Bionic nano drug-loaded vesicle as well as preparation method and application thereof

Abstract

The invention discloses a bionic nano drug-loaded vesicle as well as a preparation method and application thereof, and belongs to the technical field of nano preparations. The preparation method comprises the following steps: enabling a membrane-anchored functional photodynamic nano material (PACP) to interact with a breast cancer-derived cell membrane to realize embedding of a photosensitizer into a tumor cell membrane, collecting the PACP-modified cell membrane, mechanically extruding the PACP-modified cell membrane, and combining the PACP-modified cell membrane with a drug-loaded nanoparticle PLGA-DHA to obtain the bionic drug-loaded nano vesicle. Finally, the bionic photodynamic nano carrier-PACP modified tumor cell membrane vesicle with the breast cancer homing characteristic is prepared, the bionic nano drug-loaded vesicle PLGA-DHA (at) PACP-M is a photosensitizer modified cell membrane vesicle with tumor permeation / membrane fusion, and an endogenous ferroptosis signal can be activated at the same time.

Description

Technical Field

[0001] The present invention belongs to the technical field of nano-preparation, and in particular relates to a bionic nano-drug-loaded vesicle and a preparation method and application thereof. Background Art

[0002] Photodynamic therapy (PDT) is a treatment mode that uses a light source of a specific wavelength to activate photosensitizers and produce exogenous reactive oxygen species (ROS) to induce tumor cell death. Photosensitizers are the key components of PDT. Currently, PDT has been approved by the FDA for the clinical treatment of some tumors and symptom relief. Tumor cell death caused by PDT can change the immunogenicity of tumors and activate specific anti-tumor immune responses, that is, induce immunogenic cell death (ICD). Studies have confirmed that while the photodynamic nanotherapy platform kills tumor cells by generating ROS, it is also accompanied by the secretion and release of damage-associated molecular patterns (DAMPs) such as calreticulin (CRT), adenosine triphosphate (ATP), and high mobility group box 1 (HMGB1), thereby regulating the antigen processing and presentation process, activating specific immune responses, and inducing ICD.

[0003] The effect of conventional cytoplasmic PDT is limited due to the short half-life and limited activity radius of ROS, which in turn limits the anti-tumor immune response. In addition, the immunosuppressive effect of the tumor microenvironment makes it impossible for the PDT-mediated T cell immune response to exert a significant anti-tumor effect.

[0004] In order to enhance the PDT-mediated anti-tumor immune response, it is urgent to develop a new PDT system to efficiently induce immunogenic cell death.

[0005] Cell membrane-localized PDT can accurately deliver ROS to subcellular target sites, effectively solving the problems of short half-life and small active radius of ROS, and improving the bioavailability of ROS and the anti-tumor effect of PDT.

[0006] In addition, the specific plasma membrane damage mediated by membrane-targeted PDT can trigger the rapid release of intracellular contents, thereby promoting the effective release of immunogenic signal DAMPs and enhancing ICD induction. Summary of the invention

[0007] In view of the above-mentioned prior art, the present invention provides a bionic nano drug-loaded vesicle and a preparation method and application thereof, which solves the problems of poor solubility, short biological half-life, low bioavailability and biological barriers in the drug delivery process of dihydroartemisinin (DHA).

[0008] In order to achieve the above object, the technical solution adopted by the present invention is: to provide a method for preparing bionic nano drug-loaded vesicles, comprising the following steps:

[0009] S1: Preparation of photodynamic nanomaterial PACP with tumor cell membrane anchoring function;

[0010] S2: preparing tumor cell membrane fragments, and then co-incubating the tumor cell membrane fragments with PACP to obtain photosensitizer-modified cell membrane vesicles PACP-M with tumor permeability / membrane fusion;

[0011] S3: The dihydroartemisinin solution and the polylactic acid-glycolic acid copolymer solution are mixed and then dripped into distilled water, and PLGA-DHA is obtained by dialysis and freeze-drying; then the PLGA-DHA is mixed with the PACP-M, and then the bionic nano-drug-loaded vesicles PLGA-DHA@PACP-M are obtained by extruding a 200 nm polycarbonate membrane for 3 to 5 times.

[0012] The beneficial effects of the present invention are as follows: the present invention develops a highly efficient photodynamic nanomaterial PACP with tumor cell membrane anchoring potential, and further interacts with the cell membrane derived from breast cancer to form tumor cell membrane vesicles modified by PACP, and then combines with drug-loaded nanoparticles PLGA-DHA to obtain a bionic nano drug-loaded vesicle, because the natural product derivative DHA can inhibit breast cancer cell proliferation, and can also upregulate endogenous Fe in cells by regulating iron metabolism-related proteins and interfering with iron homeostasis. 2+ In view of this, biomimetic nanodrug-loaded vesicles PLGA-DHA@PACP-M were constructed to activate endogenous ferroptosis signals.

[0013] Based on the above technical solution, the present invention can also be improved as follows.

[0014] Further, the preparation of PACP comprises the following steps:

[0015] S11: Carboxylating the methoxy polyethylene glycol to obtain carboxylated methoxy polyethylene glycol mPEG-COOH;

[0016] S12: acetylation of chondroitin sulfate to obtain acetylated chondroitin sulfate AC-CS;

[0017] S13: mPEG-COOH, NHS and EDC were mixed in a mass ratio of 35-45:15-25:10-20, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 70-80 mg:60 mL, and the mixture was reacted at room temperature for 2-3 hours to obtain solution 1; AC-CS and DMAP were mixed in a mass ratio of 4:1, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 100 mg:70-80 mL to obtain solution 2; PpIX, NHS and EDC were mixed in a mass ratio of 15-20:70-80:55-60, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 145-150 mg:30 mL, and the mixture was reacted at room temperature for 3-4 hours to obtain solution 3;

[0018] S14: Solution 1 and solution 2 are mixed in a volume ratio of 6-7:7-8, and reacted at room temperature for 10-15 hours; then solution 3 is added to the system after the reaction, and the reaction is continued at room temperature in the dark for 45-50 hours to obtain; the volume ratio of the added solution 3 to solution 1 is 1:2-3.

[0019] The beneficial effects of adopting further technical solutions are: a photodynamic nanomaterial PACP with potential tumor cell plasma membrane anchoring function is synthesized and prepared. Compared with the free small molecule PpIX, the PACP polymer has stronger conformational flexibility with the assistance of the bulky hydrophilic PEG chain, which makes it have superior membrane anchoring affinity; and due to the steric hindrance of the hydrophilic PEG fragment, PACP has the ability to resist cellular uptake, thus bringing a good membrane anchoring effect, and the lipophilic PpIX molecule can be embedded in the phospholipid bilayer of the plasma membrane, and there is a targeting affinity between AC-CS and the cell membrane CD44 receptor. The combined effect of the three can enhance the binding ability of PACP molecules to the plasma membrane.

[0020] The preparation of tumor cell membrane fragments further comprises the following steps:

[0021] S21: Cultivate tumor cells;

[0022] S22: Collect the cultured tumor cells, place the tumor cells in a hypotonic buffer, and then ultrasonically disrupt the tumor cells 3 to 5 times in an ice bath. Centrifuge the disrupted tumor cells at 500g for 4 minutes, take the supernatant and centrifuge it at 10000g for 10 minutes, and continue to take the supernatant and centrifuge it at 100000g for 1 hour.

[0023] Furthermore, the tumor cells are mouse breast cancer cells 4T1.

[0024] Furthermore, the conditions for co-incubation of tumor cell membrane fragments with PACP are: stirring at 350-450 rpm for 30 min-1 h at room temperature of 25°C.

[0025] Furthermore, tumor cell membrane fragments were co-incubated with PACP and then extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm in sequence to obtain PACP-M.

[0026] The beneficial effects of adopting further technical solutions are: compared with traditional photodynamic nanotherapy platforms, PACP-M has both tumor permeability and membrane fusion, increases the in situ production of ROS and its bioavailability, deeply penetrates the tumor, achieves the effect of simultaneously eliminating surface and deep tumors, and efficiently induces tumor cell ICD. It is a new strategy to achieve more precise and efficient PDT therapy.

[0027] Furthermore, the solvent of the dihydroartemisinin solution is dimethyl sulfoxide, and its concentration is 10-20 mg / mL; the solvent of the polylactic acid-glycolic acid copolymer solution is acetone, and its concentration is 1-2 mg / mL; the volume ratio of the dihydroartemisinin solution to the polylactic acid-glycolic acid copolymer solution is 1:50.

[0028] Furthermore, the mass ratio of PLGA-DHA to the PACP-M is 2 to 3:1.

[0029] The invention also discloses a bionic nano drug-loaded vesicle prepared by a method for preparing the bionic nano drug-loaded vesicle.

[0030] The invention also discloses the application of bionic nano drug-loaded vesicles in preparing products for treating tumors.

[0031] The beneficial effects of the present invention are: the biomimetic nano drug-loaded vesicle PLGA-DHA@PACP-M is both a precise photosensitizer and an efficient drug carrier, which can simultaneously trigger the membrane fusion photodynamic effect and endogenous ferroptosis, promote immunogenic cell death, improve breast cancer immunogenicity, increase T cell tumor infiltration, and provide a new option for ICIs combined therapy. It is expected to improve drug delivery efficiency and enhance the efficacy of immune checkpoint inhibitors, which will help the transformation of nano drugs for clinical tumor treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The preparation flow chart of biomimetic nano drug-loaded vesicles PLGA-DHA@PACP-M;

[0033] Figure 2 The fluorescence distribution images of PACP of Example 1, ACP and PpIX of Comparative Example 1 incubated with 4T1 cells for different time periods;

[0034] Figure 3 TEM image of PACP-M of Example 1;

[0035] Figure 4This is a fluorescence microscopic image of PACP-M in Example 1 after being stained with a fluorescent probe DiO film;

[0036] Figure 5 This is a confocal fluorescence image of PACP-M in Example 1 after co-culture with 4T1 breast cancer cells for 1 hour;

[0037] Figure 6 This is a fluorescent layer scanning image after PACP-M in Example 1 was co-incubated with 4T1 cell spheres for 24 hours;

[0038] Figure 7 CLSM photo of PLGA-DHA@PACP-M of Example 1 for targeted phagocytosis of breast cancer cells;

[0039] Figure 8 This is a FACS quantitative analysis diagram of the PLGA-DHA@PACP-M of Example 1 targeted phagocytosis of breast cancer cells;

[0040] Fig. 9 It is a fluorescence intensity quantitative graph of the flow cytometry results of Experimental Example 6;

[0041] Fig.10 CLSM photos of ROS generation in breast cancer cells;

[0042] Fig.11 This is a FACS quantitative analysis of ROS production in breast cancer cells;

[0043] Fig.12 This is the statistical graph of cell survival rate in Experimental Example 7;

[0044] Fig.13 Fluorescence image of live / dead cells. DETAILED DESCRIPTION

[0045] The specific implementation modes of the present invention are described in detail below with reference to the embodiments.

[0046] Example 1

[0047] A method for preparing biomimetic nano drug-loaded vesicles, the preparation process is as follows Figure 1 As shown, the specific steps include:

[0048] (1) Preparation of photodynamic nanomaterial PACP with tumor cell membrane anchoring function:

[0049] S1: mPEG2000 (16 g, 0.003 mol), succinic anhydride (13 g, 0.03 mol) and dimethylaminopyridine (0.5 g, 0.012 mol) were dissolved in 200 mL of dried chloroform, heated to 65 ° C in an oil bath, kept warm for 48 hours, and condensed with condensed water. After the reaction, it was cooled to room temperature, filtered with filter paper, and the solvent was removed by rotary evaporation. The residual white solid was dissolved in 20 mL of 1M hydrochloric acid and extracted with chloroform. After extraction, the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was precipitated with ether and dried in vacuum to obtain mPEG-COOH;

[0050] S2: Weigh 45 mg of CS (chondroitin sulfate) and disperse it in 25 mL of formamide, and stir quickly to mix; then add 36 μL of pyridine and 22 μL of acetic anhydride to the mixed solution, and stir and react at room temperature for 24 hours until the solution becomes clear and transparent; transfer the mixed reaction solution to a dialysis bag with a molecular cutoff of 1000, and dialyze with deionized water for 4 days to obtain a purified reactant; after the dialysis is completed, the solution is freeze-dried to obtain AC-CS;

[0051] S3: 40 mg mPEG-COOH, 20 mg NHS (N-hydroxysuccinimide) and 15 mg EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) were mixed, and the mixture was dispersed in 60 mL DMSO, and reacted at room temperature for 2.5 h to obtain solution 1; 80 mg AC-CS and 20 mg DMAP were mixed, and the mixture was dispersed in 75 mL DMSO to obtain solution 2; 16 mg PpIX (protoporphyrin), 75 mg NHS and 58 mg EDC were mixed, and the mixture was dispersed in 30 mL DMSO, and stirred at room temperature for 3.5 h to obtain solution 3;

[0052] S4: Mix solution 1 and solution 2, react at room temperature for 12 hours, then add solution 3 to the system after the reaction, continue to stir and react at room temperature in the dark for 48 hours, to obtain the product.

[0053] (2) Preparation of photosensitizer-modified cell membrane vesicles PACP-M with tumor permeability / membrane fusion properties:

[0054] S1: Mouse breast cancer cells 4T1 were used as the cell membrane collection object. After the cells filled the culture bottle, the cells were collected for use. The collected 4T1 cells were placed in a 0.25M sucrose solution, and then the 4T1 cells were ultrasonically disrupted 4 times in an ice bath. The disrupted 4T1 cells were centrifuged at 500g for 4 minutes, the supernatant was centrifuged at 10000g for 10 minutes, and the supernatant was further centrifuged at 100000g for 1 hour to obtain tumor cell membranes;

[0055] S2: The tumor cell membrane was co-incubated with PACP (at 25°C, with stirring at 400 rpm for 45 min), and after the incubation, the mixture was centrifuged at 1000 g for 10 min, the lower precipitate was removed, and the cell membrane was resuspended in PBS solution to obtain the PACP-modified tumor cell membrane;

[0056] S3: The PACP-modified tumor cell membrane is passed through polycarbonate membranes with pore sizes of 400 nm and 200 nm successively to obtain PACP-M.

[0057] (3) Preparation of biomimetic nano drug-loaded vesicles PLGA-DHA@PACP-M:

[0058] S1: Mix a dimethyl sulfoxide solution (15 mg / mL) of dihydroartemisinin (DHA) and an acetone solution (1.5 mg / mL) of poly(lactic acid-co-glycolic acid) at a volume ratio of 1:50, drop into distilled water, and obtain PLGA-DHA by dialyzing and freeze-drying;

[0059] S2: PLGA-DHA and PACP-M were mixed at a mass ratio of 2.5:1, and then passed through a 200 nm polycarbonate membrane 4 times to obtain PLGA-DHA@PACP-M.

[0060] Example 2

[0061] A method for preparing biomimetic nano drug-loaded vesicles, the preparation process is as follows Figure 1 As shown, the specific steps include:

[0062] (1) Preparation of photodynamic nanomaterial PACP with tumor cell membrane anchoring function:

[0063] S1: mPEG2000 (16 g, 0.003 mol), succinic anhydride (13 g, 0.03 mol) and dimethylaminopyridine (0.5 g, 0.012 mol) were dissolved in 200 mL of dried chloroform, heated to 65 ° C in an oil bath, kept warm for 48 hours, and condensed with condensed water. After the reaction, it was cooled to room temperature, filtered with filter paper, and the solvent was removed by rotary evaporation. The residual white solid was dissolved in 20 mL of 1M hydrochloric acid and extracted with chloroform. After extraction, the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was precipitated with ether and dried in vacuum to obtain mPEG-COOH;

[0064] S2: Weigh 45 mg of CS (chondroitin sulfate) and disperse it in 25 mL of formamide, and stir quickly to mix; then add 36 μL of pyridine and 22 μL of acetic anhydride to the mixed solution, and stir and react at room temperature for 24 hours until the solution becomes clear and transparent; transfer the mixed reaction solution to a dialysis bag with a molecular cutoff of 1000, and dialyze with deionized water for 4 days to obtain a purified reactant; after the dialysis is completed, the solution is freeze-dried to obtain AC-CS;

[0065] S3: 35 mg mPEG-COOH, 25 mg NHS (N-hydroxysuccinimide) and 10 mg EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) were mixed, and the mixture was dispersed in 60 mL DMSO, and reacted at room temperature for 2 h to obtain solution 1; 80 mg AC-CS and 20 mg DMAP were mixed, and the mixture was dispersed in 70 mL DMSO to obtain solution 2; 20 mg PpIX (protoporphyrin), 80 mg NHS and 60 mg EDC were mixed, and the mixture was dispersed in 30 mL DMSO, and stirred at room temperature for 3 h to obtain solution 3;

[0066] S4: Mix solution 1 and solution 2, react at room temperature for 10 hours, then add solution 3 to the system after the reaction, continue to stir and react at room temperature in the dark for 45 hours, to obtain the product.

[0067] (2) Preparation of photosensitizer-modified cell membrane vesicles PACP-M with tumor permeability / membrane fusion properties:

[0068] S1: Mouse breast cancer cells 4T1 were used as the cell membrane collection object. After the cells filled the culture bottle, the cells were collected for use. The collected 4T1 cells were placed in a 0.25M sucrose solution, and then the 4T1 cells were ultrasonically disrupted 4 times in an ice bath. The disrupted 4T1 cells were centrifuged at 500g for 4 minutes, the supernatant was centrifuged at 10000g for 10 minutes, and the supernatant was further centrifuged at 100000g for 1 hour to obtain tumor cell membranes;

[0069] S2: The tumor cell membrane was co-incubated with PACP (at 25°C, stirred at 350 rpm for 1 h), and after the incubation, the mixture was centrifuged at 1000 g for 10 min, the lower precipitate was removed, and the cell membrane was resuspended in PBS solution to obtain the PACP-modified tumor cell membrane;

[0070] S3: The PACP-modified tumor cell membrane is passed through polycarbonate membranes with pore sizes of 400 nm and 200 nm successively to obtain PACP-M.

[0071] (3) Preparation of biomimetic nano drug-loaded vesicles PLGA-DHA@PACP-M:

[0072] S1: Mix a dimethyl sulfoxide solution (10 mg / mL) of dihydroartemisinin (DHA) and an acetone solution (1 mg / mL) of poly(lactic acid-co-glycolic acid) at a ratio of 1:50, drop into distilled water, and dialyze and freeze-dry to obtain PLGA-DHA;

[0073] S2: PLGA-DHA and PACP-M were mixed in a mass ratio of 2:1, and then passed through a 200 nm polycarbonate membrane 4 times to obtain PLGA-DHA@PACP-M.

[0074] Example 3

[0075] A method for preparing biomimetic nano drug-loaded vesicles, the preparation process is as follows Figure 1 As shown, the specific steps include:

[0076] (1) Preparation of photodynamic nanomaterial PACP with tumor cell membrane anchoring function:

[0077] S1: mPEG2000 (16 g, 0.003 mol), succinic anhydride (13 g, 0.03 mol) and dimethylaminopyridine (0.5 g, 0.012 mol) were dissolved in 200 mL of dried chloroform, heated to 65 ° C in an oil bath, kept warm for 48 hours, and condensed with condensed water. After the reaction, it was cooled to room temperature, filtered with filter paper, and the solvent was removed by rotary evaporation. The residual white solid was dissolved in 20 mL of 1M hydrochloric acid and extracted with chloroform. After extraction, the organic phase was dried with anhydrous sodium sulfate and filtered. The filtrate was precipitated with ether and dried in vacuum to obtain mPEG-COOH;

[0078] S2: Weigh 45 mg of CS (chondroitin sulfate) and disperse it in 25 mL of formamide, and stir quickly to mix; then add 36 μL of pyridine and 22 μL of acetic anhydride to the mixed solution, and stir and react at room temperature for 24 hours until the solution becomes clear and transparent; transfer the mixed reaction solution to a dialysis bag with a molecular cutoff of 1000, and dialyze with deionized water for 4 days to obtain a purified reactant; after the dialysis is completed, the solution is freeze-dried to obtain AC-CS;

[0079] S3: 45 mg mPEG-COOH, 15 mg NHS (N-hydroxysuccinimide) and 20 mg EDC (1-ethyl-(3-dimethylaminopropyl) carbodiimide) were mixed, and the mixture was dispersed in 60 mL DMSO, and reacted at room temperature for 3 h to obtain solution 1; 80 mg AC-CS and 20 mg DMAP were mixed, and the mixture was dispersed in 80 mL DMSO to obtain solution 2; 15 mg PpIX (protoporphyrin), 70 mg NHS and 55 mg EDC were mixed, and the mixture was dispersed in 30 mL DMSO, and stirred at room temperature for 4 h to obtain solution 3;

[0080] S4: Mix solution 1 and solution 2, react at room temperature for 15 hours, then add solution 3 to the system after the reaction, continue to stir and react at room temperature in the dark for 50 hours, to obtain the product.

[0081] (2) Preparation of photosensitizer-modified cell membrane vesicles PACP-M with tumor permeability / membrane fusion properties:

[0082] S1: Mouse breast cancer cells 4T1 were used as the cell membrane collection object. After the cells filled the culture bottle, the cells were collected for use. The collected 4T1 cells were placed in a 0.25M sucrose solution, and then the 4T1 cells were ultrasonically disrupted 4 times in an ice bath. The disrupted 4T1 cells were centrifuged at 500g for 4 minutes, the supernatant was centrifuged at 10000g for 10 minutes, and the supernatant was further centrifuged at 100000g for 1 hour to obtain tumor cell membranes;

[0083] S2: The tumor cell membrane was co-incubated with PACP (at 25°C, stirred at 450 rpm for 30 min), and after the incubation, the mixture was centrifuged at 1000 g for 10 min, the lower precipitate was removed, and the cell membrane was resuspended in PBS solution to obtain the PACP-modified tumor cell membrane;

[0084] S3: The PACP-modified tumor cell membrane is passed through polycarbonate membranes with pore sizes of 400 nm and 200 nm successively to obtain PACP-M.

[0085] (3) Preparation of biomimetic nano drug-loaded vesicles PLGA-DHA@PACP-M:

[0086] S1: Mix a dimethyl sulfoxide solution (20 mg / mL) of dihydroartemisinin (DHA) and an acetone solution (2 mg / mL) of poly(lactic acid-co-glycolic acid) at a ratio of 1:50, drop into distilled water, and obtain PLGA-DHA by dialyzing and freeze-drying;

[0087] S2: PLGA-DHA and PACP-M were mixed in a mass ratio of 3:1, and then passed through a 200 nm polycarbonate membrane 4 times to obtain PLGA-DHA@PACP-M.

[0088] Comparative Example

[0089] The preparation of ACP comprises the following steps:

[0090] 40 mg of AC-CS prepared in Example 1 and 60 mg of 4-dimethylaminopyridine were dispersed in 70 mL of DMSO, and then 15 mg of PpIX, 55 mg of N-hydroxysuccinimide (NHS) and 55 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) were added respectively. The mixture was stirred at room temperature in the dark until PpIX was completely reacted. The reaction mixture was then transferred to a dialysis bag with a molecular cutoff of 1000, dialyzed with deionized water for 4 days, and then freeze-dried to obtain the product.

[0091] The performance effects of the products prepared in Examples 1 to 3 are similar, and the subsequent experimental examples are the products prepared in Example 1.

[0092] Experimental Example 1

[0093] Membrane anchoring effect of PACP: 4T1 tumor cells (1×10 5 ) were inoculated into a confocal dish, and then PpIX (10 μg / mL), ACP of the comparative example, and PACP of step (1) of Example 1 were added and cultured with the cells for 30 min, 1 h, and 2 h, respectively, and finally fluorescence observation and photography were performed by CLSM. At the same time, in order to quantitatively evaluate the anchoring efficiency of PACP on the tumor cell membrane, 4T1 cells treated with PACP samples were digested and collected with EDTA-free trypsin, and then the fluorescence intensity of PpIX in the cells was detected by flow cytometry (FACS) under the 633 nm laser channel, and the relative PACP membrane anchoring efficiency was estimated by the fluorescence intensity ratio of the sample group to the blank group.

[0094] The experimental results are as follows Figure 2 As shown, after PACP self-assembled nanoparticles were engulfed by breast cancer tumor cells, most of the PpIX-labeled fluorescence was localized on the cell membrane, while ACP nanoparticles without PEG modification entered the tumor cells more, reflecting that PACP may have a potential membrane anchoring effect.

[0095] Experimental Example 2

[0096] Morphological structure analysis of PACP-M: PACP-M in step (2) of Example 1 was analyzed using a transmission electron microscope. The experimental results are as follows: Figure 3As shown, the average particle size of the prepared PACP-M is about 150nm, and it has a certain thickness of plasma membrane and hollow structure; the membrane vesicles appear non-standard round, which may be due to its hollow structure making the vesicles deformable.

[0097] Experimental Example 3

[0098] PACP-M FACS analysis: 1 mL of lipophilic membrane dye DiO (1 mg / mL) was added to 1 mL of PACP-M and stained for 15 min. Then, it was dropped onto a glass slide and placed under FM to observe the fluorescence signal on the cell membrane structure. The experimental results are shown in Figure 4 As shown, it can be found that PACP labeled with red fluorescence by PpIX and cell membrane labeled with green fluorescence by DiO appear yellow due to fluorescence superposition, proving that the polymer material PACP is attached to the PACP-M structure.

[0099] Experimental Example 4

[0100] PACP-M and breast cancer cell fusion experiment: 4T1 cells (1×10 5 ) were inoculated into the confocal dish, and then the PACP-M sample and cells were added and cultured for 1 hour, and then the lipophilic membrane dye DiO solution (10 μM) was added. After staining for 15 minutes, the fluorescence signal on the 4T1 cell membrane was observed by CLSM (PpIX: red fluorescence; DiO: green fluorescence); the experimental results are shown in Figure 5 As shown, an obvious PpIX red fluorescence signal was observed on the tumor cell membrane after PACP-M treatment, and co-localized with the membrane dye DiO green fluorescence signal, proving that the PACP-M membrane vesicles can fuse with the breast cancer cell membrane, thereby fixing the photosensitizer attached to the nanovesicles to the tumor plasma membrane, which will be conducive to the in situ generation of ROS at the membrane and realize membrane-localized photodynamic-mediated plasma membrane structure damage.

[0101] Experimental Example 5

[0102] In vitro tumor penetration experiment of PACP-M: 1% agarose solution was prepared and heated to dissolve. After high-pressure sterilization, the agarose solution was taken out and placed in a 6-well plate in a clean bench and allowed to cool and solidify. Then 4 mL of culture medium was added to each well, and 4T1 cells (1×10 6 ), placed in a cell culture incubator and continued to incubate for about 1 week to form a tumor cell sphere model; after the cell sphere is formed, the 6-well plate is taken out, and PACP-M of Example 2 is added to the wells, and the culture is continued for 48 hours. After the co-culture is completed, the distribution of PpIX red fluorescence in the 4T1 cell sphere is observed by CLSM. The experimental results are as follows Figure 6As shown, the tumor cell spheres treated with PACP-M have obvious PpIX red fluorescence distributed from the outside to the inside, indicating that the cell membrane vesicles can successfully help PACP molecules penetrate into the interior of the cell spheres, reflecting that PACP-M membrane vesicles have the potential for deep tumor penetration.

[0103] Experimental Example 6

[0104] Targeting ability test 1: The poorly soluble anticancer drug doxorubicin (DOX) was loaded through the PLGA-DHA@PACP-M assembly of step (3) of Example 1 to obtain a light-responsive nanomedicine, and the experimental subject was MCF-7 breast cancer tumor cells.

[0105] The above experimental examples prove that PACP-M can form spherical nanoparticles with uniform particle size and generate ROS under 635nm light, mediating the breakage of the polysaccharide skeleton of chondroitin sulfate, further inducing the disassembly of nanoparticles to accelerate drug release. CLSM can further reflect the targeting ability of PLGA-DHA@PACP-M, such as Figure 7 As shown in the figure, the PLGA-DHA@PACP-M targeted micelle group has a strong PpIX red fluorescence, which proves that the targeting of PLGA-DHA@PACP-M micelles to doxorubicin-resistant MCF-7 breast cancer tumor cells mediates the increase in the amount of micelle phagocytosis. In addition, the flow cytometry results are shown in Figure 8 and Fig. 9 As shown, compared with the naked drug PpIX group and the DEP non-targeted group, the PpIX fluorescence intensity of the PLGA-DHA@PACP-M micelle group was significantly increased, and its average fluorescence intensity values ​​were 4.2 times and 3.1 times that of the other two groups, respectively, both showing significant differences, further confirming the fluorescence results of CLSM, indicating that PLGA-DHA@PACP-M micelles have cell targeting capabilities.

[0106] Experimental Example 7

[0107] Targeting ability test 2: After loading the drug, PLGA-DHA@PACP-M generates ROS in cells through photodynamic conversion, thereby inducing cell death, such as Fig.10 As shown in the figure, after illumination, PLGA-DHA@PACP-M produced a strong green fluorescence signal in the doxorubicin-resistant MCF-7 breast cancer tumor cells, indicating that the intracellular PLGA-DHA@PACP-M can produce a large amount of ROS under light activation, and most of the green fluorescence signals are distributed in the cytoplasm and nucleus, suggesting that the ROS generated by light activation can not only enter the cytoplasm, but also act on the nucleus. Further flow cytometry was used to quantitatively analyze the intracellular ROS content. Fig.11It can be found that the average fluorescence intensity of ROS in the irradiated PLGA-DHA@PACP-M group is significantly stronger than that in the PpIX group, which is 4.9 times that of the PpIX group. This quantitative data strongly supports the CLSM results, both of which indicate that intracellular ACP micelles can produce a large amount of ROS under light.

[0108] according to Fig.12 and Fig.13 It can be seen that the irradiated PLGA-DHA@PACP-M produced 39.3% cytotoxicity in doxorubicin-resistant MCF-7 breast cancer tumor cells, and the number of dead cells with red fluorescence increased with the increasing concentration of micelles. These results fully prove that ROS can effectively overcome the multidrug resistance of tumors by directly causing the death of multidrug-resistant tumor cells.

[0109] Although the specific implementation of the present invention is described in detail in conjunction with the embodiments, it should not be understood as limiting the scope of protection of this patent. Within the scope described in the claims, various modifications and variations that can be made by those skilled in the art without creative work still fall within the scope of protection of this patent.

Claims

1. A method for preparing biomimetic nano drug-loaded vesicles, characterized in that: The following steps are involved: S1: Preparation of photodynamic nanomaterial PACP with tumor cell membrane anchoring function; S2: preparing tumor cell membrane fragments, and then co-incubating the tumor cell membrane fragments with PACP to obtain photosensitizer-modified cell membrane vesicles PACP-M with tumor permeability / membrane fusion; S3: The dihydroartemisinin solution and the polylactic acid-glycolic acid copolymer solution are mixed and then dripped into distilled water, and PLGA-DHA is obtained by dialysis and freeze-drying; then the PLGA-DHA is mixed with the PACP-M, and then extruded through a 200 nm polycarbonate membrane for 3 to 5 times to obtain the bionic nano drug-loaded vesicles PLGA-DHA@PACP-M.

2. The method for preparing the biomimetic nano drug-loaded vesicles according to claim 1, characterized in that: The preparation of the PACP comprises the following steps: S11: Carboxylating the methoxy polyethylene glycol to obtain carboxylated methoxy polyethylene glycol mPEG-COOH; S12: acetylation of chondroitin sulfate to obtain acetylated chondroitin sulfate AC-CS; S13: mPEG-COOH, NHS and EDC were mixed in a mass ratio of 35-45:15-25:10-20, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 70-80 mg:60 mL, and the mixture was reacted at room temperature for 2-3 hours to obtain solution 1; AC-CS and DMAP were mixed in a mass ratio of 4:1, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 100 mg:70-80 mL to obtain solution 2; PpIX, NHS and EDC were mixed in a mass ratio of 15-20:70-80:55-60, and the mixture was dispersed in an organic solvent at a solid-liquid ratio of 140-160 mg:30 mL, and the mixture was reacted at room temperature for 3-4 hours to obtain solution 3; S14: Solution 1 and solution 2 are mixed in a volume ratio of 6-7:7-8, and reacted at room temperature for 10-15 hours; then solution 3 is added to the system after the reaction, and the reaction is continued at room temperature in the dark for 45-50 hours to obtain; the volume ratio of the added solution 3 to solution 1 is 1:2-3.

3. The method for preparing the biomimetic nano drug-loaded vesicles according to claim 1, characterized in that: The preparation of the tumor cell membrane fragments comprises the following steps: S21: Cultivate tumor cells; S22: Collect the cultured tumor cells, place the tumor cells in a hypotonic buffer, and then ultrasonically disrupt the tumor cells 3 to 5 times in an ice bath. Centrifuge the disrupted tumor cells at 500g for 4 minutes, take the supernatant and centrifuge it at 10000g for 10 minutes, and continue to take the supernatant and centrifuge it at 100000g for 1 hour.

4. The method for preparing the biomimetic nano drug-loaded vesicles according to claim 3, characterized in that: The tumor cells are mouse breast cancer cells 4T1.

5. The method for preparing the biomimetic nano drug-loaded vesicles according to claim 1, characterized in that: The conditions for co-incubation of tumor cell membrane fragments with PACP are: at room temperature of 25°C, stirring at a low speed of 350-450 rpm for 30 min-1 h.

6. The method for preparing the biomimetic nano drug-loaded vesicles according to claim 5, characterized in that: Tumor cell membrane fragments were co-incubated with PACP and then extruded through polycarbonate membranes with pore sizes of 400 nm and 200 nm in sequence to obtain PACP-M.

7. The method for preparing biomimetic nano drug-loaded vesicles according to claim 1, characterized in that: The solvent of the dihydroartemisinin solution is dimethyl sulfoxide, and its concentration is 10-20 mg / mL; the solvent of the polylactic acid-glycolic acid copolymer solution is acetone, and its concentration is 1-2 mg / mL; the volume ratio of the dihydroartemisinin solution to the polylactic acid-glycolic acid copolymer solution is 1:

50.

8. The method for preparing biomimetic nano drug-loaded vesicles according to claim 1, characterized in that: The mass ratio of the PLGA-DHA to the PACP-M is 2 to 3:

1.

9. The bionic nano drug-loaded vesicles prepared by the method for preparing the bionic nano drug-loaded vesicles according to any one of claims 1 to 8.

10. Use of the bionic nano drug-loaded vesicles according to claim 9 in preparing products for treating tumors.

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