultrapure water, respectively. Dry it under vacuum at 40°C to obtain MSN. ii. Weigh MSN and disperse it in anhydrous ethanol at a ratio of 10-15 mg MSN to 1 mL anhydrous ethanol. Add 3-aminopropyltriethoxysilane and reflux at 80 °C for 24 hours under nitrogen protection. After the reaction is complete, centrifuge to collect the product, wash it three times with anhydrous ethanol to remove unreacted APTES, and dry it under vacuum at 40 °C to obtain amino-modified MSN (MSN-NH2). iii. Weigh 200 mg MSN-NH2 and disperse it in 20 mL of anhydrous ethanol. Sonicate the mixture for 10 minutes, add 3,3'-dithiodipropionic acid, and simultaneously add EDC·HCl and NHS as an activation system. Stir magnetically for 12 hours at room temperature in the dark. After the reaction is complete, collect the product by centrifugation, wash it three times alternately with deionized water and anhydrous ethanol to remove ungrafted free small molecules, and dry it under vacuum at 40 °C to obtain disulfide bond-functionalized MSN. iv. Polyhistidine (PLH) was dissolved in anhydrous dimethyl sulfoxide (DMSO), and 2-iminothione hydrochloride (20:1 molar ratio to PLH) was added. The mixture was stirred at room temperature under nitrogen protection for 2 hours to introduce thiol groups. After the reaction was completed, the reaction solution was placed in a dialysis bag with a molecular weight cutoff of 3.5 kDa and dialyzed against deionized water for 24 hours, with the water changed 6 times during the process. The dialysate was freeze-dried to obtain thiolized polyhistidine. v. Disulfide-functionalized MSN was dispersed in phosphate-buffered saline (PBS) at pH 7.4 at a concentration of 2 mg / mL. Thiolized polyhistidine (mass ratio of 1.5:1 to disulfide-functionalized MSN) was added, and the mixture was stirred at room temperature for 4 hours to allow the thiolized polyhistidine to bind to the surface of the disulfide-functionalized MSN via electrostatic adsorption. Subsequently, H2O2 aqueous solution was slowly added until the final concentration of H2O2 in the reaction solution was 0.01%. The mixture was stirred at room temperature in the dark for 2 hours to promote the formation of disulfide bond crosslinks between thiolized polyhistidine molecules through thiol oxidation, thus constructing a three-dimensional crosslinked PLH network on the MSN surface. After the reaction was completed, the mixture was purified by centrifugation, washed three times with PBS, and freeze-dried to obtain functionalized modified mesoporous silica nanoparticles.

[0023] This embodiment also provides a method for preparing pH / redox dual-responsive nanocarriers for ovarian cancer, specifically including the following steps: S1, functionalized modified mesoporous silica nanoparticles were mixed with doxorubicin hydrochloride at a mass ratio of 2:1 and magnetically stirred at room temperature for 24 hours under light-protected conditions. The drug was loaded by the dual effects of physical adsorption through mesoporous channels and chemical anchoring by disulfide bonds. After the drug loading was completed, the product was collected by centrifugation, the unloaded free drug on the surface was gently washed with deionized water, and then freeze-dried to obtain the drug-loaded nanocore. S2, the drug-loaded nanocores were dispersed in PBS at a concentration of 2 mg / mL, then mixed with functionalized biomimetic cell membrane vesicles, and repeatedly extruded 21 times using a polycarbonate membrane extruder (pore size 200 nm) to uniformly coat the surface of the nanoparticles with cell membrane. After extrusion, the product was centrifuged at 10°C and 10000×g for 10 minutes to remove uncoated free cell membrane fragments, and then resuspended and washed twice with PBS to obtain the pH / redox dual-responsive nanocarrier for ovarian cancer.

[0024] The difference between Comparative Example 1 and Example 2 is that Comparative Example 1 uses physical adsorption of PLH, while the rest is exactly the same as Example 2.

[0025] The difference between Comparative Example 2 and Example 2 is that the nanocarrier is coated with a membrane of ordinary ovarian cancer cells, while the rest is exactly the same as Example 2.

[0026] The difference between Comparative Example 3 and Example 3 is that the addition of functionalized biomimetic cell membrane vesicles was omitted, while the rest is exactly the same as Example 2.

[0027] Experimental Examples: Using the pH / redox dual-responsive nanocarriers for ovarian cancer prepared in Examples 1-3 and Comparative Examples 1-3 of this invention as test samples, the following experimental examples were conducted: 1. Cytotoxicity test: The MTT assay was used to examine the killing effect of the nano-formulation on ovarian cancer cells. SKOV-3 and A2780 ovarian cancer cells in logarithmic growth phase were collected and subjected to a cytotoxicity assay of 5 × 10⁻⁶ cells / mL. 3 Cells were seeded per well in 96-well plates. After 24 hours of culture, different concentrations (0.01–50 μg / mL, DOX as a whole) of free DOX and the test sample of this protocol were added to each well. Each group had 5 replicates, and a blank control group and a zeroing well were also included. After 48 hours of further culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the cells were incubated in the dark for 4 hours. The supernatant was discarded, and 150 μL of DMSO was added to dissolve the crystals. The absorbance was measured at 570 nm, and cell viability was calculated and IC50 was fitted. 50 Values. The results are recorded in Table 1.

[0028] 2. Drug loading and encapsulation efficiency: The doxorubicin content was determined by UV-Vis spectrophotometry. Doxorubicin hydrochloride standard was accurately weighed, and a series of standard solutions with concentrations of 1, 2, 5, 10, 20, and 50 μg / mL were prepared using PBS buffer. The absorbance was measured at 480 nm, and a standard curve was plotted. Three lyophilized drug-loaded nanoparticles, each 10 mg, were accurately weighed and dispersed in 5 mL of PBS buffer. The solutions were centrifuged at 12000 rpm for 20 minutes at 4℃, and the supernatant was used to determine the free drug content. Separately, three drug-loaded nanoparticles, each 2 mg, were accurately weighed, added to 2 mL of 0.1 M hydrochloric acid-methanol solution, and sonicated for 30 minutes to completely release the drug. After adjusting the volume with PBS, the total drug content was measured at 480 nm. The calculated drug loading and encapsulation efficiency results are recorded in Table 1.

[0029] Table 1: Test results of the pH / redox dual-responsive nanocarrier for ovarian cancer prepared in this invention.

[0030] Table 1 shows the results. Comparative Example 3 had the highest drug loading and encapsulation efficiency because it lacked a cell membrane coating layer, so the drug loading process was not affected by the membrane barrier, allowing the drug to fully enter the pores. Example 2 showed a slight decrease in drug loading and encapsulation efficiency, indicating that the GALA-CCM coating layer might cause a small loss of surface-adsorbed drug during the extrusion coating process. However, the drug loading and encapsulation efficiency remained at a high level, meeting the formulation requirements. Comparative Example 2 showed a significant decrease in drug loading and encapsulation efficiency, indicating that the ordinary CCM lacked GALA peptide intercalation, resulting in poor membrane fluidity and stability of the cell membrane vesicles. This reduced the encapsulation efficiency of the drug-loaded particles during the coating process, leading to greater drug loss. Comparative Example 1 had the lowest drug loading and encapsulation efficiency, indicating that the physically adsorbed PLH network was unstable. During the drug loading and stirring process, some PLH desorbed, resulting in a loose surface PLH layer structure, increased drug leakage during loading, and affecting the stability of the membrane layer during subsequent GALA membrane coating.

[0031] The above results indicate that the disulfide bond crosslinked PLH network is crucial for maintaining the structural stability and drug loading efficiency of the carrier; GALA embedding not only endows the membrane with fusion function, but also improves the stability and encapsulation integrity of cell membrane vesicles, thereby increasing the encapsulation efficiency.

[0032] Example 2 exhibited significantly better cytotoxicity than the comparative examples and free DOX, demonstrating that GALA-embedded cell membrane-mediated pH-responsive membrane fusion can significantly enhance the uptake efficiency of the nanocarrier by ovarian cancer cells. Comparative Example 3, lacking targeting function, had the lowest uptake efficiency and weaker cytotoxicity than free DOX, demonstrating the necessity of cell membrane coating for targeted delivery. Comparative Example 2 showed weaker cytotoxicity than Example 2, demonstrating the contribution of GALA-embedded membrane fusion function to enhanced uptake. Comparative Example 1, lacking biomimetic cell membrane coating, resulted in premature drug leakage, reducing the amount of drug delivered to the target cells, and thus exhibited weaker cytotoxicity than Example 2.

[0033] Figure 1 Zeta plots of the pH / redox dual-response nanocarrier for ovarian cancer prepared in this invention at pH=7.4 and pH=5. Examples 1-3 showed the closest potential to neutral at pH 7.4, indicating a complete multi-level modified structure, uniform surface coating, and optimal colloidal stability, effectively reducing non-specific adsorption and immune clearance in vivo. At a weakly acidic pH 5.0, the potential changed from negative to positive, demonstrating a significant protonation effect in the surface disulfide-linked polyhistidine network, inducing network relaxation and expansion, achieving a structural standby response in the tumor microenvironment. Comparative Example 1 (physically adsorbed PLH) and Comparative Example 2 (ordinary cell membrane coating group) showed significantly weaker pH response amplitudes than Example 2, with no obvious charge reversal characteristics, proving that non-crosslinked structures cannot form stable pH-responsive intelligent gating. Comparative Example 3 (bare core) showed no pH-responsive characteristics and no significant potential fluctuations. The significant differences among the groups further validated the synergistic advantages of the disulfide-linked PLH three-dimensional network and the GALA-functionalized cell membrane of this invention, which is the core structural basis for achieving precise drug release under dual conditions and avoiding drug pre-leakage.

[0034] like Figure 2 As shown, this study used confocal imaging to investigate in vitro cellular uptake in D8 ovarian cancer cells and B16F10 melanoma cells, respectively. Blue fluorescence was used to label the cell nucleus, and red fluorescence was used to label the nanocarrier of this invention. Overlay fields of view were used to visually observe the distribution and internalization of nanoparticles within the cells. Imaging results showed that bright and widely distributed red fluorescence signals were observed in ID8 ovarian cancer cells, indicating that the nanocarrier of this invention can be efficiently recognized and rapidly internalized by ovarian cancer cells. In contrast, the red fluorescence signal in B16F10 melanoma cells was weak, indicating significantly lower cellular uptake. These results confirm that the GALA peptide-functionalized biomimetic ovarian cancer cell membrane carrier of this invention can achieve specific and active targeted enrichment of ovarian cancer cells by relying on cell membrane homology recognition and Siglec-10 targeting effects, with extremely low non-specific adsorption and uptake of heterologous tumor cells.

[0035] In summary, this invention achieves intelligent and controllable drug release characteristics by constructing redox-responsive drug release sites within the carrier channels and building a dual-condition synergistic barrier structure on the particle surface, thus realizing drug locking under physiological environment stability and drug release specifically unlocked under tumor microenvironment. Simultaneously, this invention introduces a cell membrane modification process for cholesterol-anchored GALA peptides, endowing the nanocarrier with excellent deep penetration capability into the tumor matrix while retaining the advantages of natural homologous targeting, immune escape, and long circulation of ovarian cancer cell membranes. Each preparation step of this invention is layer-by-layer adapted, with each functional structure mutually constraining and synergistically enhancing the effect. The inner layer structure ensures precise intracellular drug release and drug resistance reversal, the middle network layer ensures controllable drug release and in vivo stability throughout the process, and the outer biomimetic structure ensures tumor-targeted enrichment and deep penetration performance. Overall, this invention achieves a performance upgrade of the nanocarrier throughout the entire process of in vivo circulation, tumor enrichment, microenvironment response, lesion penetration, intracellular drug release, and tumor inhibition.

[0036] The present invention and its embodiments have been described above. This description is not restrictive, and the accompanying drawings are only one embodiment of the present invention. The actual application is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar methods and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. A pH / redox dual-responsive nanocarrier for ovarian cancer, characterized in that, The raw materials include the following parts by weight: 5-15 parts of functionalized modified mesoporous silica nanoparticles, 5-15 parts of functionalized biomimetic cell membrane vesicles, and 2-8 parts of doxorubicin hydrochloride. The functionalized biomimetic cell membrane vesicles include the following raw materials: SKOV-3 human ovarian cancer cell line, GALA peptide, and cholesterol hemisuccinate. The method for preparing the functionalized biomimetic cell membrane vesicles includes the following steps: (1) SKOV-3 human ovarian cancer cell line was cultured to obtain SKOV-3 cells; (2) Take GALA peptide and cholesterol hemisuccinate, dissolve them together in anhydrous DMF solvent, add EDC / NHS activation system to carry out amidation reaction, stir at room temperature, purify, freeze dry to obtain cholesterol anchored GALA peptide; (3) Collect SKOV-3 cells, wash, lyse, centrifuge, and resuspend the supernatant in pre-cooled PBS after centrifugation to obtain cell membrane suspension; (4) Mix the cell membrane suspension with cholesterol-anchored GALA peptide, incubate on ice, centrifuge, wash, and obtain functionalized biomimetic cell membrane vesicles.

2. The nanocarrier according to claim 1, characterized in that, The mass ratio of the SKOV-3 human ovarian cancer cell line, GALA peptide, and cholesterol hemisuccinate was 1:0.05-0.2:0.

02.

3. The nanocarrier according to claim 1, characterized in that, The functionalized modified mesoporous silica nanoparticles comprise the following raw materials in parts by weight: 0.1-0.3 parts CTAB, 0.8-1.5 parts TEOS, 0.2-0.6 parts 3-aminopropyltriethoxysilane, 0.3-0.9 parts 3,3'-dithiodipropionic acid, 0.2-0.7 parts EDC·HCl, 0.1-0.4 parts NHS, and 0.5-1.2 parts polyhistidine.

4. The nanocarrier according to claim 3, characterized in that, The method for preparing the functionalized modified mesoporous silica nanoparticles includes the following steps: i. Dissolve CTAB in ultrapure water, adjust the pH, heat in a water bath, then add TEOS dropwise, centrifuge to collect the precipitate, wash, reflux to extract, and dry to obtain MSN; ii. Weigh MSN, disperse it in anhydrous ethanol, add 3-aminopropyltriethoxysilane, reflux under nitrogen protection, centrifuge, wash, and vacuum dry to obtain MSN-NH2; iii. Weigh MSN-NH2, disperse it in anhydrous ethanol, sonicate it, add 3,3'-dithiodipropionic acid, EDC·HCl and NHS at the same time, stir magnetically in the dark, centrifuge and wash, dry it to obtain disulfide bond functionalized MSN. iv. Polyhistidine was dissolved in anhydrous DMSO, 2-iminothione hydrochloride was added, and the mixture was stirred and reacted at room temperature under nitrogen protection. The mixture was purified by dialysis and freeze-dried to obtain thiolated polyhistidine. v. Disulfide-functionalized MSN was dispersed in PBS, thiolized polyhistidine was added, and the mixture was stirred at room temperature. Then, an aqueous solution of H2O2 was added, and the mixture was stirred in the dark. After centrifugation and washing, the mixture was freeze-dried to obtain functionalized modified mesoporous silica nanoparticles.

5. The nanocarrier according to claim 4, characterized in that, In step i, the ratio of CTAB to ultrapure water is 1g:450-500mL; In step ii, the ratio of MSN to anhydrous ethanol is 10-15 mg: 1 mL.

6. The nanocarrier according to claim 5, characterized in that, In step iii, the ratio of MSN-NH2 to anhydrous ethanol is 10-15 mg: 1 mL.

7. The method for preparing the nanocarrier according to any one of claims 1-6, characterized in that, Specifically, the following steps are included: S1, functionalized modified mesoporous silica nanoparticles were mixed with doxorubicin hydrochloride, stirred magnetically at room temperature in the dark, the product was collected by centrifugation, washed, and freeze-dried to obtain drug-loaded nanocores; S2, drug-loaded nanocores were dispersed in PBS, mixed with functionalized biomimetic cell membrane vesicles, extruded using a polycarbonate membrane, and then centrifuged and washed to obtain a pH / redox dual-responsive nanocarrier for ovarian cancer.

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