Immune activation anti-tumor bionic nano material as well as preparation method and application thereof

Abstract

The invention discloses an immune activation anti-tumor bionic nanomaterial and a preparation method and application thereof.The preparation method includes the steps that paclitaxel-sourced carbon dots (PCDs) are synthesized through a hydrothermal method, and the PCDs can inhibit tumor cell proliferation and migration and induce tumor cells ICD; then, assembling the M (at) P-In with Indoximod to form a nano-composite P-In; and finally, coating the P-In with a tumor cell membrane to form the bionic nano-composite material M (at) P-In. The M (at) P-In can inhibit the activity of IDO-1 while inducing ICD so as to alleviate the tumor immunosuppression microenvironment; and the nano complex is endowed with M (at) P-In tumor targeting capability and BBB penetrating capability by virtue of bionic wrapping of the cell membrane.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical and nanomaterials, and particularly relates to an immune-activated anti-tumor biomimetic nanomaterial, a preparation method and application thereof. BACKGROUND

[0002] Glioma is the most common intracranial primary malignant tumor, accounting for about 80% of all brain malignancies, and is the main cause of death from primary brain tumors. Among them, glioblastoma (GBM) is the highest degree of malignancy. Therefore, it is urgent to develop an efficient glioma treatment strategy.

[0003] Immunotherapy has become a powerful glioma treatment strategy, which can kill tumors through the host immune system. Chemotherapeutic drugs such as paclitaxel, oxaliplatin, doxorubicin and mitoxantrone can induce immunogenic cell death and release calreticulin (CRT), adenosine triphosphate (ATP) and high mobility group box 1 (HMGB1). These released molecules overactivate dendritic cells and recruit cytotoxic T lymphocytes to enhance anti-tumor immunity. However, the function of T cells will be inhibited by the immunosuppressive microenvironment mediated by the overexpression of IDO-1 in tumor tissue. Therefore, the co-delivery of ICD (immunogenic cell death) inducers and IDO-1 inhibitors can inhibit tumor growth by simultaneously enhancing immunity and intervening in the immunosuppression in the tumor microenvironment.

[0004] Paclitaxel is a first-line chemotherapeutic drug with strong anti-tumor effect. However, the water solubility and blood-brain barrier (BBB) permeability of paclitaxel limit its application in glioma treatment, and need to be modified to enhance its BBB permeability and therapeutic effect. Carbon dot modification provides a reliable solution. Carbon dots (CDs) are a new type of carbon-based nanomaterials, which have the advantages of low toxicity, strong penetration ability, easy modification, stable physical and chemical properties, and show great application prospects in the fields of treatment and drug delivery. Although some current studies have confirmed the BBB permeability of carbon dots, cell membrane-coated biomimetic nanotechnology can still prolong the circulation time of materials in the body and improve disease-related targeting. Cancer cell membrane-coated nanoparticles can utilize the adhesion molecules and specific antigens on the surface of cell membranes to assist tumor targeting through homophilic adhesion. Therefore, the combination of carbon dots and cell membrane coating can improve their targeting ability for glioma, thereby ensuring the induction of anti-tumor effects by carbon dots at the tumor site.

[0005] There is currently a way of co-delivering paclitaxel and Indoximod to the tumor site by self-assembly of modified PEG polymers. The carrier is composed of polyethylene glycol (PEG) as a hydrophilic fragment, an enzyme-cleavable Indoximod ester and an active oxygen-sensitive peroxalate ester coupling as a hydrophobic module. The copolymer can self-assemble with paclitaxel into prodrug-based nanoparticles. When the nanoparticles circulate to the tumor site, intracellular active oxygen triggers the release of paclitaxel to induce ICD and subsequently enhance the immune response. In addition, in the presence of esterase, Indoximod is removed from the polymer carrier and effectively interferes with the IDO-1-mediated immunosuppressive tumor microenvironment.

[0006] The current therapeutic effect of paclitaxel on glioma is limited by the BBB, and the paclitaxel therapeutic agent for glioma needs to be based on nanoreconstruction. For the above-mentioned nanomedicine prepared by assembling the copolymer with paclitaxel, it is developed for breast cancer treatment and it relies on the passive targeting of nanoparticles to the tumor site; and the synthesis steps of the PEG-based prodrug copolymer are relatively complicated. Therefore, its potential for glioma treatment is small.

[0007] Therefore, it is necessary to improve the prior art to provide a more reliable solution. SUMMARY

[0008] The technical problem solved by the present application is to provide an immune-activated anti-tumor biomimetic nanomaterial and its preparation method and application in view of the deficiencies in the above-mentioned prior art. The present application aims to develop carbon dots (PCDs) that can induce ICD of tumor cells and assemble with the IDO-1 inhibitor Indoximod to form a nanocomposite P-In, and finally use the tumor cell membrane to coat it to form a biomimetic nanocomposite material M@P-In. The present application will provide a new strategy for developing a new brain glioma immunotherapeutic agent based on chemotherapeutic drug-derived carbon dots.

[0009] To achieve the above-mentioned purpose, the technical scheme adopted by the present application is: the first aspect of the present application provides a preparation method of an immune-activated anti-tumor biomimetic nanomaterial, comprising the following steps: S1, preparing carbon dots: Disperse paclitaxel in ethanol, then add deionized water and ethylenediamine dihydrate, mix well; transfer the obtained solution to a reaction kettle, heat and perform hydrothermal reaction to prepare carbon dots; S2, assemble carbon dots with an IDO-1 inhibitor to prepare a carbon dot-inhibitor composite: Dissolve PCDs and an IDO-1 inhibitor in a PBS solution, stir in the dark, dialyze the obtained mixture through a dialysis bag, freeze-dry the dialysate in the dialysis bag to obtain a carbon dot-inhibitor composite, denoted as P-In; S3, coating the carbon dot-inhibitor complex with a cell membrane by co-extrusion; S3-1, providing cell membrane fragments; S3-2, co-extruding the cell membrane fragments with a polycarbonate membrane to obtain an empty cell membrane; S3-3, mixing the empty cell membrane with the carbon dot-inhibitor complex to obtain a premix, and co-extruding the premix with a polycarbonate membrane to obtain the immune-activated antitumor biomimetic nanomaterial, denoted as M@P-In.

[0010] Preferably, the IDO-1 inhibitor is Indoximod.

[0011] Preferably, step S1 is specifically: 15-60 mg of paclitaxel is dispersed in 2.5-10 mL of ethanol, followed by the addition of 12.5-50 mL of deionized water and 15-60 μL of ethylenediamine dihydrate, and ultrasonic mixing; the resulting solution is transferred to a reaction kettle, and reacted at a temperature of 140-160°C for 6-12 h, cooled to room temperature, centrifuged and filtered, and the resulting filtrate is freeze-dried to obtain carbon dots, denoted as PCDs.

[0012] Preferably, step S2 is specifically: PCDs and IDO-1 inhibitor Indoximod are dissolved in PBS solution according to a mass ratio of 0.5-2:0.5-2, the concentration of carbon dots in the PBS solution is controlled to be 0.5-2 mg / mL, and the mixture is stirred at room temperature in the dark for 12-48 hours; the resulting mixture is dialyzed in a dialysis bag with a molecular weight cutoff of 300-1000 Da in deionized water for 12-24 h, and the dialysate in the dialysis bag is freeze-dried to obtain a carbon dot-inhibitor complex, denoted as P-In.

[0013] Preferably, step S3 is specifically: S3-1, providing cell membrane fragments: the cells are washed with PSB solution and resuspended in membrane protein extraction reagent in an ice bath, and pulsed ultrasonic treatment is performed at a power of 20-80 W for 1.5-6 min under ice bath conditions; the resulting mixture is centrifuged at 0-5°C and 2000-6000 rpm for 5-20 min, the supernatant is collected, and centrifuged again at 0-5°C and 7000-25000 rpm for 15-60 min, the precipitate is resuspended in deionized water and freeze-dried to obtain cell membrane fragments; S3-2, co-extruding the cell membrane fragments with a polycarbonate membrane with a pore diameter of 200-800 nm for 10-40 times to obtain an empty cell membrane; S3-3, mixing 0.5-2 mg of the empty cell membrane with the carbon dot-inhibitor complex containing 0.5-2 mg of carbon dots to obtain a premix, and co-extruding the premix with a polycarbonate membrane with a pore diameter of 100-400 nm for 10-40 times to obtain the immunostimulatory anti-tumor biomimetic nanomaterial, denoted as M@P-In.

[0014] Preferably, the preparation method of the immunostimulatory anti-tumor biomimetic nanomaterial comprises the following steps: S1, preparing carbon dots: 30 mg of paclitaxel was dispersed in 5 mL of ethanol, followed by adding 25 mL of deionized water and 30 μL of ethylenediamine dihydrate, and ultrasonic mixing; the obtained solution was transferred to a polytetrafluoroethylene lined autoclave, and then sealed in a stainless steel reaction kettle, and reacted at 150 ℃ for 8 h, cooled to room temperature, centrifuged and filtered, and the obtained filtrate was freeze-dried to obtain carbon dots, denoted as PCDs; S2, assembling carbon dots with Indoximod to prepare a carbon dot-inhibitor complex: An equal amount of PCDs and IDO-1 inhibitor Indoximod were dissolved in a PBS solution, the carbon dot concentration was 1 mg / mL, and the mixture was stirred at room temperature in the dark for 24 hours; the obtained mixture was dialyzed in a 500 Da dialysis bag in deionized water for 12 h, and the dialysate in the dialysis bag was freeze-dried to obtain a carbon dot-inhibitor complex, denoted as P-In; S3, coating cell membranes on the carbon dot-inhibitor complex by co-extrusion: S3-1, providing cell membrane fragments: GL261 cells were washed twice with pre-cooled PSB at 4 ℃, then resuspended in membrane protein extraction reagent and ice-bathed for 20 minutes, and then subjected to pulsed ultrasonic treatment at 40 W power for 3 min under ice-bath conditions, with a pulse cycle of 6 seconds, of which the ultrasonic treatment time was 3 seconds and the intermittent time was 3 seconds; the mixture obtained by ultrasonic treatment was centrifuged at 4 ℃ and 4000 rpm for 10 minutes, the supernatant was collected, and the precipitate was resuspended in deionized water and freeze-dried to obtain cell membrane fragments; S3-2, co-extruding the cell membrane fragments with a polycarbonate membrane with a pore diameter of 400 nm for 20 times to obtain an empty cell membrane; S3-3, mixing 1 mg of the empty cell membrane with the carbon dot-inhibitor complex containing 1 mg of carbon dots to obtain a premix, and co-extruding the premix with a polycarbonate membrane with a pore diameter of 200 nm for 20 times to obtain the immunostimulatory anti-tumor biomimetic nanomaterial, denoted as M@P-In.

[0015] In a second aspect of the present application, an immunostimulatory anti-tumor biomimetic nanomaterial is provided, which is prepared by the method described above.

[0016] In a third aspect, the application provides application of the immunologically activated anti-tumor bionic nanomaterial as described above in preparation of an anti-tumor drug.

[0017] In a fourth aspect, the application provides application of the immunologically activated anti-tumor bionic nanomaterial as described above in preparation of a glioma immunotherapy agent.

[0018] In a fifth aspect, the application provides a glioma immunotherapy agent comprising the immunologically activated anti-tumor bionic nanomaterial as described above.

[0019] The application has the following beneficial effects: The application provides an immunologically activated anti-tumor bionic nanomaterial, a preparation method and application thereof. The application first synthesizes paclitaxel-derived carbon dots (PCDs) by a hydrothermal method, which can inhibit tumor cell proliferation and migration and induce ICD of tumor cells. Then the PCDs are assembled with Indoximod to form a nanocomposite P-In. Finally, the nanocomposite material M@P-In is formed by coating the nanocomposite P-In with a tumor cell membrane. The M@P-In can induce ICD and inhibit the activity of IDO-1, thereby reducing the tumor immunosuppressive microenvironment. The bionic coating of the cell membrane endows the nanocomposite M@P-In with tumor targeting ability and BBB penetration ability. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 TEM images and particle size distribution diagrams of PCDs and M@P-In; Figure 2 Fluorescence spectrum of PCDs; Figure 3 BBB penetration test results of PCDs and M@P-In; Figure 4 Inhibition rate test results of glioma cells by different formulations; Figure 5 Cloning number test results after incubation of different formulations; Figure 6 Cell migration and invasion test results after treatment of different formulations; Figure 7 Test results of CRT exposure and ATP release of tumor cells induced by treatment of different formulations; Figure 8 Test results of activation of DCs by tumor cells pretreated by different formulations; Figure 9 Kyn content in supernatant after treatment of tumor cells by different formulations; Figure 10 Test results of in vivo anti-tumor effects of different formulations. DETAILED DESCRIPTION

[0021] The application will be further described in detail below with reference to the examples so that those skilled in the art can implement the application according to the description.

[0022] It should be understood that the terms such as "have", "contain" and "include" used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0023] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are commercially available unless otherwise specified. The specific conditions not specified in the following examples are carried out under conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be purchased on the market.

[0024] Example 1 An immune-activated anti-tumor biomimetic nanomaterial, the preparation method thereof comprises the following steps: S1, preparing carbon dots: 30 mg of paclitaxel was dispersed in 5 mL of ethanol, then 25 mL of deionized water and 30 μL of ethylenediamine dihydrate were added, and ultrasonic mixing was performed; the obtained solution was transferred to a polytetrafluoroethylene-lined autoclave, then sealed in a stainless steel reaction kettle, and reacted at 150 ℃ for 8 h, cooled to room temperature, centrifuged and filtered, and the obtained clear brown filtrate was freeze-dried to obtain carbon dots, denoted as PCDs; S2, assembling carbon dots with Indoximod to prepare carbon dot-inhibitor complex: An equal amount of PCDs and IDO-1 inhibitor Indoximod were dissolved in PBS solution, the final concentration of carbon dots was 1 mg / mL, and the mixture was stirred at room temperature for 24 hours in the dark. The obtained mixture was dialyzed in deionized water for 12 hours by using a 500 Da dialysis bag to remove the un-assembled Indoximod, and the dialysate in the dialysis bag was freeze-dried to obtain the carbon dot-inhibitor complex, denoted as P-In; S3, coating cell membrane on the carbon dot-inhibitor complex by co-extrusion method: S3-1, providing cell membrane fragments: GL261 cells were collected by cell scraper and washed twice with pre-cooled PSB at 4 ℃, the collected cells were resuspended in membrane protein extraction reagent and ice-bathed for 20 minutes, and pulsed ultrasonic treatment was performed at 40 W power under ice-bath condition for 3 min, the pulse cycle was 6 seconds, and the ultrasonic treatment time was 3 seconds and the intermittent time was 3 seconds; the mixture obtained by ultrasonic treatment was centrifuged at 4 ℃ and 4000 rpm for 10 minutes, the supernatant was collected, and the precipitate was resuspended in deionized water and freeze-dried to obtain cell membrane fragments; The membrane protein extraction reagent is specifically cell membrane protein extraction reagent A (Biyun Tian, P0033), and 1% benzylsulfonyl fluoride is added thereto, S3-2, the cell membrane fragments are co-extruded with a polycarbonate membrane with a pore diameter of 400 nm for 20 times to obtain the empty cell membrane; S3-3, 1 mg of the empty cell membrane is mixed with a carbon dot-inhibitor complex containing 1 mg of carbon dots to obtain a premix, and the premix is co-extruded with a polycarbonate membrane with a pore diameter of 200 nm for 20 times to obtain the immune-activated anti-tumor biomimetic nanomaterial, denoted as M@P-In.

[0025] Example 2 Transmission electron microscopy imaging of PCDs and M@P-In (1) Sample preparation: The PCDs or M@P-In solution prepared in Example 1 is added dropwise to a copper grid (sample grid) covered with a carbon film on the surface, and then a culture dish is placed on the sample grid, and the sample is naturally air-dried at room temperature. Carefully rinse the sample grid with deionized water, then rinse with ethanol, and finally dry the excess ethanol with filter paper.

[0026] (2) Sample imaging: Turn on the transmission electron microscope, and measure the sample by following the steps of sample loading, sample feeding, sample height adjustment, instrument adjustment, and sample imaging. Select an appropriate magnification to have a large number of nanoparticles in the field of view, and image the carbon dot sample in different fields of view. Figure 1 The results show that the average particle size of PCDs (A and B in Figure 1 is 1.67 nm, which is conducive to the delivery, bioavailability and BBB penetration of carbon dots in vivo. The cell membrane-coated nanocomposite constructed by the liposome extruder is spherical in structure, and the black particles inside the structure indicate the successful packaging of P-In (C and D in Figure 1 ). In contrast, the average particle size of P-In (4.96±1.12 nm) is larger than that of PCDs, indicating the successful loading of Indoximod to produce a new nanocomposite.

[0027] Example 3 Application potential of PCDs in biological imaging The PCDs solution can emit bright blue-green fluorescence under ultraviolet lamp irradiation (A in Figure 2 ). The fluorescence properties of PCDs were analyzed by a fluorescence spectrometer. The results show that the maximum excitation wavelength of the carbon dots is 415 nm, and the maximum emission wavelength is 492 nm, which has application potential for live cell imaging (A in Figure 2 ). Under irradiation at different excitation wavelengths, the emission intensity changes significantly, but the emission wavelength does not shift significantly, indicating that the emission wavelength of the carbon dots is non-excitation dependent (B in Figure 2 ).

[0028] Example 4 BBB penetration ability of PCDs and M@P-In The BBB penetration rate of PCDs and M@P-In was tested by Transwell and using the photoluminescence properties of PCDs. The cultured bEnd.3 cells were seeded into the upper layer of the Transwell insert at 2 x 105cells per well. When the cells grew into a continuous dense layer on the membrane, PCDs and M@P-In were added to the top chamber. After 12 hours of incubation, the culture medium in the bottom chamber was collected and the fluorescence intensity was measured by a microplate reader. The permeability of the BBB model was determined by quantifying the relative fluorescence intensity of PCDs and M@P-In penetrating into the bottom chamber. The test results are shown in 5 The upper layer of the Transwell insert was seeded with 2 x 105cells per well. When the cells grew into a continuous dense layer on the membrane, PCDs and M@P-In were added to the top chamber. After 12 hours of incubation, the culture medium in the bottom chamber was collected and the fluorescence intensity was measured by a microplate reader. The permeability of the BBB model was determined by quantifying the relative fluorescence intensity of PCDs and M@P-In penetrating into the bottom chamber. The test results are shown in Figure 3 It can also be seen that the coating of the cell membrane improves the BBB permeability of PCDs.

[0029] Example 5 Anti-tumor activity of PCDs and M@P-In (1) The glioma cells cultured in T25 were digested, and 5000 cells were resuspended in 100 μL of culture medium and seeded into a 96-well cell culture plate, and incubated in a 37°C incubator overnight. The cell culture medium was discarded and replaced with culture medium containing 50 μg / mL Indoximod, PCDs or M@P-In, respectively, with 3 replicate wells for each group, and incubated for another 48 hours. After the incubation, the cell culture medium in each well was replaced with culture medium containing 10 μL of WST-1, and incubated at 37°C for 1 hour. The absorbance at 450 nm of each well was measured, and blank wells (without cells, with culture medium and WST-1 solution) and control wells (with cells, without drugs, with culture medium and WST-1 solution) were also set. The cell activity of tumor cells after treatment with carbon dots was calculated using the following formula: Cell activity = [(OD experimental group - OD blank well) / (OD control group - OD blank well)] x 100% The inhibition of different preparations on the brain glioma cell line GL261 is shown in Figure 4 It can be seen that M@P-In has the relatively optimal inhibitory effect.

[0030] (2) GL261 cells were seeded in 6-well plates and incubated overnight at 37 °C after adding culture medium. The culture medium was replaced with culture medium containing 50 ug / mL different formulations or fresh regular cell culture medium and continued to culture for 24 hours. Then replaced with complete culture medium without drug and continued to culture for 14 days. After the end of culture, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS for 3 times, stained with 1% crystal violet for 10 minutes, finally washed with PBS to remove excess crystal violet dye, and photographed to record the number of clones generated. From the test results, it can be seen that the control group and Indoximod treated cells formed 138.3 ± 2.35 and 135.3 ± 6.34 clones, respectively, while PCDs and M@P-In exposure caused the number to drop to 48 ± 4.97 and 30.67 ± 2.05, indicating the significant anti-tumor proliferation ability of PCDs and the enhanced anti-proliferative activity of M@P-In.

[0031] (3) GL261 cells were seeded in the upper layer of transwell inserts in 24-well plates at a density of 10000 per well and added with 50 ug / mL different formulations; 600 μL complete culture medium was added to the lower layer of the insert. After 48 hours, the transwell was removed, the cells in the upper layer of the transwell insert were scraped off with a cotton swab, and the cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature. After washing with PBS for 3 times, stained with 1% crystal violet for 10 minutes, finally washed with PBS to remove excess crystal violet dye, observed the cell migration under an inverted microscope, counted the number of purple cells in each field, and calculated the effect of carbon dots on cell migration and invasion (Figs. 5A and 5B). Figure 6 From the test results, it can be seen that PCDs and M@P-In both significantly inhibited the migration and invasion ability of tumor cells.

[0032] Example 6 Performance test of PCDs and M@P-In in activating anti-tumor immunity by inducing ICD (1) 5 x 10 4 GL261 cells were seeded in 24-well plates and incubated with different formulations for 48 hours. For CRT detection, GL261 cells were treated with 4% paraformaldehyde and permeabilized with 0.1% Triton x-100, then stained with CRT antibody overnight at 4 °C. After incubation with a fluorescently labeled secondary antibody, cell images were acquired using a confocal microscope. Meanwhile, the supernatant was collected to determine the ATP content in the supernatant using an ATP detection kit. As Figure 7As shown, PCDs and M@P-In treated cells exposed more CRT signals; in addition, the ATP content in PCDs and M@P-In group culture supernatant was significantly higher than that of control group and Indoximod group. These results showed that PCDs and M@P-In induced ICD of tumor cells.

[0033] (2) To detect the maturation of DCs in vitro, in this embodiment, the marrow-derived dendritic cells (BMDCs) were isolated from the femur and tibia of mice and cultured in 1640 medium (containing 10% FBS, 20 ng / mL GM-CSF, 10 ng / mL IL-4) for 7 days to obtain immature DCs. Then these DCs were co-cultured with GL261 cells pretreated with different agents for 24 hours, and then the proportion of mature DCs (CD80 + , CD86 + ) cells was analyzed by FCM. As shown in Figure 8 , compared with the control group and Indoximod treatment group, PCDs and M@P-In treated cells induced a higher proportion of DCs maturation.

[0034] (3) The inhibitory effect of M@P-In on IDO-1 was tested by colorimetry. 5x10 4 GL261 cells were inoculated in a 24-well plate and cultured with complete medium containing 100 ng / mL IFN-γ and 100 μM L-tryptophan. When the confluence reached 80%, the cells were incubated with medium containing PCDs, Indoximod and M@P-In for 72 h. 1 mL of supernatant was mixed with 100 μL of 30% trichloroacetic acid and incubated in a 50 ℃ oven for 30 min. The supernatant was centrifuged at 10000xg for 10 min to obtain the supernatant, and the supernatant was mixed with an equal volume of Ehrlich reagent and reacted at room temperature for 30 min, and the absorbance of the sample at 492 nm was measured by a microplate reader. The measured absorbance value was brought into the standard curve to calculate the inhibition rate. The inhibition of IDO-1 is shown in Figure 9 , from the test results, it can be seen that Indoximod significantly reduces the level of kynurenine in the supernatant of cell culture medium, and PCDs also reduce the production of kynurenine, which may be the result of inhibiting cell proliferation. M@P-In combines both mechanisms, showing the lowest kynurenine content.

[0035] Example 7 In vivo anti-tumor effect of PCDs and M@P-In 6-8 week old male C57BL / 6J mice were selected for the experiment, and were raised in a SPF environment, maintaining a 12 h day-night cycle. After a week of adaptation, 1.5x10 5A GL261-Luci cell was suspended in 3 μΐ PBS solution and injected into the striatum of each mouse (coordinates: 0.8 mm anterior, 2.0 mm lateral, 3.5 mm ventral) by stereotactic technique. Two weeks after injection, bioluminescence imaging was detected using IVIS Lumina III live imaging system. The successfully modeled mice were randomly divided into four groups, and then PCDs, Indoximod, M@P-In or normal saline (10 mg / kg) were injected through the tail vein every two days for 14 days. Live imaging was performed at specific time points to monitor tumor growth. The tumor growth of mice in different groups is shown in FIG. 6B. Figure 10 As can be seen from the test results, compared with the PBS control group and the Indoximod group, both PCDs and M@P-In significantly inhibited tumor growth.

[0036] Although embodiments of the present application have been disclosed as above, they are not limited only to the uses listed in the specification and embodiments, and can be fully applied to various fields suitable for the present application, and additional modifications can be easily made by those skilled in the art, and therefore the present application is not limited to specific details, without departing from the general concept defined by the claims and the equivalent scope.

Claims

1. A preparation method of an immune-activated anti-tumor biomimetic nanomaterial, characterized in that, The method comprises the following steps: S1, preparing carbon dots: Paclitaxel is dispersed in ethanol, then deionized water and ethylenediamine dihydrate are added, and mixed uniformly; the obtained solution is transferred into a reaction kettle, and a hydrothermal reaction is carried out by heating to prepare carbon dots; S2, assembling the carbon dots and the IDO-1 inhibitor to prepare a carbon dot-inhibitor complex: The carbon dots and the IDO-1 inhibitor are dissolved in a PBS solution, stirred in the dark, the obtained mixture is dialyzed through a dialysis bag, the dialysate in the dialysis bag is freeze-dried to obtain a carbon dot-inhibitor complex, denoted as P-In; S3, coating a cell membrane on the carbon dot-inhibitor complex by using a co-extrusion method: S3-1, providing cell membrane fragments; S3-2, co-extruding the cell membrane fragments and a polycarbonate membrane to obtain an empty cell membrane; S3-3, mixing the empty cell membrane and the carbon dot-inhibitor complex to obtain a premix, and co-extruding the premix and a polycarbonate membrane to obtain the immunostimulating antitumor biomimetic nanomaterial, denoted as M@P-In.

2. The method for preparing the immune-activating antitumor biomimetic nanomaterial according to claim 1, characterized in that, Wherein, The IDO-1 inhibitor is an IDO-1 inhibitor Indoximod.

3. The method for preparing the immune-activated antitumor biomimetic nanomaterial according to claim 1, characterized in that, Step S1 is specifically: 15-60 mg of paclitaxel is dispersed in 2.5-10 mL of ethanol, then 12.5-50 mL of deionized water and 15-60 μL of ethylenediamine dihydrate are added, and ultrasonic mixing is performed; the obtained solution is transferred into a reaction kettle, and a hydrothermal reaction is carried out at a temperature of 140-160℃ for 6-12 h, cooled to room temperature, centrifuged and filtered, and the obtained filtrate is freeze-dried to obtain carbon dots, denoted as PCDs.

4. The method for preparing the immune-activated antitumor biomimetic nanomaterial according to claim 3, characterized in that, Step S2 is specifically: PCDs and the IDO-1 inhibitor Indoximod are dissolved in a PBS solution according to a mass ratio of 0.5-2:0.5-2, the concentration of carbon dots in the PBS solution is controlled to be 0.5-2 mg / mL, stirring is carried out in the dark at room temperature for 12-48 hours, the obtained mixture is dialyzed in deionized water through a dialysis bag with a molecular weight cut-off of 300-1000 Da for 12-24 h, the dialysate in the dialysis bag is freeze-dried to obtain a carbon dot-inhibitor complex, denoted as P-In.

5. The method for preparing the immune-activated antitumor biomimetic nanomaterial according to claim 1, characterized in that, Step S3 is specifically: S3-1, providing cell membrane fragments: after the cells are washed with a PBS solution, resuspended in a membrane protein extraction reagent, and subjected to pulse ultrasonic treatment at a power of 20-80 W under ice bath conditions for 1.5-6 min; the obtained mixture is centrifuged at 0-5℃ and 2000-6000 rpm for 5-20 min, the supernatant is collected, centrifuged at 0-5℃ and 7000-25000 rpm for 15-60 min again, and the precipitate is resuspended in deionized water and freeze-dried to obtain cell membrane fragments; S3-2, co-extruding the cell membrane fragments and a polycarbonate membrane with a pore diameter of 200-800 nm for 10-40 times to obtain an empty cell membrane; S3-3, mixing 0.5-2 mg of the empty cell membrane and the carbon dot-inhibitor complex containing 0.5-2 mg of carbon dots to obtain a premix, and co-extruding the premix and a polycarbonate membrane with a pore diameter of 100-400 nm for 10-40 times to obtain the immunostimulating antitumor biomimetic nanomaterial, denoted as M@P-In.

6. The method for preparing the immune-activated antitumor biomimetic nanomaterial according to claim 1, characterized in that, The method comprises the following steps: S1, preparing carbon dots: 30 mg of paclitaxel was dispersed in 5 mL of ethanol, then 25 mL of deionized water and 30 μL of ethylenediamine dihydrate were added, and the mixture was ultrasonically mixed; the resulting solution was transferred to a stainless steel reactor, and reacted at 150°C for 8 h, cooled to room temperature, centrifuged and filtered, and the resulting filtrate was freeze-dried to obtain carbon dots, denoted as PCDs; S2, the carbon dots were assembled with Indoximod to prepare a carbon dot-inhibitor complex: An equal mass of PCDs and an IDO-1 inhibitor Indoximod were dissolved in a PBS solution, the carbon dot concentration was 1 mg / mL, and the mixture was stirred at room temperature in the dark for 24 h; the resulting mixture was dialyzed in a 500 Da dialysis bag in deionized water for 12 h, and the dialysate in the dialysis bag was freeze-dried to obtain a carbon dot-inhibitor complex, denoted as P-In; S3, a cell membrane was coated on the carbon dot-inhibitor complex by co-extrusion: S3-1, cell membrane fragments were provided: GL261 cells were washed twice with pre-cooled PBS at 4°C, then resuspended in a membrane protein extraction reagent and ice-bathed for 20 min, and then subjected to pulsed ultrasonic treatment at 40 W for 3 min under ice-bath conditions, with a pulse cycle of 6 s and an ultrasonic treatment time of 3 s and an intermittent time of 3 s; the mixture obtained by ultrasonic treatment was centrifuged at 4°C and 4000 rpm for 10 min, the supernatant was collected, and the precipitate was resuspended in deionized water and freeze-dried to obtain cell membrane fragments; S3-2, the cell membrane fragments were co-extruded with a polycarbonate membrane with a pore diameter of 400 nm for 20 times to obtain empty cell membranes; S3-3, 1 mg of empty cell membranes were mixed with a carbon dot-inhibitor complex containing 1 mg of carbon dots to obtain a premix, and the premix was co-extruded with a polycarbonate membrane with a pore diameter of 200 nm for 20 times to obtain the immunostimulatory anti-tumor biomimetic nanomaterial, denoted as M@P-In.

7. An immunostimulatory antitumor biomimetic nanomaterial, characterized in that, The immunostimulatory anti-tumor biomimetic nanomaterial is prepared by the method of any one of claims 1-6.

8. Use of the immunostimulatory anti-tumor biomimetic nanomaterial of claim 7 in the preparation of an anti-tumor drug.

9. Use of the immunostimulatory anti-tumor biomimetic nanomaterial of claim 7 in the preparation of a brain glioma immunotherapeutic agent.

10. A brain glioma immunotherapeutic agent, characterized by, The immunostimulatory anti-tumor biomimetic nanomaterial of claim 7.

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