PH-sensitive targeted nano drug delivery system containing Survivin siRNA and ZnO

Abstract

The invention discloses a siRNA delivery carrier CCM-CS / ZnO (at) siSurvivin with pH responsiveness and tumor targeting property, a preparation method thereof, physicochemical properties such as form and structure thereof, encapsulation efficiency and drug release behavior thereof, cell uptake efficiency, tumor targeting ability, lysosome escape ability, gene silencing efficiency, apoptosis promoting ability, tumor cell growth inhibition ability thereof, and application of the siRNA delivery carrier CCM-CS / ZnO (at) siSurvivin. The invention discloses the synergistic anti-tumor capability of Survivin siRNA and ZnO, and further discloses the in-vivo targeting property of the Survivin siRNA and ZnO, the effect of inhibiting the growth of BALB / c tumor-bearing nude mouse tumors and the in-vivo safety of the Survivin siRNA and ZnO. Therefore, the target delivery vector has important application in the field of gene therapy of tumors.

Description

Technical Field

[0001] The present invention relates to a pH-sensitive targeted nano-drug delivery system CCM-CS / ZnO@siSurvivin containing Survivin siRNA and ZnO, belonging to the field of biomedicine. Background Art

[0002] Gene therapy has become a powerful tool for treating various diseases including cancer. During the occurrence and development of cancer, Survivin protein is highly expressed and is closely related to tumor proliferation, differentiation, invasion and metastasis. It has been reported that silencing Survivin protein by small interfering RNA (siRNA) has the prospect of inhibiting cancer development. However, there are still problems in the successful clinical application of siRNA.

[0003] Zinc oxide nanoparticles (ZnO NPs) are a kind of material with good biocompatibility, chemical stability and pH sensitivity. It has been reported that ZnO NPs can be used as a pH-responsive material to control the release of drugs in acidic environments. In addition, the Zn released by ZnO NPs in the acidic lysosomal microenvironment 2+ will also damage the structure of lysosomes. All these properties make ZnONPs a promising material for siRNA escape and pH-responsive release. In addition, it has been reported that ZnO NPs are a kind of nanozyme similar to catalase, which can amplify the ROS level, thereby inducing oxidative stress in tumor tissues. Therefore, ZnO NPs can also be used as an effective anti-tumor agent to play a synergistic anti-tumor role together with Survivin siRNA.

[0004] Nanoparticles coated with cell membranes can significantly prolong the circulation time of nanoparticles, reduce immunogenicity, and improve the stability of nanoparticles in vivo. Compared with other cell membranes, cancer cell membranes inherit the homologous targeting effect from the source tumor cells. Nanoparticles coated with cancer cell membranes are expected to become an effective tumor-targeted therapy.

[0005] In the present invention, a cancer cell membrane-coated pH-responsive nanoparticle based on the acid-dissolution property of ZnO NPs is developed, so as to help Survivin siRNA escape from lysosomes and inhibit tumor growth through the synergistic effect of gene drugs and ZnO NPs. When taken up by tumor cells, ZnO NPs will dissolve in acidic lysosomes, releasing Survivin siRNA into the cytoplasm. ZnO NPs will also induce apoptosis by enhancing the level of oxidative stress in tumor cells and play an anti-tumor role together with Survivin siRNA.

[0006] In summary, the fabricated nano-drug delivery system provides an effective strategy for improving the delivery efficiency of siRNA and also offers a new approach for synergistic cancer therapy. Summary of the Invention

[0007] The first objective of the present invention is to provide a pH-sensitive targeted nano-drug delivery system containing Survivin siRNA and ZnO, abbreviated as: CCM-CS / ZnO@siSurvivin.

[0008] The drug delivery system described in the present invention is mainly prepared from the anti-tumor gene therapy drug Survivin siRNA, chitosan oligosaccharide (CS), zinc oxide nanoparticles (ZnO), and 4T1 tumor cell membrane (CCM).

[0009] The drug delivery system CCM-CS / ZnO@siSurvivin of the present invention has a nanostructure.

[0010] Another objective of the present invention is to provide a preparation method for the drug delivery system CCM-CS / ZnO@siSurvivin.

[0011] The preparation method described in the present invention includes the following steps:

[0012] Prepare ZnO NPs by the sol-gel method, combine ZnO NPs with CS to encapsulate Survivin siRNA, and then coat CCM by the liposome extrusion method.

[0013] Specifically, the preparation method of the drug delivery system described in the present invention includes the following steps:

[0014] (1) Prepare ZnO NPs as follows: Heat and reflux to prepare ethanol solutions of zinc acetate and sodium hydroxide respectively, add the ethanol solution of sodium hydroxide dropwise to the ethanol solution of zinc acetate, stir rapidly, and finally precipitate the nanoparticles with n-hexane;

[0015] (2) Prepare the composite nanoparticles CS / ZnO@siSurvivin of CS and ZnO NPs loaded with Survivin siRNA as follows: Mix Survivin siRNA with the DMSO solution of CS, incubate and then add ZnO NPs, and stir to form composite nanoparticles;

[0016] (3) Prepare the nanoparticles CCM-CS / ZnO@siSurvivin coated with tumor cell membrane as follows: Extract 4T1 tumor cell membrane by gradient centrifugation, and then coat the cell membrane on the surface of the nanoparticles through a liposome extruder.

[0017] Preferably, the preparation method of the drug-loaded system CCM-CS / ZnO@siSurvivin of the present invention comprises the following steps:

[0018] 1) Prepare ZnO NPs as follows:

[0019] (1) Take ZnO(OAc)·2H2O and add anhydrous ethanol; take sodium hydroxide and add anhydrous ethanol;

[0020] (2) Under the conditions of oil bath heating and condenser reflux, stir the above solutions until the ethanol boils and then continue heating until the solute is completely dissolved, and then stop heating and cool to room temperature;

[0021] (3) Under the ice bath condition, rapidly stir the ethanol solution of ZnO(OAc)·2H2O, and drip the ethanol solution of NaOH into the above ethanol solution of ZnO(OAc)·2H2O through a microporous filter membrane, and continuously stir rapidly to ripen the seeds. After the reaction ends, the ethanol solution is clear and transparent;

[0022] (4) Add n-hexane to the ethanol solution, observe that the solution gradually changes from clear to turbid. After the ZnO NPs are completely precipitated, centrifuge to make the nanoparticles precipitate to the bottom of the centrifuge tube, and then add anhydrous ethanol and ultrasonically disperse to obtain an ethanol solution of ZnO NPs.

[0023] 2) Prepare the composite nanoparticles CS / ZnO@siSurvivi n of CS and ZnO NPs loaded with Survivin siRNA as follows:

[0024] (1) Prepare a 20mg / mL chitosan DMSO solution,; prepare a 1M NaOH solution,; take the 1M NaOH solution and slowly add it drop by drop to the above chitosan solution under ultrasonic conditions; take the chitosan solution and dilute it with DMSO; take the siRNA solution and mix it with the chitosan solution at room temperature and perform water bath ultrasound;

[0025] (2) Take the ZnO NPs prepared above and add them drop by drop to the mixed solution of chitosan and siRNA under the condition of water bath ultrasound, stir, centrifuge, discard the supernatant to obtain a white precipitate, and disperse the precipitate in deionized water.

[0026] 3) Prepare the nanoparticles CCM-CS / ZnO@siSurvivin coated with tumor cell membranes as follows,

[0027] (1) Discard the cell culture medium, wash the cell surface, and add PBS buffer; scrape the cells with a cell scraper; after collecting the cells from 3 dishes, sonicate under the probe sonication of a cell disruptor; centrifuge the cell debris suspension; discard the precipitate, and centrifuge the supernatant; discard the supernatant, redisperse the precipitate in deionized water, and sonicate with the probe to obtain a 4T1 tumor cell membrane solution;

[0028] (2) Under the condition of water bath sonication, slowly add the above-synthesized CS / ZnO@siSurvivin NPs to the extracted 4T1 tumor cell membrane solution; take out the liposome extruder, repeatedly extrude the above liquid, after the extrusion is completed, centrifuge to remove the blank vesicles in the supernatant, and redisperse the precipitate in deionized water to obtain CCM-CS / ZnO@siSurvivin NPs.

[0029] Further preferably, the preparation method of the drug-loaded system CCM-CS / ZnO@siSurvivin of the present invention includes the following steps:

[0030] 1) Prepare ZnO NPs

[0031] (1) Weigh 1.09 g of zinc acetate dihydrate (ZnO(OAc)2·2H2O) and add 50 mL of absolute ethanol. Weigh 0.386 g of sodium hydroxide (NaOH) and add 26 mL of absolute ethanol.

[0032] (2) Under the conditions of oil bath heating and condenser reflux, stir the above solution at 800 rpm until the ethanol boils and then continue heating for 2 h. Stop heating after the solute is completely dissolved and cool to room temperature.

[0033] (3) Under the ice bath condition, quickly stir the ethanol solution of ZnO(OAc)·2H2O, and drop the ethanol solution of NaOH into the above ethanol solution of ZnO(OAc)·2H2O through a 0.22 mm microporous filter membrane, and continuously stir quickly for 4 h to ripen the seeds.

[0034] (4) Add 228 mL of n-hexane to the ethanol solution. Centrifuge at 10000 rpm for 1 min, discard the supernatant, add 50 mL of absolute ethanol, and disperse by sonication to obtain an ethanol solution of ZnO NPs.

[0035] 2) Prepare CS and ZnO NPs composite nanoparticles CS / ZnO@siS urvivin loaded with Survivin siRNA

[0036] (1) Prepare a 20 mg / mL chitosan DMSO solution and ultrasonicate it in water for 10 min. Prepare a 1 M NaOH solution and ultrasonicate it for 3 min. Take 200 μL of the 1 M NaOH solution and slowly add it drop by drop to the above 2.145 mL of 20 mg / mL chitosan solution under ultrasonic conditions. Take 60 μL of the chitosan solution, add 540 μL of DMSO to dilute it to 1 mg / mL. Take 10 μL of a 0.3 μg / μL siRNA solution and mix it with 600 μL of the chitosan solution at room temperature, then ultrasonicate it in water for 15 min and stir vigorously for 30 min.

[0037] (2) Take 100 μL of the ZnO NPs prepared above and slowly add it drop by drop to the mixed solution of chitosan and siRNA under ultrasonic conditions in water, and continue to stir vigorously at a speed of 1500 rpm for 4 h. After the reaction, centrifuge at 4 °C and 12000 g for 10 min, discard the supernatant to obtain a white precipitate, and disperse the precipitate in 1 mL of deionized water.

[0038] 3) Prepare nanoparticles CCM-CS / ZnO@siSurvivin coated with tumor cell membranes

[0039] (1) Discard the cell culture medium, wash the cell surface twice, and then add 500 μL of PBS buffer. Scrape the cells with a cell scraper. After collecting the cells from 3 dishes, ultrasonicate them for 12 min under the probe sonication of a cell disruptor. Centrifuge the cell debris suspension at 4 °C and 700 g for 10 min. Discard the precipitate, and centrifuge the supernatant at 4 °C and 13000 g for 30 min. Discard the supernatant, redisperse the precipitate in 2 mL of deionized water, and ultrasonicate it with the probe for 2 min to obtain a 4T1 tumor cell membrane solution.

[0040] (2) Under ultrasonic conditions in water, slowly add 1 mL of the synthesized CS / ZnO@siSurvivin NPs above to the extracted 2 mL of 4T1 tumor cell membrane solution. Take out the liposome extruder, install a 400 nm polycarbonate membrane and gasket according to the instructions, and repeatedly extrude the above liquid 31 times. After the extrusion, centrifuge at 4 °C and 12000 g for 15 min to remove the blank vesicles in the supernatant, and redisperse the precipitate in 3 mL of deionized water to obtain CCM-CS / ZnO@siSurvivin NPs.

[0041] Among them, the mass ratio of Survivin siRNA to CS is 3:1 (μg / mg).

[0042] The present invention also aims to provide the application of the drug delivery system CCM-CS / ZnO@siSurvivin in the preparation of drugs for treating or preventing tumors.

[0043] The present invention also aims to provide the application of CCM-CS / ZnO@siSurvivin in the preparation of drugs for inhibiting the proliferation of tumor cells.

[0044] The present invention also aims to evaluate the homologous targeting effect of CCM-CS / ZnO@siSurvivin.

[0045] The present invention also aims to evaluate the drug uptake efficiency of CCM-CS / ZnO@siSurvivin.

[0046] The present invention also aims to evaluate the cell inhibitory effect of CCM-CS / ZnO@siSurvivin.

[0047] The present invention also aims to evaluate the gene silencing effect of CCM-CS / ZnO@siSurvivin.

[0048] The present invention also aims to evaluate the effect of CCM-CS / ZnO@siSurvivin in inducing oxidative stress.

[0049] The present invention also aims to evaluate the effect of CCM-CS / ZnO@siSurvivin in inhibiting the tumor growth of mice.

[0050] The present invention discloses the siRNA delivery carrier CCM-CS / ZnO@siSurvivin with pH responsiveness and tumor targeting, discloses its preparation method, discloses its physical and chemical properties such as morphology and structure, discloses its encapsulation efficiency and drug release behavior, discloses its cell uptake efficiency, tumor targeting ability, lysosome escape ability, gene silencing efficiency, pro-apoptotic ability, tumor cell growth inhibition ability, discloses the synergistic anti-tumor ability of Survivin siRNA and ZnO, and further discloses its in vivo targeting property, the effect of inhibiting the tumor growth of BALB / c nude mice bearing tumors and its in vivo safety. Furthermore, it clarifies that this targeted delivery carrier has important applications in the field of gene therapy for tumors.

[0051] The present invention uses chitosan with good biocompatibility as the gene carrier, and introduces ZnO NPs with pH-responsive ability, which plays a role in promoting the release of siRNA and synergistic anti-tumor. The tumor cell membrane coating in the present invention improves the stability and targeting of drug delivery and enhances the gene delivery efficiency. Compared with other carriers, the CCM-CS / ZnO@siSurvivin of the present invention takes into account the release of siRNA and the treatment effect while solving the problem of precise drug delivery, and has the advantages of strong targeting, high efficiency and good effect.

[0052] The following terms appearing in the specification are further explained:

[0053] CS: Chitosan

[0054] CCM: Cancer cell membrane

[0055] ZnO NPs: Zinc oxide nanoparticles

[0056] siSurvivin: Small interfering RNA of Survivin gene

[0057] PDI: Polydispersity index

[0058] ELISA: Enzyme-linked immunosorbent assay

[0059] RPMI 1640: Roswell Park Memorial Institute cell culture medium

[0060] EDTA: Ethylenediaminetetraacetic acid

[0061] MTT: 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

[0062] NC: Non-homologous siRNA

[0063] DiO: Cell membrane green fluorescent probe

[0064] DCFH-DA: 2,7-Dichlorofluorescein diacetate

[0065] WB: Western Blot protein immunoblotting experiment

[0066] Real Time-qPCR: Real-time quantitative reverse transcription-polymerase chain reaction

[0067] SOD: Superoxide dismutase

[0068] GSH: Glutathione

[0069] FITC: Fluorescein isothiocyanate

[0070] PI: Propidium iodide

[0071] H&E: Hematoxylin-eosin staining method Description of the drawings

[0072] Figure 1 . Schematic diagram of the preparation and mechanism of CCM-CS / ZnO@siSurvivin

[0073] Figure 2 . Particle size determination of ZnO NPs

[0074] Figure 3 . Fluorescence spectrum characterization of ZnO NPs

[0075] Figure 4 .Characterization of the ultraviolet absorption spectrum of ZnO NPs.

[0076] Figure 5 .Transmission electron microscope images of ZnO NPs.

[0077] Figure 6 .Particle size changes of nanoparticles before and after coating.

[0078] Figure 7 .Changes in Zeta potential during the synthesis process. ZnO(a), CS / ZnO(b), CS / ZnO@siRNA(c), CCM-CS / ZnO@siRNA(d).

[0079] Figure 8 .Determination of the optimal ratio of chitosan and siRNA by agarose gel electrophoresis.

[0080] Figure 9 .Transmission electron microscope images of each nanoparticle during the synthesis process.

[0081] Figure 10 .Elemental distribution of O, P, and Zn in CCM-CS / ZnO@siSurvivin NPs.

[0082] Figure 11 .Detection of protein concentration by BCA method.

[0083] Figure 12 .Characterization of protein species changes by SDS-PAGE.

[0084] Figure 13 .Characterization of the protection ability of nanoparticles for siRNA by agarose gel electrophoresis.

[0085] Figure 14 .Release at different time points under the conditions of pH = 7.4 and pH = 5.5.

[0086] Figure 15 .Release at 10 min under different pH conditions.

[0087] Figure 16 .DCF fluorescence spectra of solutions at different time points.

[0088] Figure 17 .Storage stability of nanoparticles.

[0089] Figure 18 .Observation of the positions of each component of CCM-CS / ZnO@siSurvivin NPs by laser confocal microscopy.

[0090] Figure 19. Observe the uptake effect of Survivin siRNA by cells using laser confocal microscopy.

[0091] Figure 20 . Observe the uptake effect of Survivin siRNA by cells over time using laser confocal microscopy.

[0092] Figure 21 . Characterize the uptake efficiency of Survivin siRNA by cells using flow cytometry.

[0093] Figure 22 Observe the uptake of siRNA by 4T1 cells, RAW 264.7 cells, and NCTC-929 cells.

[0094] Figure 23 . Characterize the uptake efficiency of Survivin siRNA by cells using flow cytometry.

[0095] Figure 24 . Detect the cytotoxicity of CCM-CS / ZnO@siSurvivin to 4T1 cells by MTT assay (intra-group control)

[0096] Figure 25 . Detect the cytotoxicity of CCM-CS / ZnO@siSurvivin to 4T1 cells by MTT assay (inter-group control)

[0097] Figure 26 . Detect the gene silencing efficiency at the mRNA level by qPCR

[0098] Figure 27 . Detect the gene silencing efficiency at the protein level by WB

[0099] Figure 28 . Detect the intracellular ROS level by DCFH-DA

[0100] Figure 29 . Detect the apoptosis of cells by live / dead cell staining and semi-quantitative statistics

[0101] Figure 30 . Detect the apoptosis of cells by live / dead cell staining

[0102] Figure 31 . Detect the apoptosis of cells by flow cytometry

[0103] Figure 32 . Detect the apoptosis rate of cells by flow cytometry

[0104] Figure 33 . Observe the siRNA distribution in excised organs and tumors at different time points by small animal fluorescence imaging

[0105] Figure 34. Observation of siRNA distribution in excised tumors at each time point by small animal fluorescence imaging

[0106] Figure 35 . Schematic diagram of the administration cycle of the mouse tumor model

[0107] Figure 36 . Changes in the average tumor volume of mice in each group during the administration process

[0108] Figure 37 . Changes in the tumor volume of each mouse during the administration process

[0109] Figure 38 . Mouse tumor images after the administration ends

[0110] Figure 39 . Ratio of tumor mass to body weight of mice after the administration ends

[0111] Figure 40 . TUNEL staining of mouse tumor tissue sections

[0112] Figure 41 . HE staining of mouse tumor tissue sections

[0113] Figure 42 . Immunofluorescence staining of Survivin protein in mouse tumor tissue sections

[0114] Figure 43 . Elisa detection of Survivin protein in mouse tumor homogenates

[0115] Figure 44 . ROS staining of mouse tumor tissue sections

[0116] Figure 45 . Detection of GSH concentration in mouse tumor tissues

[0117] Figure 46 . Detection of SOD concentration in mouse tumor tissues

[0118] Figure 47 . HE staining of major organs of mice after the administration ends

[0119] Figure 48 . Changes in the average body weight of mice in each group during the administration cycle

[0120] Figure 49 . Ratio of the mass of major organs of mice to body weight after the administration ends Detailed implementation manners

[0121] To further illustrate the present invention, a series of embodiments are given below. These embodiments are completely illustrative and are only used to specifically describe the present invention and should not be construed as a limitation to the present invention.

[0122] Example 1 Preparation and Related Characterization of ZnO NPs

[0123] 1. Synthesis of ZnO NPs

[0124] (1) Take 1.09 g of zinc acetate dihydrate (ZnO(OAc)2·2H2O) and add 50 mL of absolute ethanol. Take 0.386 g of sodium hydroxide (NaOH) and add 26 mL of absolute ethanol.

[0125] (2) Under the conditions of oil bath heating and reflux condensation, stir the above solution at 800 rpm until the ethanol boils and then continue heating for 2 h, and cool to room temperature.

[0126] (3) Under the ice bath condition, rapidly stir the ethanol solution of ZnO(OAc)2·2H2O, and drop the ethanol solution of NaOH into the above ethanol solution of ZnO(OAc)2·2H2O through a 0.22 mm microporous filter membrane, and continuously stir rapidly for 4 h to ripen the seeds.

[0127] (4) Add 228 mL of n-hexane to the reaction solution, centrifuge at 10000 rpm for 1 min, discard the supernatant, add 50 mL of absolute ethanol, and disperse ultrasonically to obtain an ethanol solution of ZnO NPs.

[0128] 2. Particle Size Measurement of ZnO NPs

[0129] Experimental Procedure Measure the particle size of the ethanol solution of ZnO NPs prepared above using a nanoparticle size analyzer. Set the solvent as ethanol before testing.

[0130] Experimental Results The measured particle size of ZnO NPs is 7.791 ± 0.228 nm, PDI = 0.105 ± 0.006, the particles are uniform, and the dispersibility is good, which is in line with the particle size of ZnO NPs described in the literature.

[0131] 3. Fluorescence Spectrum Characterization of ZnO NPs

[0132] Experimental Procedure Place the obtained ethanol solution of ZnO NPs under an ultraviolet lamp for observation, and use a fluorescence spectrophotometer to detect its excitation spectrum and emission spectrum.

[0133] Experimental Results The fluorescence spectrum of ZnO NPs shows that ZnO NPs have characteristic peaks at λex = 320 nm and λem = 520 nm, which is in line with the characteristic fluorescence spectrum of ZnO NPs described in the literature.

[0134] 4. Ultraviolet Absorption Spectrum Characterization of ZnO NPs

[0135] Experimental Procedure Use an ultraviolet spectrophotometer to detect the ultraviolet absorption spectrum of the ZnO NPs solution.

[0136] The UV absorption spectrum of ZnO NPs shows that ZnO has a characteristic absorption at 320 nm.

[0137] 5. Transmission electron microscopy (TEM) characterization of ZnO NPs

[0138] Experimental procedure: Take ZnO NPs and observe their morphology with a transmission electron microscope.

[0139] Experimental results: TEM observations revealed that ZnO NPs are spherical particles with a particle size of about 5 nm, smooth surfaces, and uniform sizes. The electron diffraction pattern of ZnO NPs shows obvious characteristic lattice fringes, indicating that the synthesized ZnO NPs have a crystal structure.

[0140] Example 2. Preparation and related characterization of CCM-CS / ZnO@siSurvivin nanoparticles

[0141] 1. Synthesis of CS / ZnO@siSurvivin nanoparticles

[0142] (1) Prepare a DMSO solution of chitosan at 20 mg / mL and a 1 M NaOH solution.

[0143] (2) Take 200 μL of the 1 M NaOH solution and slowly add it drop by drop to the above 2.145 mL of 20 mg / mL chitosan solution under a water bath ultrasonic environment.

[0144] (3) When preparing the nanoparticles, take 60 μL of the chitosan solution prepared above, add 540 μL of DMSO to dilute it to 1 mg / mL. Take 10 μL of a 0.3 μg / μL siRNA solution, mix it with 600 μL of 1 mg / mL chitosan solution at room temperature, then place it in a water bath for ultrasonic treatment for 15 min, and after ultrasonic treatment, place it in a strong stirring for 30 min.

[0145] (4) Take 100 μL of the ZnO NPs prepared above and add it drop by drop to the mixed solution of chitosan and siRNA under the condition of water bath ultrasonic treatment, and the solution continues to be strongly stirred for 4 h.

[0146] (5) After the reaction is completed, centrifuge at 12000 g for 10 min at 4 °C, discard the supernatant to obtain a white precipitate, and disperse the precipitate in 1 mL of deionized water to obtain CS / ZnO@siSurvivin nanoparticles.

[0147] 2. Synthesis of CCM-CS / ZnO@siSurvivin nanoparticles

[0148] (1) Discard the cell culture medium, scrape the cells from 3 dishes, and ultrasonicate for 12 min under the probe sonication of the cell disruptor.

[0149] (2) Centrifuge the cell debris suspension at 700 g for 10 min at 4 °C, discard the precipitate, and take the supernatant and centrifuge at 13000 g for 30 min at 4 °C. Discard the supernatant, redisperse the precipitate in 2 mL of deionized water, and ultrasonicate with the probe for 2 min under ice-water bath conditions.

[0150] (3) Under the condition of water bath sonication, slowly add 1 mL of CS / ZnO@siSurvivin NPs synthesized above to the extracted 2 mL of 4T1 tumor cell membrane solution. Take out the liposome extruder, install a 400 nm polycarbonate membrane and gasket according to the instructions, and repeatedly extrude the above liquid 31 times to obtain uniform nanoparticles encapsulated with 4T1 cell membrane. Centrifuge at 12000 g for 15 min at 4 °C to remove the blank vesicles in the supernatant, and redisperse the precipitate in 3 mL of deionized water to obtain CCM-CS / ZnO@siSurvivin NPs.

[0151] 3. Experimental steps for measuring the particle sizes of CS / ZnO@siSurvivin and CCM-CS / ZnO@siSurvivin Use a nanoparticle size analyzer to measure the particle sizes of the uncoated nanoparticles and the coated nanoparticles prepared from the same batch. Experimental results The particle size changes of the nanoparticles before and after coating were measured by a nanoparticle size analyzer. As Figure 6 shown, the particle size changed from 105.7 nm to 122.4 nm after coating with the cell membrane, an increase of 17.4 nm. This is in line with the thickness of the cell membrane described in the literature.

[0152] 4. Determination of Zeta potential during the synthesis process

[0153] Experimental steps Measure the Zeta potential of ZnO NPs, CS / ZnO NPs, CS / ZnO@siSurvivin NPs, and CCM-CS / ZnO@siSurvivin NPs.

[0154] Experimental results Detect the changes in Zeta potential of each part during the synthesis process. The potential of the synthesized zinc oxide nanoparticles is 14.2 mV. After complexing with the positively charged chitosan, the potential is 23.1 mV. After loading the negatively charged siRNA, the potential becomes lower, 17.5 mV. After externally coating with the tumor cell membrane, the potential reverses to -18.0 mV, indicating that the cell membrane is successfully coated. By detecting the changes in Zeta potential at each step, it can be proved that the loading of ZnO, Survivin siRNA, and chitosan is successful.

[0155] 5. Determination of drug loading and encapsulation efficiency of Survivin siRNA

[0156] Measurement of drug loading of Survivin siRNA by agarose gel electrophoresis

[0157] Experimental procedure

[0158] (1) Preparation of samples with different mass ratios of Survivin siRNA to CS: CS / ZnO@siSurvivin NPs were synthesized according to the mass ratios of siRNA to CS of 0.25:1, 0.5:1, 1:1, 2:1, 3:1, 4:1 (μg:mg).

[0159] (2) Prepare a 10 mg / mL heparin sodium solution. Take 10 μL of the nanoparticle dispersions of each group with different ratios above, add 10 μL of heparin sodium solution, vortex and mix well, then incubate on a shaker for 30 min. Centrifuge at 4°C and 12,000 g for 10 min, take 10 μL of the supernatant free siRNA, add 2 μL of RNA loading buffer (5×), mix well, and then take 10 μL each for separation and visualization by agarose gel electrophoresis.

[0160] The experimental results showed that from 0.25:1 - 3:1 (μg / mg), as the siRNA:CS mass ratio gradually increased, the band color gradually became brighter, indicating that the maximum drug loading of siRNA had not been reached yet. When the siRNA:CS feeding ratio reached 3:1, the brightness reached the maximum, and further increasing the siRNA feeding amount would not increase the encapsulated siRNA, indicating that the drug loading of siRNA reached the maximum when the siRNA:CS mass ratio was 3:1 (μg / mg).

[0161] Measurement of encapsulation efficiency of Survivin siRNA by fluorescence spectrophotometry

[0162] Experimental procedure

[0163] (1) Take 600 μL of the 1 mg / mL chitosan solution described above, add 10 μL of 0.3 μg / mL Cy5-SurvivivinsiRNA solution, and synthesize CS / ZnO@siSurvivin NPs under dark conditions according to the previous steps. Centrifuge at 4°C and 12,000 g for 10 min, discard the supernatant, and redisperse the precipitate in 1 mL of ultrapure water.

[0164] (2) Prepare standard solutions of Cy5-Survivivin siRNA with concentrations of 0.001, 0.005, 0.010, 0.002 μg / mL by adding DEPC water.

[0165] (3) Detect the fluorescence intensity of Cy5 using a fluorescence spectrophotometer (λex = 650 nm, λem = 670 nm), draw a standard curve using the standard solution, and calculate the encapsulation efficiency of Survivin siRNA.

[0166] The experimental results showed that after loading Cy5-fluorescently labeled Survivin siRNA into the nanoparticles, the fluorescence intensities in the nanoparticles and the Cy5-Survivin siRNA standard were measured. Substituting the fluorescence intensity of the nanoparticles into the standard curve, it was found that when the feeding amount was 10 μL of 0.3 μg / μL, the encapsulation efficiency of siRNA was 62.1%.

[0167] 6. Determination of the drug loading and encapsulation efficiency of ZnO

[0168] Experimental procedure

[0169] (1) Dissolve the zinc acetate standard in deionized water and gradient dilute it to prepare standard solutions with different zinc element concentrations.

[0170] (2) Take 600 μL of concentrated hydrochloric acid and place it in a vial, add 200 μL of concentrated nitric acid (add concentrated hydrochloric acid first and then concentrated nitric acid), and then add 200 μL of the synthesized CCM-CS / ZnO@siSurvivin NPs dispersion, ZnO NPs solution, and standard solution respectively. Digest in the fume hood in the dark for 2 days.

[0171] (3) Pipette 200 μL of the digested solution, add deionized water, dilute to 10 mL, and use ICP-OES to measure the zinc element concentration in the standard and sample, draw a standard curve, and calculate the ZnO content in the ZnO solution and the nanoparticles.

[0172] Experimental results: Use ICP-OES to measure the ZnO content in CCM-CS / ZnO@siSurvivin NPs and the ZnO solution. Draw a standard curve through the zinc acetate standard, substitute the sample into the standard curve, calculate the ZnO content and the encapsulation efficiency of ZnO. The ZnO concentration in the ZnO solution was calculated to be 5667.2 mg / L, and the zinc oxide concentration in the CCM-CS / ZnO@siSurvivin NPs sample was 82.4 mg / L. The calculated encapsulation efficiency of zinc oxide was 81.5%.

[0173] 7. Detect changes in the types and concentrations of tumor cell membrane proteins coated

[0174] Experimental procedure

[0175] (1) Extract the total protein of 4T1 tumor cells:

[0176] Add 500 μL of cell lysis buffer to a large dish, shake the dish repeatedly to allow the lysis buffer to come into full contact with the cells for 3 - 5 min. Scrape the cells with a cell scraper and collect them into a centrifuge tube. Incubate on ice for 30 min. Centrifuge at 12,000 g for 5 min at 4 °C, and collect the supernatant, which is the total protein solution.

[0177] (2) Extract the membrane proteins of 4T1 tumor cells:

[0178] According to the above method for extracting cell membranes, first extract the cell membranes from a large dish. Add 500 μL of lysis buffer to the extracted cell membranes, place on ice bath for 30 min, vortex for 30 s every 5 min, centrifuge (4 °C, 12,000 rpm, 10 min), and the supernatant is the cell membrane protein product.

[0179] (3) Extract the proteins in CCM - CS / ZnO@siSurvivin

[0180] Take the CCM - CS / ZnO@siSurvivin nanoparticles coated with cell membranes extracted from the cells in a large dish, add 500 μL of lysis buffer, place on ice bath for 30 min, vortex for 30 s every 5 min, centrifuge (4 °C, 12,000 rpm, 10 min), and transfer the supernatant to a new EP tube to obtain the protein product in the nanoparticles.

[0181] (4) Detect the protein concentration by BCA method

[0182] Prepare the protein standard solution according to the instructions and store it sealed at -20 °C. Mix reagent A and B of BCA in a ratio of 50:1 to make the BCA working solution. Note that the BCA working solution should be prepared freshly before use. Take a 96 - well plate, add 10 μL of the protein standard solution and the test solution to the wells in sequence, and finally add 200 μL of the BCA working solution to each well. Incubate at 37 °C for 30 min. Measure the absorbance at a wavelength of 562 nm using an enzyme - linked immunosorbent assay (ELISA) reader, record the values, plot the standard curve and calculate the protein concentration in each sample.

[0183] (5) Characterize the changes in protein types by SDS - PAGE

[0184] ① Protein denaturation: Preheat the metal bath at 100 °C to mix the protein solution evenly with the reducing loading buffer. Put the protein into the metal bath, boil at 100 °C at 400 rpm for 5 min, cool to room temperature on ice, and store at -20 °C in the refrigerator for later use.

[0185] ② Sample loading and electrophoresis: Prepare the electrophoresis solution: Take 1 packet of electrophoresis solution powder, add 1 L of deionized water, fully dissolve and mix evenly to prepare 1 L of electrophoresis solution, and store it at room temperature for later use. Melt the protein sample and 1× Loading buffer at room temperature or 37 °C, vortex and mix well, and then centrifuge with a hand-held centrifuge. Tear off the bottom strip of the precast gel, with the long side of the gel facing outwards and the short side facing inwards, and clamp it firmly. Fill the inner electrophoresis tank with electrophoresis solution, gently and carefully pull out the comb, and use a pipette to add an appropriate amount of sample to the lane. Supplement the electrophoresis solution in the outer tank to the scale line of the electrophoresis tank. Electrophorese at 110 V - 150 V until the bands are developed to the desired position.

[0186] ③ Coomassie Brilliant Blue staining: Carefully take out the gel after electrophoresis, add Coomassie Brilliant Blue staining solution to cover the entire gel, incubate on a shaker for 4 h to stain the protein. Prepare the decolorizing solution: Mix 250 mL of methanol and 80 mL of acetic acid, and then add deionized water to make up to 1000 mL to obtain the Coomassie Brilliant Blue decolorizing solution. Immerse the gel in the decolorizing solution and decolorize on a shaker for 1 hour, and protein bands can be observed. Carefully discard the decolorizing solution and observe and take pictures.

[0187] The experimental results measured the changes in protein concentration and types of total protein in 4T1 cells, membrane protein in 4T1 cells, and the protein in the preparation encapsulated with 4T1 tumor cell membrane by BCA method and SDS-PAGE. We found that the protein concentration and types in the extracted membrane protein of 4T1 cells and the protein in the preparation encapsulated with 4T1 tumor cell membrane were relatively similar, indicating that during the synthesis process, the protein loss of the cell membrane was less, and it could better play the subsequent immune escape and tumor targeting roles.

[0188] 8. Characterization of the siRNA protection ability of CCM-CS / ZnO@siSurvivin

[0189] Experimental procedures

[0190] (1) Prepare solutions: 5 mg / mL proteinase K solution, 10 mg / mL heparin sodium solution, TBE working solution.

[0191] (2) Prepare samples: Take 5 μL each of the sample solution and free gene solution with the same gene concentration, 5 replicates each. Add 2 μL of RNase A solution (10 μL / mL) respectively, and place the samples in a 37 °C constant temperature incubator for enzymatic digestion. Add 2 μL of proteinase K solution to the samples at specific times to terminate the digestion of RNase A. After all the digestions are terminated, add 10 μL of 10 μL / mL heparin sodium solution to each sample tube, incubate on a shaker for 30 min to displace the genes in the carrier. Centrifuge the samples at 4 °C and 12000 g for 5 min, take 10 μL of the supernatant, and add 2 μL of RNALoading buffer (5×) to prepare for sample loading.

[0192] (3) Agarose gel electrophoresis: Perform electrophoresis according to the method described above and develop the film.

[0193] The experimental results show that the band brightness of free siRNA without carrier protection disappeared after being digested by RNase for 2 h, indicating that siRNA had been degraded by RNase. However, the siRNA in CCM-CS / ZnO@siSurvivin NPs still maintained its brightness after being digested by RNase for 6 h, indicating that the synthesized nanoparticle carrier had good siRNA protection ability, could avoid the phenomenon of siRNA being digested during body fluid transportation, and ensure that Survivin siRNA could be better transported to the target site to exert an effective RNAi effect.

[0194] 9. Experimental steps for the morphology and element distribution of CCM-CS / ZnO@siSurvivin nanoparticles: Place CS / ZnO@siSurvivin NPs, CCM blank vesicles, CCM-CS / ZnO@siSurvivin NPs, and CCM-CS / ZnO@siSurvivin NPs (pH = 5.5) under an electron microscope for observation.

[0195] The experimental results: The morphology of each nanoparticle was observed by transmission electron microscopy. The chitosan and zinc oxide composite nanoparticles loaded with genes were uniform spheres. The extruded blank cell membranes were in the shape of vacuoles. For the nanoparticles coated with cell membranes, spherical particles with a size of about 100 nm could be observed, with clear edges and an obvious core-shell structure. When the pH of the nanoparticles was adjusted with dilute hydrochloric acid, the spherical structure of the nanoparticles was observed to disassemble. This indicates that the synthesized CCM-CS / ZnO@siSurvivin NPs have acid-responsive ability. The nanoparticles maintain a good shape under the physiological pH condition of pH = 7.4, and when in the lysosomal pH environment (pH = 5.5), ZnO disassembles and the nanoparticles lose their original spherical morphology.

[0196] Energy spectrum scanning of the transmission electron microscope found that elements O, P, and Zn were concentrated inside the nanoparticles. As the representative element of ZnO, Zn was widely distributed in the nanoparticles, indicating that ZnO was successfully loaded. P, as the representative element of siRNA and cell membranes, was also widely distributed in the nanoparticles. This indicates that the structure of CCM-CS / ZnO@siSurvivin NPs is complete and all parts have been successfully loaded.

[0197] 10. In vitro release of CCM-CS / ZnO@siSurvivin

[0198] Experimental steps

[0199] (1) Release at different time points under pH = 7.4 and pH = 5.5 conditions: Prepare CCM-CS / ZnO@siSurvivin NPs as described above, divide them into two portions, and adjust the pH of one portion to 5.5 with dilute hydrochloric acid. Divide the two solutions into 8 portions each, incubate them on a water bath shaker at 37 °C at 200 rpm, take them out at specific times, centrifuge at 12000 g for 10 min, take 10 μL of the supernatant, add 2 μL of RNA Loading Buffer (5×), and perform electrophoresis according to the method of agarose gel electrophoresis, and develop the film.

[0200] (2) Release at 10 min under different pH conditions: Prepare CCM-CS / ZnO@siSurvivin NPs as described above, divide them into seven portions, and adjust the pH to 4.5, 5.0, 5.5, 6.0, 6.5, 7.0 with dilute hydrochloric acid respectively. Place the samples in a water bath shaker at 37 °C, incubate at 200 rpm for 10 min, take out the samples, centrifuge at 12000 g for 10 min, take out 10 μL of the supernatant, add 2 μL of RNA Loading Buffer (5×), and perform electrophoresis according to the method of agarose gel electrophoresis, and develop the film.

[0201] Experimental results The experimental results show that with the decrease of pH and the extension of time, more and more siRNA is released, and pH-sensitive release can also be achieved in vivo. The synthesized CCM-CS / ZnO@siSurvivin NPs can remain stable in the body fluid environment and release rapidly in the lysosome environment.

[0202] 11. Characterization of the ability of CCM-CS / ZnO@siSurvivin to promote the accumulation of reactive oxygen species (ROS) in vitro

[0203] Experimental procedure

[0204] (1) Preparation of DCFH-DA working solution: Take 2.5 mg of H2DCFDA, add 865 μL of absolute ethanol to prepare a DCFH-DA stock solution. Take 34.6 μL of the stock solution, add 1384 μL of 0.01 M NaOH solution, and incubate on a shaker at room temperature for 30 min. After the incubation, add 7 mL of PBS buffer solution to prepare a 29 μM DCF solution, store it in the dark at -20 °C sealed for later use.

[0205] (2) Prepare CCM-CS / ZnO@siSurvivin according to the above steps, add 4 mL of DCF solution, incubate in the dark on a water bath shaker at 37 °C, take it out at 2 h, 4 h, 8 h, 20 h, and collect the DCF fluorescence spectrum in the wavelength range of 500 - 700 nm using 490 nm excitation light on a fluorescence spectrophotometer.

[0206] The experimental results were obtained by staining the reactive oxygen species in the CCM-CS / ZnO@siSurvivin NPs solution with the DCF reactive oxygen fluorescence probe. As time extended, the fluorescence peak value of DCF continuously increased, indicating that CCM-CS / ZnO@siSurvivin could promote the accumulation of reactive oxygen species at the in vitro solution level.

[0207] 12. Characterization of the storage stability of CCM-CS / ZnO@siSurvivin

[0208] Experimental procedure: The synthesized encapsulated CCM-CS / ZnO@siSurvivin and unencapsulated CS / ZnO@siSurvivin were dispersed in RPMI 1640 medium and stored at 4°C. The changes in their particle size and PDI were detected at regular intervals.

[0209] Experimental results: The storage stabilities of the encapsulated nanoparticles CS / ZnO@siSurvivin NPs and the unencapsulated nanoparticles CCM-CS / ZnO@siSurvivin NPs at 4°C were characterized respectively. As Figure 19 shown, it was found that both the particle size and PDI of the unencapsulated group increased significantly after 5 days, while the particle size and PDI of the encapsulated group showed no obvious changes within 30 days, indicating that the encapsulation of the cell membrane contributed to the stability of the nanoparticles.

[0210] Example 3. Evaluation of the transfection effect of the CCM-CS / ZnO@siSurvivin gene

[0211] 1. Laser confocal microscopy to observe the positions of each component of CCM-CS / ZnO@siSurvivin NPs during cell uptake

[0212] Experimental procedure

[0213] (1) Preparation of DiO-stained CCM-CS / ZnO@Cy5-siSurvivin NPs: The selected DiO dye was a DMSO solution with a concentration of 1 mM. CCM-CS / ZnO@Cy5-siSurvivin NPs were synthesized according to the steps described above. The obtained nanoparticles were dispersed in 3 mL of deionized water, and then 15 μL of DiO solution was added. They were incubated in a water bath shaker at 37°C for 20 min. Centrifuged at 12,000 g for 10 min at 4°C, and the supernatant was discarded.

[0214] (2) Cell administration: The cells in the confocal dish were administered at a gene concentration of 5 nM. After the cells adhered to the wall, the original medium was aspirated, washed twice, and then the medium containing the drug was added, and the cells were cultured for another 4 h.

[0215] (3) Observe under a confocal microscope. Set the excitation wavelength of Cy5 to 650 nm and the emission wavelength to 670 nm; set the excitation wavelength of ZnO to 320 nm and the emission wavelength to 520 nm; set the excitation wavelength of DiO to 484 nm and the emission wavelength to 501 nm.

[0216] The experimental results are as Figure 20 shown. It can be observed that the fluorescence parts of red siRNA, blue ZnO, and green CCM almost completely overlap during cell uptake. This indicates that during cell uptake, the components of the nanoparticles remain intact and enter the cells as a whole to play their roles.

[0217] 2. Laser confocal observation of the uptake effect of Survivin siRNA by cells

[0218] Experimental steps

[0219] (1) Cell administration: Seed the cells on confocal dishes according to the above method. Set the free genome group, Lipo group, and CCM-CS / ZnO@Cy5-siSurvivin NPs group, control the gene concentration to be the same at 5 nM, and administer the drugs according to the instructions.

[0220] (2) Cell staining: Stain the cell nuclei and lysosomes according to the instructions of Hoechst 33342 and Lyso-Tracker.

[0221] The experimental results are as Figure 19 shown. There is almost no red siRNA in the cells of the free group. This may be because siRNA itself has a negative charge and a relatively large molecular weight, making it difficult to penetrate the cell membrane and be taken up by cells. In addition, siRNA without carrier protection is also easily digested by enzymes during uptake. In the Lipo group, scattered red fluorescence can be seen, indicating that Lipo can be used as a carrier to transfect siRNA into cells. In the CCM-CS / ZnO@siSurvivin group, obvious red siRNA aggregation in the cells can be observed, and the transfection effect is better than that of the positive control Lipo group. This may be because the 4T1 tumor cell membrane covering the surface of the nanoparticles improves their targeting and uptake ability.

[0222] 3. Laser confocal observation of the uptake effect of Survivin siRNA by cells over time

[0223] The experimental steps are according to the above laser confocal experimental method to observe the uptake of cells at different times.

[0224] The experimental results of laser confocal microscopy for observing the uptake of Survivin siRNA by cells over time showed that after 0.5 h of administration, there was little co-localization of Cy5-siRNA in tumor cells, and lysosomes were labeled as green spots. However, as more and more CCM-CS / ZnO@siSurvivin NPs entered the cells over time, the fluorescent spots in lysosomes became blurred and the fluorescence intensity of lysosomes gradually decreased. This may be because the zinc oxide nanoparticles in CCM-CS / ZnO@siSurvivin would release Zn 2+ in acidic lysosomes, disrupting the lysosomes and promoting the escape of Survivin siRNA.

[0225] 4. Characterization of the uptake efficiency of Survivin siRNA by cells using flow cytometry

[0226] Experimental procedures

[0227] (1) Seed the cells in a 6-well plate and administer the drug according to the steps for observing the uptake of Survivin siRNA by cells using laser confocal microscopy as described above.

[0228] (2) After 4 h of administration, gently digest the cells in each well with trypsin, re-disperse them in 0.5 mL of PBS, and use a flow cytometer to detect the intensity of Cy5 fluorescence in the cells.

[0229] Experimental results Cy5-Survivin siRNA was used for administration, and the Cy5 fluorescence intensity in the cells was detected using a flow cytometer. A graph was plotted with the fluorescence intensity on the x-axis and the cell number on the y-axis. As Figure 23 shown, the fluorescence peak of free siRNA was similar to that of the control group, while the peaks of the Lipo group and the formulation group shifted significantly to the right, indicating that the cells in the formulation group had a higher uptake of siRNA and could better introduce siRNA into the cells.

[0230] Example 4. Evaluation of the homologous targeting effect of CCM-CS / ZnO@siSurvivin

[0231] 1. Laser confocal microscopy for observing the uptake of Survivin siRNA by mouse breast cancer cell line 4T1 cells, mouse macrophage cell line RAW 264.1 cells, and mouse fibroblast cell line NCTC-929 cells

[0232] Experimental procedures

[0233] (1) Cultivation of each cell line: Mouse macrophage cell line RAW 264.1 cells were cultured in DMEM complete medium (10% FBS and 1% double antibody), and mouse fibroblast cell line NCTC-929 cells were cultured in MEM complete medium (10% FBS and 1% double antibody). Other cultivation methods were the same as those of 4T1 cells described above.

[0234] (2) Cell seeding in confocal dishes: According to the above method, each cell was seeded on a confocal dish and placed in a CO2 incubator for 24 h to allow the cells to adhere to the dish for standby.

[0235] (3) Cell administration: Each cell was administered at a concentration of 5 nM of gene concentration. After 4 h of administration, the medium containing the drug was discarded, and the nuclei and lysosomes were stained with Hoechst 33342 and Lyso-Tracker Green, and observed under a laser confocal microscope.

[0236] For the uptake experiment using Cy5-labeled Survivin siRNA, it was found that there was less distribution of red siRNA in the cytoplasm of NCTC-929 cells, there were scattered red siRNA distributions in the cytoplasm of RAW 264.7 cells, while the red siRNA in 4T1 cells was significantly and evenly distributed in the cytoplasm, indicating that the uptake amount of siRNA by 4T1 cells was different from that of other cell lines, indicating that CCM-CS / ZnO@siSurvivin had targeting to homologous 4T1 tumor cells.

[0237] 2. Flow cytometry was used to characterize the uptake experiment steps of Survivin siRNA by mouse breast cancer cell line 4T1 cells, mouse macrophage cell line RAW 264.1 cells, and mouse fibroblast cell line NCTC-929 cells. Each cell was seeded in a 6-well plate and administered according to the steps of the above laser confocal observation of the uptake effect of Survivin siRNA by cells. After 4 h of administration, the cells in each well were gently digested with trypsin and redispersed in 0.5 mL of PBS, and the intensity of Cy5 fluorescence in the cells was detected using a flow cytometer.

[0238] For the experimental results, Cy5-Survivin siRNA was used for administration, and the Cy5 fluorescence intensity in the cells was detected using a flow cytometer. A graph was plotted with the fluorescence intensity as the abscissa and the cell number as the ordinate. As Figure 25As shown, the red peak is the control group without drug administration, and the blue peak is the experimental group after 4 h of administration of CCM-CS / ZnO@siSurvivin. The experimental results show that there is little difference in the peak shape and peak position between the drug-administered group and the control group of NCTC-929 cells and RAW 264.7 cells, indicating that the uptake of Cy5-Survivin siRNA by NCTC-929 cells and RAW264.7 cells is small. However, the experimental group peak of 4T1 cells shifted significantly to the right, indicating that the content of Cy5-Survivin siRNA in the experimental group cells is relatively high, that is, the uptake of Cy5-Survivin siRNA by 4T1 cells is relatively high. Similar to the results observed by laser confocal microscopy, it shows that CCM-CS / ZnO@siSurvivin has homologous targeting to 4T1 tumor cells.

[0239] Example 5. Evaluation of the in vitro anti-proliferation effect of CCM-CS / ZnO@siSurvivin

[0240] Experimental procedure: Seed the cells in a 96-well plate and administer the drug at the corresponding concentration after culturing for 24 h. After 24 h of drug administration, add the MTT working solution and continue to incubate for 4 h. Take out the 96-well plate, discard the liquid in the wells, add 200 μL DMSO to each well, and shake vigorously at 37 °C for 15 - 20 min. It can be seen that the crystals in the cells are fully dissolved in DMSO. Set the detection wavelength of the microplate reader to 570 nm and measure the OD value of each well. Calculate the cell survival rate of each well according to

[0241] the following formula.

[0242] Experimental results: Lipo@siSurvivin and CCM-CS / ZnO@NC were set as controls. The Lipo@siSurvivin group uses Lipo 2000, a commercial siRNA carrier. Setting this group can characterize the effect of Survivin siRNA. CCM-CS / ZnO@NC replaces the gene carried by the carrier with a non-functional NC gene to characterize the killing effect of ZnO. As shown in the figure, when comparing within the group, it was found that each group of drugs could inhibit the growth of 4T1 cells at a certain concentration and showed concentration dependence. When comparing between groups, it was found that Lipo@siSurvivin had the least inhibitory effect on the growth of 4T1 cells, followed by CCM-CS / ZnO@NC, while the CCM-CS / ZnO@siSurvivin group had a good inhibitory effect on the growth of 4T1 cells, indicating that ZnO and Survivin siRNA have a synergistic tumor suppression effect.

[0243] Example 6. Experimental procedure for evaluating the gene silencing efficiency of CCM-CS / ZnO@siSurvivin at the mRNA level

[0244] (1) According to the culture method described above, inoculate the cells into a 6-well plate. After the cells adhere to the wall, administer the drug at a concentration of 5 nM according to the gene concentration. After 6 hours of drug administration, discard the drug-containing medium and replace it with an equal volume of complete medium, and continue to incubate in the incubator for 42 hours.

[0245] (2) Take out the 6-well plate, discard the culture solution, add 1 mL of Trizol lysis solution to each well, let it stand for 10 minutes, then pipette and blow several times to transfer the liquid in the well to a nuclease-free centrifuge tube, and let it stand for 5 minutes to fully decompose the nucleic acid-protein complex.

[0246] (3) Pipette 0.2 mL of chloroform into the nuclease-free centrifuge tube and shake vigorously for 15 seconds. Let it stand at room temperature for 3 minutes and then centrifuge. Centrifugation conditions: 4 °C, 12000 rpm, 15 minutes. After centrifugation, the suspension is divided into three layers: the lower layer is the red phenol-chloroform layer, the middle layer is the white protein complex, and the top layer is the aqueous phase. RNA exists in the aqueous phase, accounting for about 50% of the total volume.

[0247] (4) Transfer the upper aqueous phase to another nuclease-free centrifuge tube, add 0.5 mL of isopropanol to each tube, mix well, then let it stand at room temperature for 10 minutes, and then centrifuge. Centrifugation conditions: 4 °C, 12000 rpm, 15 minutes, and a white precipitate can be seen.

[0248] (5) Aspirate the supernatant, add 0.5 mL of 75% ethanol (V 乙醇 / V DEPC = 3:1). Wash the RNA precipitate, shake and mix well, then centrifuge. Centrifugation conditions: 4 °C, 12000 rpm, 20 minutes, repeat the washing three times, pour out the supernatant, and air-dry for later use.

[0249] (6) Pipette 20 μL of DEPC water to resuspend the RNA, pipette and blow 4 times repeatedly, sonicate to completely dissolve the RNA, incubate in a water bath at 55 °C for 15 minutes, let it stand at room temperature for 20 minutes after the water bath, transfer it to a nuclease-free centrifuge tube, and use NanoDrop to detect the concentration of the obtained RNA.

[0250] (7) Sample preparation: The reverse transcription RNA loading amount is 2 μg. Calculate the volume of RNA required for reverse transcription from the known RNA concentration. Pipette 10 μL of 2×RT Buffer, 1 μL of RT EnzymeMix and the corresponding volume of RNA solution into the PCR reaction tube, and then pipette the corresponding volume of DEPC water to make the volume in each well 20 μL. Put the PCR reaction tube into the PCR System 9700, and set the reverse transcription conditions as: 37 °C for 60 minutes, 95 °C for 5 minutes, 4 °C ∞.

[0251] (8) The loading amount of each group of cDNA was 50 ng, and the volume of the cDNA sample to be added was calculated from the measured cDNA concentration. Add 25 μL of Gene Expression Master Mix, 2.5 μL of the primer labeled with the probe (target gene or reference gene GAPDH), and the calculated volume of the cDNA sample into the PCR reaction tube, and then add the corresponding volume of DEPC water to make the volume of each reaction well 50 μL. Centrifuge at 4 °C and 1000 rpm for 3 min. Place the PCR reaction tube into the Real-Time PCR System 7500, and set the conditions as Hold: 50 °C for 2 min, 95 °C for 10 min, Cycle (40 cycles), 95 °C for 15 s, 60 °C for 1 min for amplification. After the amplification is completed, calculate its relative content according to the Ct value.

[0252] The experimental results of RT-PCR showed that the expression rate of Survivin mRNA in the free Survivin siRNA treatment group only decreased slightly to 0.98 ± 0.10. While the expression rate in the CCM-CS / ZnO@siSurvivin treatment group was significantly reduced to 0.40 ± 0.05, even higher than that in the positive control group (0.63 ± 0.10, Lipo@siSurvivin), indicating that CCM-CS / ZnO@siSurvivin has good Survivin gene silencing efficiency at the mRNA level. Example 7. Evaluation of the gene silencing efficiency of CCM-CS / ZnO@siSurvivin at the protein level

[0253] Experimental procedures

[0254] (1) Protein extraction: The protein extraction method was the same as that in the previous SDS-PAGE for protein extraction. After extracting the protein, detect the protein concentration using a BCA kit.

[0255] (2) Protein immunoblotting (Western Blot, WB)

[0256] Electrophoresis: Separate the protein bands according to the electrophoresis method described in the previous SDS-PAGE experiment.

[0257] Transfer membrane: Take 50 mL of rapid transfer membrane solution (20×), add 850 mL of ultrapure water and 100 mL of absolute ethanol, and prepare 1 L of 1× rapid transfer membrane solution for storage at room temperature for later use. Moisten the sponge and filter paper with the transfer membrane solution, and insert the filter paper vertically into the transfer membrane solution to eliminate air bubbles using the siphon phenomenon. Cut a 0.22 μm polyvinylidene fluoride (PVDF) membrane to an appropriate size with a paper cutter, and place it in absolute methanol to activate it for dozens of seconds. Pry open the precast gel with tweezers and cut off the excess gel that does not need to be transferred with a gel cutter. Place the black side of the transfer membrane carbon plate facing down, and arrange the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in order, and use a roller or the like to eliminate air bubbles. Set a constant current of 400 mA and transfer the membrane for 30 min.

[0258] Blocking: Prepare TBST. Transfer the completed PVDF membrane into a suitable container with tweezers, and add an appropriate volume of rapid blocking solution according to the size of the membrane to completely immerse and cover the membrane. Place it on a horizontal shaker and incubate it with shaking at room temperature for 10 min. Take out the blocked membrane and rinse it 2 - 3 times with TBST.

[0259] (3) Antibody incubation: Dilute the primary antibody with the antibody dilution solution. Place the PVDF membrane in an antibody hybridization bag, and add the primary antibody to the hybridization bag with a pipette at a dosage of about 6 mL of the primary antibody per whole membrane. After removing air bubbles, seal it with a sealer and incubate it at 4°C overnight. Store the remaining primary antibody at 4°C for later use. Add an appropriate volume of the secondary antibody to the PVDF membrane to basically immerse and cover the membrane, and incubate it on a shaker at 15 rpm at room temperature for 1 h. Discard the secondary antibody, place it on a side-swing shaker at 37 rpm, and wash it 3 times with 1×TBST, 8 min each time.

[0260] (4) Development: Prepare it immediately before use. Mix solution A and solution B in the ECL chemiluminescence hypersensitive color development kit evenly at a ratio of 1:1, and keep it in the dark at 4°C. Preheat the Bio-Rad gel imager to the detection temperature, place the PVDF membrane in the machine, drop an appropriate amount of developer, and select the exposure duration and method according to the situation for exposure imaging. When the content of the internal reference protein β-actin is almost the same in the experimental results, the Survivin protein band in the free gene administration group is slightly reduced compared with the Control group. Lipo@siSurvivin is used as a positive control, and its Survivin protein band is significantly reduced compared with the Control group. In the experimental group CCM-CS / ZnO@siSurvivin, the Survivin protein band is significantly reduced, and its effect is even more obvious than that of the positive control Lipo@siSurvivin group. This may be because the targeting effect of CCM and the ZnO-promoted lysosomal escape effect greatly enhance the silencing effect of Survivin siRNA.

[0261] Example 8: Evaluation of the Promotion of Reactive Oxygen Species Accumulation in 4T1 Cells by CCM-CS / ZnO@siSurvivin

[0262] Experimental procedure: Seed 4T1 cells in a 6-well plate according to the method described above. After the cells adhere, administer the drug. The drug concentration is 5 nM according to the gene concentration. After 6 hours of drug administration, stain with DCFH-DA probe and observe under a fluorescence microscope.

[0263] Experimental results: DCFH-DA fluorescence probe detected that both CCM-CS / ZnO@NC and CCM-CS / ZnO@siSurvivin could promote the increase of intracellular ROS level, while Lipo@sivivivin had no effect on intracellular ROS level. The results showed that Survivin siRNA in CCM-CS / ZnO@siSurvivin did not have the ability to increase intracellular reactive oxygen species level, while ZnO could promote intracellular reactive oxygen species level.

[0264] Example 9: Evaluation of the Promotion of Apoptosis in 4T1 Cells by CCM-CS / ZnO@siSurvivin

[0265] Experimental procedure

[0266] (1) Detect the apoptosis of cells by live / dead cell staining: Seed 4T1 cells in a 6-well plate according to the method described above. After 6 hours of drug administration, take out the cells. Stain live cells with Calcein-AM and dead cells with PI according to the usage method of the live / dead cell staining kit. Observe under a fluorescence microscope.

[0267] (2) Detect the apoptosis rate of 4T1 cells by flow cytometry: Seed 4T1 cells in a 6-well plate according to the method described above. After 6 hours of drug administration, take out the 6-well plate and collect the cells in the wells. Stain according to the kit instructions. Set the excitation wavelength of the flow cytometer to 488 nm and the emission wavelength to 530 nm. Calculate the apoptosis rate of 4T1 cells in each group.

[0268] Experimental results

[0269] (1) Detection of cell apoptosis by live / dead cell staining showed that there were more green fluorescence regions and fewer red fluorescence regions in the Lipo@siSurvivin group, indicating a lower apoptosis rate of cells. In the CCM-CS / ZnO@NC group, the green fluorescence decreased while the red region increased, indicating that ZnO could effectively promote the apoptosis of 4T1 cells. This apoptosis might be due to ZnO promoting the accumulation of intracellular reactive oxygen species and inducing oxidative stress, which led to cell apoptosis. In the experimental group of CCM-CS / ZnO@siSurvivin, the green fluorescence region significantly decreased while the red region increased, indicating that CCM-CS / ZnO@siSurvivin had a better ability to promote the apoptosis of 4T1 cells. Moreover, the pro-apoptotic effect of the CCM-CS / ZnO@siSurvivin group was better than that of Lipo@siSurvivin and CCM-CS / ZnO@NC, indicating that ZnO and Survivin siRNA had a synergistic ability to promote the apoptosis of 4T1 cells.

[0270] (2) On the scatter plot of the dual-variable flow cytometer, the lower left quadrant represents live cells, (Alexa Fluor488- / PI-); the upper right quadrant represents mid- to late-stage apoptotic cells, (Alexa Fluor 488+ / PI+); the lower right quadrant represents early apoptotic cells, (Alexa Fluor 488+ / PI-); and the upper left quadrant represents dead cells, (Alexa Fluor 488- / PI+). As shown in the figure, due to the pro-apoptotic effect of ZnO NPs, the apoptosis and mortality rate of CCM-CS / ZnO@NC cells were 23.22% ± 1.07%, and the apoptosis and mortality rate of CCM-CS / ZnO@siSurvivin increased to 86.45% ± 2.46%, which was due to the synergistic effect of ZnO and Survivin siRNA.

[0271] Example 10. Evaluation of the in vivo targeting of CCM-CS / ZnO@siSurvivin

[0272] Experimental procedure

[0273] (1) Establishment of a tumor-bearing mouse model: Inoculate 4T1 cells in T75 culture flasks in advance, about 15 flasks in total, and change the culture medium one day before inoculation. Depilate the mice one day in advance. Digest the cells in 15 T75 flasks, centrifuge each tube, and wash the cells twice with PBS. Resuspend the washed cells in 7 mL of blank RPMI 1640 culture medium, count, and obtain a concentration of 2.5×10 7Cell suspension at / mL was mixed evenly and transported on ice. The exposed skin of the mice was disinfected with alcohol cotton balls, and 100 μL of the cell suspension was injected. Blisters could be seen bulging on the skin surface. After the injection, wait for about 10 s and then slowly rotate the needle and pull it out. Observe every day after inoculation and record the tumor volume and body weight. The tumor volume was calculated according to the formula (long diameter × short diameter 2 ) / 2.

[0274] (2) Evaluation of the in vivo targeting of CCM-CS / ZnO@siSurvivin by small animal fluorescence imaging: When the tumors of the tumor-bearing mice reached 100 mm 3 , the animals were taken out. Administration was carried out at the corresponding time points. The dosage was 100 μL / animal, ZnO was 4 mg / kg, and Cy5-siSurvivin was 32 μg / kg. The mice were sacrificed, and their internal organs and tumors were dissected and stored on ice in the dark for observation using a small animal fluorescence imaging system.

[0275] For the experiment results, fluorescence genes labeled with Cy5 were used for administration, and ex vivo photographs were taken at 6 h, 12 h, and 24 h respectively. As shown in the figure, the fluorescence at the tumor site in the nanoparticle group without cell membrane coating reached the highest level 6 h after administration and was gradually cleared at 12 h. However, for the nanoparticles coated with tumor cell membranes, the fluorescence was distributed throughout the tumor site 12 h after administration, and the fluorescence accumulation continued to increase 24 h after administration, indicating that coating with tumor cell membranes is beneficial to the distribution and accumulation of the preparation at the tumor site.

[0276] Example 11. In vivo pharmacodynamic evaluation of CCM-CS / ZnO@siSurvivin

[0277] Experimental procedures

[0278] (1) Evaluation of the in vivo anti-tumor activity of CCM-CS / ZnO@siSurvivin

[0279] The tumor-bearing mouse model was constructed according to the steps described in the previous in vivo targeting evaluation. When the tumors of the tumor-bearing mice reached 100 mm 3 , the animals were randomly divided into 5 groups with 6 animals in each group. Administration was carried out according to the corresponding groups. The dosage was 100 μL / animal, ZnO was 4 mg / kg, and Survivin siRNA was 32 μg / kg. The administration frequency was once every 2 days, and the changes in tumor volume were recorded. After the administration cycle ended, the mice were sacrificed, the main organs and tumor sites were dissected, weighed and recorded, and the tumors were photographed. The tumor tissues of the mice were sectioned and stained with TUNEL and H&E.

[0280] (2) Evaluation of the in vivo gene silencing efficiency of CCM-CS / ZnO@siSurvivin

[0281] After the administration cycle ended, the tumor tissues of the mice were sectioned and subjected to immunofluorescent staining for Survivin protein. Part of the tumor tissues were homogenized, and after extracting the tissue proteins, the expression of Survivin protein was characterized by ELISA.

[0282] (3) Evaluation of the promotion of oxidative stress by CCM-CS / ZnO@siSurvivin in vivo

[0283] After the administration cycle ended, the amount of GSH, the activity of SOD, and the level of reactive oxygen species in the tumor tissues of the mice were detected.

[0284] Experimental results

[0285] (1) Detect the anti-tumor effect of CCM-CS / ZnO@siSurvivin in mice. Model mice were respectively given 0.9% sodium chloride solution (control), naked Survivin siRNA, CCM-CS / ZnO@NC, CS / ZnO@siSurvivin, and CCM-CS / ZnO@siSurvivin. The results showed that the inhibitory effect of naked siSurvivin on tumors was limited. When encapsulated with CS / ZnO@siSurvivin, the anti-tumor effect of Survivin siRNA was not enhanced. This may be because nanoparticles without CCM coating are prone to aggregation in vivo, making it difficult for Survivin siRNA to penetrate tumor tissues. At the same time, CCM-CS / ZnO@NC also has an anti-tumor effect due to the apoptosis-promoting effect of zinc oxide nanoparticles. More notably, CCM-CS / ZnO@siSurvivin has the best tumor suppression effect. After administering 6 doses, the tumors were isolated, and it could be observed with the naked eye that the treatment effect of CCM-CS / ZnO@siSurvivin was the best. The ratios of the isolated tumor mass to body weight for the control, Survivin siRNA, CCM-CS / ZnO@NC, CS / ZnO@siSurvivin, and CCM-CS / ZnO@siSurvivin were 0.062±0.043, 0.052±0.0070, 0.046±0.0040, 0.040, 0.055±0.0067, and 0.023±0.0021, respectively, which also demonstrated the anti-tumor effect of CCM-CS / ZnO@siSurvivin. TUNEL immunofluorescence staining and H&E histopathological staining were used to study the structure and necrosis of tumors after administration. As shown in the figure, due to the function of ZnO NPs, CCM-CS / ZnO@NC would induce tumor cell necrosis, and a few dead cells were stained with green fluorescence. While CCM-CS / ZnO@siSurvivin would cause a large number of cell deaths due to the synergistic effect of ZnO NPs and Survivin siRNA and the targeting effect of CCM. The H&E staining results confirmed that both CCM-CS / ZnO@NC and CCM-CS / ZnO@siSurvivin could loosen the tumor tissue structure. In addition, the tumor cells treated with CCM-CS / ZnO@siSurvivin were arranged more loosely than those treated with CCM-CS / ZnO@NC, further demonstrating the synergistic effect of ZnO NPs and siSurvivin.

[0286] (2) Detect the expression of Survivin protein by immunofluorescence staining. Due to the lack of Survivin siRNA (CCM-CS / ZnO@NC) or the inability of Survivin siRNA to penetrate tumor tissues (naked Survivin siRNA), the silencing effect on Survivin protein is limited. However, for the CCM-CS / ZnO@siSurvivin treatment group, very little Survivin protein was detected, indicating that the nanoparticles can effectively silence the Survivin gene in vivo. Detect the Survivin protein level in the experimental group by ELISA ( Figure 43 ), which further confirmed that CCM-CS / ZnO@siSurvivin has a significant gene silencing effect.

[0287] (3) Immunofluorescence staining experiments confirmed the ROS induction effect of ZnO NPs in the system. The ROS levels in tumor tissues treated with CCM-CS / ZnO@NC and CCM-CS / ZnO@siSurvivin were almost the same, significantly higher than those in the control group and tumor tissues treated with naked Survivin siRNA. In addition, the ROS level in the CS / ZnO@siSurvivin treatment group only increased slightly, which was related to the aggregation of nanoparticles. The oxidative stress level of tumor tissues was characterized by evaluating the amount of GSH and the activity of SOD, and the results were consistent with the ROS immunofluorescence staining. CCM-CS / ZnO@NC and CCM-CS / ZnO@siSurvivin had the same effect on reducing the GSH content and SOD activity, while the effect of CS / ZnO@siSurvivin was limited. When cells are in a state of oxidative stress, the excessive accumulation of ROS can induce apoptosis of tumor cells by activating apoptotic genes, endonucleases and lipid peroxidation. It is suggested that in addition to the gene silencing effect of Survivin siRNA, the oxidative stress level is also involved in the anti-tumor effect of CCM-CS / ZnO@siSurvivin.

[0288] Example 12. In vivo safety evaluation of CCM-CS / ZnO@siSurvivin

[0289] The experimental procedure was to administer drugs according to the drug administration procedure of the previous in vivo pharmacodynamic evaluation and record the body weight changes of each group of mice. After the last drug administration, the main organs of the mice were dissected and weighed, the ratio of the mass of each organ to the body weight of the mice was calculated, and the sections were stained with H&E.

[0290] After the administration, H&E staining of the major organs of the mice did not show obvious abnormalities, and there were no significant differences in the body weight of the mice during the administration process and the ratio of each organ to the body weight of the mice after administration, indicating that the administration did not cause obvious harm to the mice, and CCM-CS / ZnO@siSurvivin has certain in vivo safety.

[0291] The above are only the preferred embodiments of the present invention. It should be noted that the embodiments of the present invention are not limited by the described embodiments. Without departing from the principle of the present invention, several changes, modifications, substitutions, combinations, and simplifications can be made, all of which should be equivalent replacement methods, and these should also be regarded as the protection scope of the present invention.

Claims

1. A pH-sensitive targeted nano-drug delivery system containing Survivin siRNA and ZnO, abbreviated as: CCM-CS / ZnO@siSurvivin.

2. The targeted nano-drug delivery system according to claim 1, characterized in that, It is mainly prepared from the anti-tumor gene therapy drug Survivin siRNA, chitosan oligosaccharide (CS), zinc oxide nanoparticles (ZnO), and 4T1 tumor cell membrane (CCM).

3. The drug delivery system according to claim 1, wherein It has a nanostructure.

4. The drug delivery system according to claim 1, wherein, The preparation method of this system is as follows: Prepare ZnO NPs by the sol-gel method, combine ZnO NPs with CS to encapsulate Survivin siRNA, and then coat CCM by the liposome extrusion method.

5. The preparation method of the drug-loading system according to claim 1, characterized in that, It includes the following steps: (1) Prepare ZnO NPs. The method is as follows: Heat and reflux to prepare ethanol solutions of zinc acetate and sodium hydroxide respectively. Drop the ethanol solution of sodium hydroxide into the ethanol solution of zinc acetate, stir rapidly, and finally precipitate the nanoparticles with n-hexane. (2) Prepare the CS and ZnO NPs composite nanoparticles CS / ZnO@siSurvivin loaded with Survivin siRNA. The method is as follows: Mix Survivin siRNA with the DMSO solution of CS, incubate, add ZnO NPs, and stir to form composite nanoparticles. (3) Prepare the nanoparticles CCM-CS / ZnO@siSurvivin coated with tumor cell membrane. The method is as follows: Extract 4T1 tumor cell membrane by gradient centrifugation, and then coat the cell membrane on the surface of the nanoparticles through a liposome extruder.

6. The preparation method according to claim 5, wherein the ZnO NPs are prepared as follows: (1) Take ZnO(OAc)·2H2O and add anhydrous ethanol; take sodium hydroxide and add anhydrous ethanol. (2) Under the conditions of oil bath heating and condensation reflux, stir the above solutions until the ethanol boils and then continue heating until the solute is completely dissolved, and then stop heating and cool to room temperature. (3) Under the ice bath condition, rapidly stir the ethanol solution of ZnO(OAc)·2H2O, and drop the ethanol solution of NaOH through a microporous filter membrane into the above ethanol solution of ZnO(OAc)·2H2O, and continuously stir rapidly to ripen the seeds. After the reaction ends, the ethanol solution is clear and transparent. (4) Add n-hexane to the ethanol solution, observe that the solution gradually changes from clear to turbid. After the ZnO NPs are completely precipitated, centrifuge to make the nanoparticles precipitate to the bottom of the centrifuge tube, and then add anhydrous ethanol and ultrasonically disperse to obtain the ethanol solution of ZnO NPs.

7. The preparation method according to claim 5, characterized in that, The method for preparing the CS and ZnO NPs composite nanoparticles CS / ZnO@siSurvivin loaded with Survivin siRNA is as follows: (1) Prepare a 20mg / mL chitosan DMSO solution; prepare a 1M NaOH solution; take the 1M NaOH solution and slowly add it dropwise to the above chitosan solution under ultrasonic conditions; take the chitosan solution, add DMSO for dilution; take the siRNA solution and mix it with the chitosan solution at room temperature and perform water bath ultrasound. (2) Take the ZnO NPs prepared above, and dropwise add them into the mixed solution of chitosan and siRNA under the condition of water bath sonication. Stir, centrifuge, discard the supernatant, and obtain a white precipitate. Disperse the precipitate in deionized water.

8. The preparation method according to claim 5, characterized in that, The method for preparing the nanoparticles CCM-CS / ZnO@siSurvivin coated with tumor cell membranes is as follows: (1) Discard the cell culture medium, wash the cell surface, and add PBS buffer; scrape the cells with a cell scraper; after collecting the cells in 3 dishes, sonicate under the probe sonication of a cell disruptor; centrifuge the cell debris suspension; discard the precipitate, centrifuge the supernatant; discard the supernatant, redisperse the precipitate in deionized water, and perform probe sonication to obtain a 4T1 tumor cell membrane solution; (2) Under the condition of water bath sonication, slowly add the above-synthesized CS / ZnO@siSurvivin NPs to the extracted 4T1 tumor cell membrane solution; take out the liposome extruder, repeatedly extrude the above liquid, after the extrusion is completed, centrifuge to remove the blank vesicles in the supernatant, and redisperse the precipitate in deionized water to obtain CCM-CS / ZnO@siSurvivin NPs.

9. The preparation method according to any one of claims 5-8, characterized in that, Among them, The mass ratio of Survivin siRNA to CS is 3:1 (μg / mg).

10. Use of the drug delivery system according to claim 1 in the preparation of a drug for treating or preventing tumors.

Images