Functional nanomaterials based on choline phosphate-cell membrane interaction and applications thereof

Abstract

The present application relates to the technical field of medicine, in particular to a functional nanomaterial based on choline phosphate-cell membrane interaction and application thereof.The present application modifies manganese mineralized black phosphorus nanomaterial by bio-inspired polymer pGluCP adsorption, which retains the excellent biocompatibility and photothermal performance of BP, and at the same time, pGluCP enhances the 'chassis' effect of the material, captures tumor-related antigens, and can simultaneously capture water-soluble antigens and water-insoluble membrane antigens and form pathogen-like micro-nanoparticles, which is helpful for antigen delivery and APC presentation, and provides a feasible tool for capturing non-water-soluble membrane antigens, which is a major challenge.MnBP and pGluCP synergistically act, and can realize the personalized conversion of autologous tumors to vaccine production and delivery.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a functional nanomaterial based on choline phosphate-cell membrane interaction and its application. BACKGROUND

[0002] In recent years, tumor immunotherapy has become a transformative force in clinical oncology and a focus of innovative cancer treatment. Among them, tumor vaccine therapy has unique advantages: specific killing of tumor cells, small side effects; the immune system throughout the body can capture metastatic tumor cells in time; the immune memory function induced by the vaccine can prevent tumor recurrence. However, traditional tumor vaccine development has many difficulties such as difficulty in identifying tumor antigens, complicated preparation process, lack of broad spectrum due to tumor heterogeneity, etc. In recent years, the rise of tumor in situ vaccine provides a new idea to solve the limitations of conventional tumor vaccine. Tumor in situ vaccine refers to killing tumor cells in situ, releasing a large amount of tumor antigens, attracting and activating antigen presenting cells (APC), realizing antigen presentation, and triggering specific anti-tumor immune response, aiming to realize the individualization of autologous tumor vaccine production and delivery. Tumor in situ vaccine has the advantages of simplicity, time saving, economy, universality and small side effects, and has become a research hotspot in the field of anti-tumor. However, the immune effect induced by tumor in situ vaccine is weak, which is difficult to cure tumor. The reason is that the vaccine cascade immune response process is complex, restricted by multiple limiting factors, resulting in low efficiency. Among them, the presentation of tumor antigens by antigen presenting cells is the key limiting step of vaccine cascade reaction. Studies have found that free antigens released during tumor cell death are easily quickly cleared and not effectively presented by APC. Therefore, in the complex tumor microenvironment, how to improve the antigen capture and presentation efficiency of APC is crucial to start specific anti-tumor immune response.

[0003] In recent years, research progress has focused on the importance of improving tumor antigen capture and presentation. Researchers have designed functional nanocarriers with protein adsorption groups such as amino groups that can bind to tumor antigens and transport them to lymph nodes, thereby improving antigen presentation. Some researchers have proposed that three-dimensional networks based on hydrogels are effective antigen traps that can retain and concentrate antigens at the injection site, promoting APC capture of antigens. It is worth noting that capturing water-soluble and water-insoluble membrane antigens to simulate pathogen-like particles can significantly improve antigen presentation efficiency. However, capturing water-insoluble membrane antigens is a major challenge. These antigens are usually embedded in the liquid bilayer of tumor cells, making them difficult to extract and transport. Although progress has been made in functional nanocarriers and hydrogel networks, there are still limited reports on successful strategies for effectively capturing and transporting these challenging antigens. Further research and development of innovative methods to handle water-insoluble tumor cell membrane antigens are needed, which will greatly improve the efficacy of cancer immunotherapy. Summary of the Invention

[0004] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and provide a nanomaterial based on choline phosphate-cell membrane interaction to enhance antigen capture function.

[0005] Another object of the present invention is to provide the application of the nanomaterials based on the synergistic effect of choline phosphate and cell membrane to enhance antigen capture function.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] A functional nanomaterial MnBP@pGluCP based on choline phosphate-cell membrane interaction is prepared from manganese mineralized black phosphorus nanosheets MnBP and polyglutamic acid-choline phosphate pGluCP.

[0008] Preferably, the mass ratio of MnBP to pGluCP is 1:6-24. More preferably, the ratio of MnBP to pGluCP is 1:6.

[0009] Preferably, the preparation method of the functional nanomaterial MnBP@pGluCP based on choline phosphate-cell membrane interaction includes the following steps:

[0010] (1) Preparation of black phosphorus nanosheets;

[0011] Bulk black phosphorus crystals were dispersed in N-methylpyrrolidone, sonicated under ice bath conditions, and then the suspension was centrifuged, washed, and the supernatant was collected to obtain black phosphorus nanosheet suspension I.

[0012] (2) Preparation of manganese mineralized black phosphorus nanosheets;

[0013] The black phosphorus nanosheet suspension obtained in step (1) was dispersed in N-methylpyrrolidone to obtain black phosphorus nanosheet suspension II. NH3•H2O was added, and the mixture was stirred at 40°C. Mn was then added dropwise. 2+ The solution was stirred in the dark, centrifuged, and washed to obtain manganese-mineralized black phosphorus nanosheets.

[0014] (3) The functional nanomaterial based on choline phosphate-cell membrane interaction is obtained by mixing and stirring manganese mineralized black phosphorus nanosheet suspension and polyglutamic acid-choline phosphate solution.

[0015] Preferably, in step (1), the ratio of N-methylpyrrolidone to black phosphorus is 1 mL to 1 mg. That is, 1 mg of BP blocky black phosphorus crystals are mixed per milliliter of NMP.

[0016] Preferably, in step (1), the ultrasonic power is 600w and the ultrasonic time is 18h under ice bath conditions.

[0017] In black phosphorus nanosheet suspension II, based on the original amount of black phosphorus used, the concentration of black phosphorus is 50 μg / mL, and the mass percentage concentration of added NH3•H2O is 1.9-2.0%; the volume of added NH3•H2O is 0.1% of the volume of black phosphorus nanosheet suspension II. For example, in one embodiment of the present invention, NH3•H2O is prepared by adding 3.87 mL of 25% NH3•H2O to 50 mL of deionized water.

[0018] Preferably, in step (2), Mn 2+ Mn in solution 2+ The concentration is 0.7-0.8 mmol / L, and the volume is 1 mL. Mn 2+ The solution can be MnCl 2、 MnSO4, etc.

[0019] Preferably, in step (3), the mixing and stirring reaction temperature is room temperature and the reaction time is 4-8 hours.

[0020] Preferably, the washing in steps (2) and (3) involves washing the precipitate until the supernatant is clear and transparent.

[0021] The functional nanomaterial MnBP@pGluCP of this invention can enhance the antigen capture function of the material through choline phosphate-cell membrane synergy, and can capture not only water-soluble antigens, but also water-insoluble membrane antigens.

[0022] This invention also provides the application of the aforementioned functional nanomaterials in the preparation of antigen-capturing materials and their use in in situ tumor vaccines.

[0023] The application is achieved through the following steps: the aforementioned functional nanomaterial MnBP@pGluCP based on choline phosphate-cell membrane interaction is injected into tumors to construct an in situ vaccine platform. First, black phosphorus exhibits excellent photothermal effects, effectively eliminating tumor cells and inducing immunogenic cell death, thereby leading to the release of tumor antigens. Subsequently, pGluCP modification enables the simultaneous capture of both water-soluble and water-insoluble membrane antigens, forming pathogen-like micro / nanoparticles, which are then assembled in situ into a tumor vaccine. Finally, the unique interaction between CP and pGluCP enhances the uptake of tumor antigens by pGluCP, thereby enhancing antigen presentation and vaccine efficacy.

[0024] In some embodiments, the tumor is a mouse breast cancer tumor.

[0025] The present invention has the following advantages and effects compared with the prior art:

[0026] (1) Synthetic manganese-mineralized BP (MnBP) retains the inherent properties of BP (excellent biocompatibility and photothermal properties) and endows it with highly promising anti-tumor therapeutic effects, providing a broad-spectrum strategy for the surface functionalization of BP. Poly(glutamate-choline phosphate) (pGluCP), a high-molecular-weight polymer with unique cell membrane adhesion capabilities, enhances the "chassis" function of the material, captures tumor-associated antigens, and the CP-PC interaction provides a robust platform for vaccine assembly and delivery. MnBP and pGluCP work synergistically to achieve personalized transformation of autologous tumors into vaccine production and delivery.

[0027] (2) Capturing water-insoluble membrane antigens is difficult and rarely reported. This invention uses bio-inspired polymer pGluCP to adsorb modified manganese mineralized black phosphorus nanomaterials, which can simultaneously capture water-soluble antigens and water-insoluble membrane antigens and form micro-nano particles similar to pathogens. This helps with antigen delivery and APC presentation, providing a feasible tool for the major challenge of capturing insoluble membrane antigens and breaking through the efficiency bottleneck of antigen delivery in the immune cascade reaction. This will break the bottleneck of low antigen presentation efficiency in the immune cascade reaction. Attached Figure Description

[0028] Figure 1 This study focuses on the preparation and mechanism of action of nanomaterials that enhance antigen capture function based on the synergistic effect of choline phosphate and cell membrane.

[0029] Figure 2 Figures show the characterization results of MnBP and MnBP@pGluCP; where a is a high-resolution transmission electron microscope image of MnBP; b is the EDS elemental analysis result of MnBP; c is the potential change of BP and MnBP; d is the infrared absorption spectrum of BP under different synthesis conditions; e is the ultraviolet absorption spectrum of BP and MnBP; and f is the XPS spectrum of BP and MnBP.

[0030] Figure 3 The results of experiments on the saturation of MnBP and pGluCP binding at different ratios;

[0031] Figure 4 It is the synergistic effect of MnBP and pGluCP that enables antigen release and in-situ retention; where 'a' represents laser irradiation (808 nm, 1.5 W / cm²). 2 (b) Infrared thermal images of MnBP and MnBP@pGluCP; (b) Thermal images of MnBP and MnBP@pGluCP after exposure to an 808 nm laser (808 nm, 1.5 W / cm²). 2 Apoptosis of 4T1 cells treated with the tumor injection site. c shows the in vivo fluorescence imaging results at the tumor injection site; d shows the average fluorescence intensity at the tumor injection site.

[0032] Figure 5This is an assessment of antigen capture capacity; a) SDS-PAGE results of 4T1 cell lysates after co-incubation with MnBP and MnBP@pGluCP, respectively; b) Total protein captured by different nanoparticles after incubation of MnBP or MnBP@pGluCP with 4T1 lysates; c) Changes in zeta potential after incubation of MnBP or MnBP@pGluCP with 4T1 cell lysates.

[0033] Figure 6 This study examines the effects of MnBP@pGluCP capturing different types of antigens on the maturation and antigen presentation efficiency of BMDCs; (a) shows the superior antigen capture ability of MnBP@pGluCP for water-insoluble membrane antigens; (b) shows representative flow cytometry analysis of BMDCs activated by MnBP@pGluCP capturing different types of antigens; (c) shows the quantitative analysis of the expression levels of MHC I, (d) CD80, and (e) CD86 on BMDCs activated after MnBP@pGluCP captures different types of antigens.

[0034] Figure 7 This study investigates the immunomodulatory effects of nanomaterials that enhance antigen capture capacity through choline phosphate-cell membrane synergy in a primary tumor model. Specifically, 'a' represents subcutaneous inoculation of mice with 4T1 tumor cells, followed by in situ treatment with a specified formulation on days 7, 11, and 15, and subsequent treatment with an 808 nm laser (1.5 W / cm²) on days 8, 12, and 16. 2 a) Schematic diagram of the treatment plan (5 min); b) Microscopic image of tumor cell-specific killing; c) Quantitative data of spleen cell-specific killing ability in mice of different treatment groups; d and e) CD8 in the spleen + Flow cytometry representation and quantitative data of T cells; f and g represent CD8+ in tumor-draining lymph nodes, respectively. + Flow cytometry representation and quantitative data of T cells;

[0035] Figure 8 This study describes the antitumor effect of nanomaterials with enhanced antigen-capturing capacity based on the synergistic effect of choline phosphate and cell membrane in a distal tumor model. In the figures, a is a schematic diagram of the bilateral tumor model protocol; b shows the changes in primary tumor volume monitored during three inoculations; c is the growth curve of the distal tumor in the mice during the experiment; d shows the changes in mouse body weight; e and f are images and weights of the primary tumor in vitro; and g and h are images and weights of the distal tumor in vitro. Detailed Implementation

[0036] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0037] In the description of this invention, unless otherwise explicitly defined, terms such as heating, cleaning, weighing, and freezing should be interpreted broadly. Those skilled in the art can reasonably determine the specific meaning of the above terms in this invention in conjunction with the specific content of the technical solution.

[0038] In the description of this invention, references to terms such as "some embodiments" and "examples" indicate that the specific methods or materials described in connection with that embodiment or example are included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the specific methods and materials described may be combined in any suitable manner in one or more embodiments or examples.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0040] Unless otherwise specified, all reagents, materials, and equipment used in the embodiments of this invention are commercially available; unless otherwise specified, all test methods are conventional test methods in the field.

[0041] The pGluCP polymer involved in the embodiments of the present invention can be prepared according to the method described in Chinese patent application (application number 201810948438, entitled "A poly(glutamic acid-choline phosphate) and its application as a vaccine adjuvant").

[0042] The ovalbumin (OVA) used in the embodiments of this invention was purchased from Sigma.

[0043] Example 1: Preparation of Nanomaterials with Enhanced Antigen Capture Function Based on Choline Phosphate-Cell Membrane Synergistic Effect

[0044] refer to Figure 1 a) mainly includes the following steps:

[0045] (1) Preparation of black phosphorus nanosheets (BP)

[0046] 20 mg of blocky BP crystals were dispersed in 20 mL of N-methylpyrrolidone (NMP) and sonicated at 600 W for 18 h under ice bath conditions. After sonication, the suspension was centrifuged at 9000 rpm for 10 min to remove excessively large products, and the supernatant was collected and stored at 4 °C for subsequent use.

[0047] (2) Preparation of manganese mineralized black phosphorus nanosheets MnBP

[0048] 10 mL of BP solution was dispersed in NMP, with a BP concentration of 50 μg / mL. 0.1% (v / v) NH3•H2O was added (NH3•H2O was prepared by adding 3.87 mL of 25% NH3•H2O to 50 mL of deionized water). After stirring at 40 °C for 2 h, 1 mL of MnCl2 aqueous solution (obtained by dissolving 1.4 mg of MnCl2•4H2O in 10 mL of deionized water) was added dropwise to the above mixture. The mixture was stirred at 350 rpm for 12 h in the dark. Finally, the product was collected, washed three times with ethanol and deionized water, and then freeze-dried for storage.

[0049] (3) Synthesis of MnBP@pGluCP

[0050] The MnBP suspension was mixed with the pGluCP solution at a mass ratio of 1 / 6, 1 / 12 or 1 / 24, and stirred at room temperature for 6 h. After the reaction was completed, the mixture was washed with water 2-3 times and then freeze-dried to obtain MnBP@pGluCP.

[0051] I. Characterization of MnBP and MnBP@pGluCP

[0052] The shape, size, and elemental composition of MnBP nanoparticles were determined using energy-dispersive X-ray spectroscopy (EDS) combined with high-resolution transmission electron microscopy (HRTEM). The chemical composition of MnBPs was determined using X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR). The ultraviolet absorption spectra of BPs and MnBPs were also analyzed. The potential changes of MnBP and MnBP@pGluCP were detected using a Malvern particle size analyzer.

[0053] The results are as follows Figure 2 As shown, MnBP exhibits a plate-like morphology with a lateral dimension of 300-400 nm. Figure 2 a) EDS proves that MnP was successfully mineralized on the surface of BPs ( Figure 2 b). After MnP mineralization, the Zeta potential of BP increased from -31.9 mV to -19.1 mV, reflecting the neutralizing effect of Mn on phosphate anions ( Figure 2c). d) Figures at 509, 547, 1025, and 1394 cm -1 The broad absorption band at 3430 cm⁻¹ is related to the stretching of PO and P=O. -1 The absorption band at the point originates from OH- extension. Compared with the "BPs+ammonia" group, the OH band intensity of MnBPs is increased and broadened, confirming the formation of intermolecular and weakly hydrogen-bonded OH groups in MnP. XPS results show that Mn is present in MnBP ( Figure 2 e), all the above results indicate the successful preparation of MnBP. The UV-Vis absorption spectra of BPs and MnBPs show broad-spectrum absorption, with MnBP showing stronger absorption (e). Figure 2 f). MnBP and pGluCP were prepared via simple and efficient charge adsorption, and the conversion of charge from negative to positive confirmed the successful preparation of MnBP@pGluCP. Based on the saturation experiment of pGluCP binding, the optimal mass ratio of MnBP to pGluCP was determined to be 1:6. Figure 3 The MnBP@pGluCP complex prepared according to this ratio was used in subsequent experiments.

[0054] II. Antigen release and in situ accumulation based on MnBP@pGluCP

[0055] First, the photothermal effects of MnBP and MnBP@pGluCP were investigated using infrared photothermal imaging. Apoptosis of 4T1 cells under different photothermal conditions was detected. Subsequently, in vivo imaging in small animals was used to investigate the in situ accumulation of MnBP@pGluCP in tumors. Specifically, BALB / c mice (4-6 weeks old, 18-20 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into two groups (n=3), 1×10 6 Cells per mouse were seeded on the back of mice at a density of 1 cell per mouse, and the tumors were allowed to grow to 50 mm. 3 MnBP / Cy5.5-OVA (Cy5.5 fluorescently labeled OVA, purchased from Xi'an Qiyue Biotechnology Co., Ltd.) or MnBP@pGluCP / Cy5.5-OVA (5 mg of MnBP or MnBP@pGluCP nanoparticles were added to 1 mL of Cy5.5-OVA solution (300 µg, prepared with physiological saline), stirred at 300 rpm for 12 h at room temperature to obtain MnBP / Cy5.5-OVA or MnBP@pGluCP / Cy5.5-OVA) were injected into mouse tumors. The fluorescence intensity of the model antigen at the tumor site was observed using a small animal bioluminescence imaging system.

[0056] The results are as follows Figure 4As shown, the infrared photothermal imager also verified the photothermal conversion capability of MnBP and MnBP@pGluCP in the near-infrared region. Figure 4 a). Figure 4 b shows that the apoptosis rate of 4T1 cells induced by PTT based on MnBP@pGluCP was 46.38%, while the apoptosis rate of 4T1 cells mediated by MnBP under near-infrared irradiation was 23.47%. This further proves that the modification of MnBP with pGluCP significantly enhances the tumor killing ability and induces tumor cell apoptosis, which will promote the release of tumor-associated antigens.

[0057] To investigate the retention of materials at the tumor site, when the 4T1 tumor in mice reached 50 mm... 3 At that time, the OVA-Cy5.5 assembled material was randomly injected in situ into the tumor site, and fluorescence imaging was performed at 1, 2, 4, 8, 10, 12, 24, 36, and 48 h after administration. The results are as follows: Figure 4 CD40 analysis showed that MnBP and MnBP@pGluCP remained at the tumor site, with fluorescence slowly decreasing over time. Compared to MnBP, MnBP@pGluCP exhibited stronger tumor accumulation capacity, attributed to the cell membrane adhesion of pGluCP. This membrane adhesion prolonged the retention time of the antigen at the tumor site, thereby generating a sustained and potent anti-tumor immune response.

[0058] III. Capture of water-soluble and water-insoluble membrane antigens by MnBP@pGluCP

[0059] To evaluate the simultaneous capture of water-soluble and water-insoluble membrane antigens by MnBP@pGluCP, 4T1 cells were lysed using RIPA buffer containing protease and phosphatase inhibitors. Solutions containing water-soluble protein antigens and water-insoluble membrane antigens were collected, and 1 mg of MnBP and MnBP@pGluCP were added, respectively. After incubation overnight at 4°C, the precipitates were collected by centrifugation at 12,000 rpm for 10 min. The resulting antigen nanocomplexes were used for downstream analysis. For quantitative analysis, the total protein content was analyzed using a BCA assay kit. The potential changes of the antigen nanocomplexes were measured using DLS. Simultaneously, the antigen-material complexes were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to assess the purity and integrity of the captured antigens.

[0060] The results are as follows Figure 5 As shown, SDS-PAGE confirmed the excellent antigen capture ability of MnBP@pGluCP. The results showed that the protein content in the lysate captured by MnBP@pGluCP was richer than that captured by MnBP. Figure 5 a). For example Figure 5As shown in Figure b, MnBP@pGluCP can rapidly and effectively capture 4T1 cell lysates, capturing approximately 1.8 times the total amount of protein compared to MnBP alone. This enhanced capture ability caused the zeta potential of MnBP@pGluCP to significantly change from positive to negative (Figure 5c). This change in zeta potential suggests that MnBP@pGluCP has the potential to capture a wider variety of antigens, which may be beneficial for its application in immunotherapy strategies.

[0061] IV. The effect of MnBP@pGluCP capturing multiple types of antigens on antigen presentation efficiency

[0062] 4T1 cells were lysed using RIPA buffer containing protease and phosphatase inhibitors, and the lysate was centrifuged at 12,000 rpm for 10 min at 4 °C. The supernatant containing water-soluble proteins was collected. Particles containing cell debris and water-insoluble membrane components were resuspended in PBS. MnBP@pGluCP was added to the supernatant and resuspended particles to capture antigens. The mixture was gently stirred and incubated at room temperature for 1 h. The pGluCP antigen complex in the supernatant was collected by centrifugation to obtain a water-soluble antigen / nanomaterial complex. For the resuspended particles, the membrane antigen coated with pGluCP was allowed to precipitate under gravity. The captured antigen was washed with PBS and then used for downstream analysis. To verify the ability to capture water-insoluble membrane antigens, purified cancer cell membranes were incubated with MnBP@pGluCP and analyzed by SDS-PAGE gel electrophoresis. Subsequently, the expression of BMDC surface markers after capturing different types of antigens was further investigated.

[0063] The results are as follows Figure 6 The results showed that the proteomic profile of pGluCP was highly similar to that of purified cancer cell membranes, strongly demonstrating that MnBP@pGluCP can efficiently capture insoluble tumor cell membrane antigens. This superior antigen capture capability enhances antigen diversity, providing a wider range of antigenic peptides for T cell recognition and promoting a more comprehensive immune response. Figure 6 As shown in bc, compared with adsorbing water-soluble or water-insoluble membrane antigens alone, the MnBP@pGluCP nanocomposite, which simultaneously captures both water-insoluble and water-soluble antigens, exhibits the highest SIINFEKL peptide level, confirming its significantly improved cross-presentation efficiency. More importantly, in terms of simultaneously inducing DC maturation and enhancing antigen presentation capabilities, the nanocomposite capturing multiple types of antigens is superior to the single-antigen nanocomposite. Figure 6 b, de)

[0064] Example 2: Application of nanomaterials with choline phosphate-cell membrane synergistic antigen capture function in in situ tumor vaccines.

[0065] I. Immunological Effects of Nanomaterials with Enhanced Antigen Capture Function Based on Choline Phosphate-Cell Membrane Synergistic Effect in Primary Tumor Models

[0066] On day 0, 5 × 10⁴ 4T1 tumor cells (purchased from the First Military Hospital of Guangzhou) were subcutaneously injected. 5 Cells / mouse were transferred to the right posterior side of female BALB / c mice (4-6 weeks old, 18-20 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.) until they grew to 100 mm. 3 Subsequent experiments will be conducted when the left and right sides are in motion.

[0067] On days 7, 11, and 15, 100 μL of physiological saline (Saline), MnBP, and MnBP@pGluCP (100 ug / mouse) were administered, with a power of 1.5 W / cm² the day after each administration. 2 The mice were irradiated with an 808 nm laser for 5 minutes. After 21 days of treatment, the mice were sacrificed, and their spleens and lymph nodes were collected for subsequent experiments.

[0068] The spleen was ground into a single-cell suspension of spleen cells. The resulting spleen cells and tumor cells were seeded at a ratio of 25:1 in 96-well plates and cultured for 24 h. Cell status was observed under an inverted fluorescence microscope, and tumor cell viability was detected using CCK-8 assay. Additionally, the spleen and lymphocytes were ground into single-cell suspensions, and the resulting spleen cells and lymphocytes (1×10⁻⁶ cells / well) were... 6 (1 cell / mouse) was stained with fluorescently labeled antibodies (APC-anti-CD3, PerCP-Cy5.5-anti-CD8a, FITC-anti-CD4) for 30 minutes and detected by flow cytometry.

[0069] Experimental results are as follows Figure 7 As shown, 4T1 tumor cells were subcutaneously inoculated into the right posterior back on day 0, and allowed to grow to 100 mm. 3 Mice were given intratumoral injections on days 7, 11, and 15, and laser irradiation on days 8, 12, and 16. Figure 7 a). The experimental results showed that, compared with the saline group, the MnBP+L group exhibited certain tumor killing, but the tumor killing efficiency of the MnBP@pGluCP+L group was significantly higher than that of other groups, providing strong evidence for antigen-specific CTL responses. Figure 7 bc). In addition, CD8 in the spleen and lymph nodes + T cell detection results as follows Figure 7 As shown in dg, compared with the saline and MnBP+L groups, the MnBP@pGluCP+L group exhibited the highest CD8 concentration. +T cell infiltration was observed in the MnBP@pGluCP+L group. + The T cell infiltration level was significantly higher in the MnBP+L group than in the MnBP+L group, thanks to its superior antigen capture and presentation capabilities.

[0070] These results indicate that nanomaterials based on the synergistic effect of choline phosphate and cell membrane to enhance antigen capture function can kill tumor cells under photothermal action, and capture tumor-associated antigens during tumor cell death through the "chassis" of pGluCP-enhanced materials, thereby recruiting more dendritic cells (DCs) and significantly enhancing the vaccine effect.

[0071] II. Antitumor Effects of Nanomaterials with Enhanced Antigen Capture Function Based on Choline Phosphate-Cell Membrane Interactions in Distant Tumor Models

[0072] To further investigate the antitumor effect of the in situ tumor vaccine formulation on a mouse model with distal tumors, a bilateral tumor model was constructed. Mice were subcutaneously injected with 4T1 cells (5 × 10⁻⁶) into the right posterior side. 5 / each) (primary tumor), 4 days later, 4T1 cells (5×10) were subcutaneously injected into the left posterior side. 5 / mouse) (distal tumor). When the tumor on the right side of the mouse reached approximately 50 mm 3 Mice were randomly divided into groups (n=5). The primary tumor site was treated with the same form of anti-tumor therapy as in the primary tumor model. Tumor volume and body weight were measured every two days (including on the day of inoculation). Tumor volume = (width + weight) / (body weight + weight). 2 (×length) / 2.

[0073] The results are as follows Figure 8 As shown, 4T1 tumor cells were subcutaneously inoculated into the right posterior side of mice on day 0, and into the left posterior side on day 4. When the tumor on the right side of the mouse reached approximately 50 mm... 3 At that time, the drug was injected into the tumor once every two days, followed by laser irradiation the next day, for a total of three times. Figure 8 a). Tumor growth curves and images showed that, compared with the saline group, the MnBP+L group significantly inhibited the growth of primary 4T1 tumors and also had a significant inhibitory effect on the growth of distant tumors. Figure 8 b, ef), while the MnBP@pGluCP+L group showed more significant effects in inhibiting tumors and reducing tumor weight (b, ef), while the MnBP@pGluCP+L group had more significant effects in inhibiting tumors and reducing tumor weight (b, ef), Figure 8 c, gh). Meanwhile, the mice's body weight did not change significantly during the treatment period, demonstrating the good biocompatibility of the drug. Figure 8 d).

[0074] In summary, for reference Figure 1(b) As can be seen, this invention, through the adsorption and modification of manganese-mineralized black phosphorus nanomaterials by the bio-inspired polymer pGluCP, retains the excellent biocompatibility and photothermal properties of BP. Simultaneously, pGluCP acts as a reinforcing "chassis" for the material, capturing tumor-associated antigens. It can simultaneously capture both water-soluble and water-insoluble membrane antigens, forming pathogen-like micro / nanoparticles, which facilitates antigen delivery and APC presentation, providing a feasible tool to address the significant challenge of capturing insoluble membrane antigens. The synergistic effect of MnBP and pGluCP enables personalized transformation of autologous tumors into vaccine production and delivery.

[0075] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A functional nanomaterial based on choline phosphate-cell membrane interaction, characterized in that, The functional nanomaterial is prepared from manganese-mineralized black phosphorus nanosheets (MnBP) and polyglutamic acid-choline phosphate (pGluCP); the mass ratio of MnBP to pGluCP is 1:6-24. The method for preparing the functional nanomaterials includes the following steps: (1) Preparation of black phosphorus nanosheets; Bulk black phosphorus crystals were dispersed in N-methylpyrrolidone, sonicated under ice bath conditions, and then the suspension was centrifuged, washed, and the supernatant was collected to obtain black phosphorus nanosheet suspension I. (2) Preparation of manganese mineralized black phosphorus nanosheets; The black phosphorus nanosheet suspension obtained in step (1) was dispersed in N-methylpyrrolidone to obtain black phosphorus nanosheet suspension II. NH3•H2O was added, and the mixture was stirred at 40°C. Mn was then added dropwise. 2+ The solution was stirred in the dark, centrifuged, and washed to obtain manganese-mineralized black phosphorus nanosheets. (3) The functional nanomaterial based on choline phosphate-cell membrane interaction is obtained by mixing and stirring a suspension of manganese mineralized black phosphorus nanosheets and a polyglutamic acid-choline phosphate solution. In step (2), the concentration of black phosphorus nanosheet suspension II is 50 μg / mL based on the original amount of black phosphorus used, and the mass percentage concentration of added NH3•H2O is 1.9-2.0%; the volume of added NH3•H2O is 0.1% of the volume of black phosphorus nanosheet suspension II.

2. The functional nanomaterial according to claim 1, characterized in that, In step (1), N-methylpyrrolidone: black phosphorus = 1 mL: 1 mg.

3. The functional nanomaterial according to claim 1, characterized in that, In step (1), the ultrasonic power is 600w and the ultrasonic time is 18h under ice bath conditions.

4. The functional nanomaterial according to claim 1, characterized in that, In step (2), Mn 2+ Mn in solution 2+ The concentration is 0.7-0.8 mmol / L, and the volume is 1 mL.

5. The functional nanomaterial according to claim 1, characterized in that: In step (3), the mixing and stirring reaction temperature is room temperature, and the reaction time is 4-8 hours.

6. The functional nanomaterial according to claim 1, characterized in that: In step (2), the washing process involves washing the precipitate until the supernatant is clear and transparent.

7. The use of the functional nanomaterials as described in any one of claims 1-6 in the preparation of antigen-capturing functional materials or in in situ tumor vaccines.

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