Functional nano material based on choline phosphate-cell membrane interaction and application thereof

Abstract

The invention relates to the technical field of medicines, in particular to a functional nano material based on choline phosphate-cell membrane interaction and application thereof. According to the invention, the biologically inspired polymer pGluCP adsorbs the modified manganese mineralized black phosphorus nano material, on one hand, the excellent biocompatibility and photo-thermal performance of BP are retained, and on the other hand, the pGluCP enhances the'chassis' effect of the material, captures tumor-associated antigens, and can capture water-soluble antigens and water-insoluble membrane antigens at the same time and form micro-nano particles similar to pathogens; and antigen delivery and APCs presentation are facilitated, and a feasible tool is provided for a major challenge of capturing a water-insoluble membrane antigen. MnBP and pGluCP have a synergistic effect, so that personalized transformation from autologous tumor to vaccine production and delivery can be realized.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to a functional nano material based on choline phosphate-cell membrane interaction and application thereof. Background Art

[0002] In recent years, tumor immunotherapy has become a revolutionary force in clinical oncology and a focus of innovative cancer treatment. Among them, tumor vaccine therapy has unique advantages: specific killing of tumor cells with few side effects; the immune system throughout the body can instantly capture metastatic tumor cells; and the immune memory function induced by the vaccine can prevent tumor recurrence for a long time. However, the development of traditional tumor vaccines has many problems, such as the difficulty in identifying tumor antigens, the cumbersome preparation process, and the lack of broad spectrum due to tumor heterogeneity. In recent years, the rise of tumor in situ vaccines has provided a new idea for solving the limitations of conventional tumor vaccines. Tumor in situ vaccines refer to in situ killing of tumor cells, releasing a large number of tumor antigens, attracting and activating antigen presenting cells (APCs), achieving antigen presentation, and inducing specific anti-tumor immune responses, aiming to achieve personalized transformation of vaccine production and delivery for autologous tumors. Tumor in situ vaccines have significant advantages such as simplicity, time saving, economy, universality and few side effects, and have become a research hotspot in the field of anti-tumor. However, the immune effect induced by tumor in situ vaccines is weak and it is difficult to cure tumors. The reason is that the vaccine cascade immune response process is complex, trapped by multiple restrictive factors, and leads to low efficiency. Among them, the presentation of tumor antigens by antigen-presenting cells is the key rate-limiting step in the vaccine cascade reaction. Studies have found that free antigens released during tumor cell death are easily and quickly cleared and are not effectively presented by APCs. Therefore, in the complex tumor microenvironment, how to improve the antigen capture and presentation efficiency of APCs is crucial for initiating specific anti-tumor immune responses.

[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, which can bind to tumor antigens and transport them to lymph nodes, thereby improving antigen presentation. Other researchers have proposed that hydrogel-based three-dimensional networks are effective antigen capture devices that can retain and concentrate antigens at the injection site and promote antigen capture by APCs. It is worth noting that capturing water-soluble and water-insoluble membrane antigens to simulate pathogen-like particles can significantly improve the efficiency of antigen presentation. However, capturing water-insoluble membrane antigens is a major challenge. These antigens are often embedded in the liquid bilayer of tumor cells and are difficult to extract and transport. Despite advances in functional nanocarriers and hydrogel networks, there are currently limited reports on successful strategies for effectively capturing and delivering these challenging antigens. Further research is needed to develop innovative methods for handling water-insoluble tumor cell membrane antigens, which will greatly improve the efficacy of cancer immunotherapy. Summary of the invention

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

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

[0006] The purpose of the present invention is achieved through the following technical solutions:

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

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

[0009] Preferably, the method for preparing the functional nanomaterial MnBP@pGluCP based on choline phosphate-cell membrane interaction comprises the steps of:

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

[0011] The blocky black phosphorus crystals are dispersed in N-methylpyrrolidone, and ultrasonically treated in an ice bath, and then the suspension is centrifuged, washed, and the supernatant is collected to obtain a 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 a black phosphorus nanosheet suspension II, NH3·H2O was added, and the mixture was stirred at 40°C. Mn 2+ The solution was stirred in the dark, centrifuged, and washed to obtain manganese-mineralized black phosphorus nanosheets;

[0014] (3) The manganese-mineralized black phosphorus nanosheet suspension and the polyglutamic acid-choline phosphate solution are mixed and stirred to obtain the functional nanomaterial based on choline phosphate-cell membrane interaction.

[0015] Preferably, in step (1), N-methylpyrrolidone: black phosphorus = 1 mL: 1 mg, that is, 1 mg BP block black phosphorus crystals are mixed with 1 mL NMP.

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

[0017] In the black phosphorus nanosheet suspension II, the concentration of black phosphorus is 50 μg / mL, calculated based on the original amount of black phosphorus, and the mass percentage concentration of the added NH3·H2O is 1.9-2.0%; the volume of the added NH3·H2O is 0.1% of the volume of the 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 0.7-0.8mmol / L and the dosage is 1mL. 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) is to wash the precipitate until the supernatant is clear and transparent.

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

[0022] The invention also provides the use of the functional nano material in preparing materials with antigen capture function and in in situ tumor vaccines.

[0023] The application is achieved through the following steps: the above-mentioned functional nanomaterial MnBP@pGluCP based on choline phosphate-cell membrane interaction is injected into the tumor to construct an in situ vaccine platform. First, black phosphorus has an excellent photothermal effect, which effectively eliminates tumor cells and induces immunogenic cell death, thereby leading to the release of tumor antigens. Subsequently, the water-soluble antigens and water-insoluble membrane antigens are simultaneously captured through pGluCP modification to form micro-nanoparticles similar to pathogens, which are assembled into tumor vaccines in situ. Then, through the unique interaction between CP-PC, the uptake of tumor antigens by APC is enhanced, thereby enhancing antigen presentation and vaccine effect.

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

[0025] Compared with the prior art, the present invention has the following advantages and effects:

[0026] (1) Synthetic manganese-mineralized BP (MnBP) retains the inherent properties of BP (excellent biocompatibility and photothermal properties) and endows it with highly potential anti-tumor efficacy, providing a broad-spectrum strategy for the surface functionalization of BP. Poly(glutamic acid-choline phosphate) (pGluCP) with unique cell membrane adhesion ability enhances the "chassis" effect of the material and captures tumor-associated antigens. 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) The capture of water-insoluble membrane antigens is difficult and rarely reported. The present invention uses the bioinspired polymer pGluCP to adsorb modified manganese-mineralized black phosphorus nanomaterials, which can simultaneously capture water-soluble antigens and water-insoluble membrane antigens and form pathogen-like micro-nanoparticles, which are helpful for antigen delivery and presentation by APCs, and provide a feasible tool for the major challenge of capturing water-insoluble membrane antigens, breaking through the bottleneck of low efficiency of antigen delivery in the immune cascade reaction. This will break the bottleneck of low efficiency of antigen presentation in the immune cascade reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is the preparation and mechanism of nanomaterials based on the synergistic effect of choline phosphate and cell membrane to enhance antigen capture function;

[0029] Figure 2 are the relevant characterization results of MnBP and MnBP@pGluCP; among them, a is the high-resolution transmission electron microscopy image of MnBP; b is the EDS elemental analysis result of MnBP; c is the potential change of BP, MnBP and MnBP@pGluCP; d is the infrared absorption spectrum of BP under different synthesis conditions; e is the ultraviolet absorption spectrum of BP and MnBP; f is the XPS spectrum of BP and MnBP;

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

[0031] Figure 4 MnBP and pGluCP work together to achieve antigen release and in situ retention; a is laser irradiation (808 nm, 1.5 W / cm 2 ) under infrared thermal imaging of MnBP and MnBP@pGluCP; b is infrared thermal imaging of MnBP and MnBP@pGluCP under ... 2 )-treated 4T1 cells. c is the animal fluorescence in vivo imaging result of the tumor injection site; d is the average fluorescence intensity of the tumor injection site;

[0032] Figure 5is the evaluation of antigen capture ability; a is the SDS-PAGE result of 4T1 cell lysate incubated with MnBP and MnBP@pGluCP respectively; b is the total amount of protein captured by different nanoparticles after MnBP or MnBP@pGluCP was incubated with 4T1 lysate; c is the change of zeta potential after MnBP or MnBP@pGluCP was incubated with 4T1 cell lysate;

[0033] Figure 6 (a) The effect of MnBP@pGluCP capturing different types of antigens on BMDCs maturation and antigen presentation efficiency; (b) Representative flow cytometric analysis of BMDCs activated by MnBP@pGluCP capturing different types of antigens; (c) Quantitative analysis of the expression levels of MHC I, (d) CD80, and (e) CD86 on BMDCs activated by MnBP@pGluCP capturing different types of antigens;

[0034] Figure 7 The immunological effect of nanomaterials based on the synergistic effect of choline phosphate-cell membrane to enhance antigen capture ability in primary tumor models; a was subcutaneously inoculated with 4T1 tumor cells in mice, in situ therapeutic inoculation with the specified preparation on days 7, 11, and 15, and treated with 808nm laser (1.5W / cm 2 , 5min) scheme schematic diagram; b is a microscope image of tumor cell specific killing; c is a quantitative data diagram of the specific killing ability of spleen cells in mice in different treatment groups; d and e are CD8 + Representative flow cytometry images and quantitative data of T cells; f and g are CD8 + Representative flow cytometry images and quantitative data of T cells;

[0035] Figure 8 It is the anti-tumor effect of nanomaterials based on the synergistic effect of choline phosphate-cell membrane to enhance antigen capture ability in a distal tumor model; wherein, a is a schematic diagram of the bilateral tumor model; b is the monitoring of the volume change of the primary tumor of mice during three inoculations; c is the growth curve of the distal tumor of mice during the experiment; d is the weight change of the experimental mouse; e and f are the in vitro tumor images and weights of the primary tumor, respectively; g and h are the in vitro tumor images and weights of the distal tumor, respectively. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0037] In the description of the present invention, unless otherwise clearly defined, terms such as heating, cleaning, weighing, freezing, etc. should be understood in a broad sense, and technicians in the relevant technical field can reasonably determine the specific meanings of the above terms in the present invention in combination with the specific content of the technical solution.

[0038] In the description of the present invention, the description with reference to the terms "some embodiments", "examples", etc. means that the specific methods and materials described in conjunction with the embodiments or examples are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific methods and materials described can be combined in any one or more embodiments or examples in a suitable manner.

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

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

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

[0042] The ovalbumin (OVA) involved in the examples of the present invention was purchased from Sigma.

[0043] Example 1 Preparation of nanomaterials based on choline phosphate-cell membrane synergistic effect to enhance antigen capture function

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

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

[0046] 20 mg of bulk BP crystals were dispersed in 20 mL of N-methylpyrrolidone (NMP) and ultrasonically treated at 600 W for 18 h under ice bath conditions. After the ultrasonic treatment, the above material suspension was centrifuged at 9000 rpm for 10 min to remove the oversized product, and the supernatant was collected and stored at 4°C for subsequent use.

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

[0048] 10mL BP solution was dispersed in NMP, where the BP concentration was 50μg / mL, and 0.1% (V / V) NH3·H2O (NH3·H2O was prepared by adding 3.87mL 25% NH3·H2O to 50mL deionized water); after stirring at 40°C for 2h, 1mL MnCl2 aqueous solution (MnCl2 aqueous solution was prepared by dissolving 1.4mg MnCl2·4H2O in 10mL deionized water) was dripped into the above mixed solution dropwise, and stirred at 350rpm for 12h in the dark. Finally, the product was collected, washed three times with ethanol and deionized water, respectively, and freeze-dried for storage.

[0049] (3) Synthesis of MnBP@pGluCP

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

[0051] 1. Characterization of MnBP and MnBP@pGluCP

[0052] Energy dispersive X-ray spectroscopy (EDS) combined with high-resolution transmission electron microscopy (HRTEM) was used to detect the shape and size of MnBP nanoparticles and elemental analysis. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared (FTIR) spectroscopy were used to determine the chemical composition of MnBPs. The UV absorption spectra of BPs and MnBPs were detected. 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 is in a flake-like morphology with a lateral size of 300-400 nm ( Figure 2 a), EDS proved 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 neutralization of phosphate anions by Mn ( Figure 2 c) Figure d shows the peaks at 509, 547, 1025 and 1394 cm-1 The broad absorption band at 3430 cm-1 is associated with the stretching of PO and P=O. -1 The absorption band at is derived from OH-stretching. Compared with the “BPs+ammonia” group, the OH band intensity of MnBPs is increased and broadened, confirming the formation of intermolecular and weak hydrogen-bonded OH in MnP. XPS results show that Mn exists in MnBP ( Figure 2 e), all of the above results indicate that MnBP was successfully prepared. The UV-visible absorption spectra of BPs and MnBPs show broad-spectrum absorption, among which the absorption of MnBP is stronger ( Figure 2 f). MnBP and pGluCP were prepared by simple and efficient charge adsorption, and the charge conversion from negative to positive confirmed the successful preparation of MnBP@pGluCP. According to the saturation experiment of pGluCP binding, the optimal mass ratio of MnBP and pGluCP was determined to be 1:6 ( Figure 3 ). The MnBP@pGluCP complex prepared according to this ratio was used in subsequent experiments.

[0054] 2. Antigen release and in situ accumulation based on MnBP@pGluCP

[0055] First, infrared photothermal imaging was used to study the photothermal effects of MnBP and MnBP@pGluCP. The apoptosis of 4T1 cells was detected under different photothermal conditions. Subsequently, in vivo imaging of small animals was used to study the accumulation of MnBP@pGluCP in situ in the tumor. Specifically, BALB / c mice (4-6 weeks, 18-20g, purchased from Beijing Huafukang Biotechnology Co., Ltd.) were randomly divided into two groups (n=3), 1×10 6 The cells were implanted on the back of mice at a density of 10 cells / mouse. When the tumor grew 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 MnBP or MnBP@pGluCP nanoparticles were added to 1 mL Cy5.5-OVA solution (300 μg, prepared with normal saline) and stirred at 300 rpm for 12 h at room temperature to obtain MnBP / Cy5.5-OVA or MnBP@pGluCP / Cy5.5-OVA, and MnBP / Cy5.5-OVA or MnBP@pGluCP / Cy5.5-OVA were injected into mouse tumors, respectively. 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 4 As shown, the infrared thermal imager also verified the photothermal conversion ability 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%, which further proves that the modification of pGluCP on MnBP significantly improves the tumor killing ability and causes tumor cell apoptosis, which will promote the release of tumor-associated antigens.

[0057] To investigate the retention of the material at the tumor site, mice were inoculated with 4T1 tumors up to 50 mm. 3 The OVA-Cy5.5 assembly materials were randomly injected into the tumor site in situ, and fluorescence imaging was performed at 1, 2, 4, 8, 10, 12, 24, 36, and 48 h after administration. Figure 4 CD showed that MnBP and MnBP@pGluCP could be retained at the tumor site, and the fluorescence slowly decreased over time. Compared with MnBP, MnBP@pGluCP had a stronger tumor accumulation ability, which was attributed to the cell membrane adhesion of pGluCP. This membrane adhesion prolonged the retention time of the antigen at the tumor site, thus generating a sustained and strong anti-tumor immune response.

[0058] 3. 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 lysis buffer containing protease and phosphatase inhibitors. The solution containing water-soluble protein antigens and water-insoluble membrane antigens was collected, and 1 mg of MnBP and MnBP@pGluCP were added, respectively. After incubation at 4°C overnight, the precipitate was collected by centrifugation at 12,000 rpm for 10 min, and the resulting antigen nanocomplex was used for downstream analysis. For quantitative analysis, the total protein amount was analyzed using a BCA detection kit. The potential change of the antigen nanocomplex was measured by DLS. At the same time, the antigen-material complex was also subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to evaluate the purity and integrity of the captured antigen.

[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 of the antigen captured by MnBP ( Figure 5 a). Figure 5As shown in b, MnBP@pGluCP can quickly and effectively capture 4T1 cell lysate, and the total amount of protein captured is about 1.8 times that of MnBP alone. The improvement in capture capacity causes the zeta potential of MnBP@pGluCP to change significantly from positive to negative ( Figure 5 c) This change in zeta potential suggests that MnBP@pGluCP has the potential to capture a wider variety of antigens, which may facilitate its application in immunotherapy strategies.

[0061] 4. Effect of MnBP@pGluCP on antigen presentation efficiency after capturing multiple types of antigens

[0062] 4T1 cells were lysed using RIPA buffered lysis 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. The pellet containing cell debris and water-insoluble membrane components was resuspended in PBS. MnBP@pGluCP was added to the supernatant and resuspended pellet to capture the antigen. The mixture was incubated at room temperature for 1 h with gentle stirring. The pGluCP antigen complex in the supernatant was collected by centrifugation to obtain the water-soluble antigen / nanomaterial complex. For the resuspended pellet, the membrane antigen coated with pGluCP was allowed to precipitate under gravity. The captured antigen was rinsed 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 its capture of different types of antigens was further studied.

[0063] The results are as follows Figure 6 a shows that the protein spectrum of pGluCP is very similar to that of purified cancer cell membranes, which strongly proves that MnBP@pGluCP can efficiently capture water-insoluble tumor cell membrane antigens. This excellent antigen capture ability increases the diversity of antigens, provides a wider range of antigen peptides for T cell recognition, and promotes a more comprehensive immune response. Figure 6 As shown in Fig. 2a, compared with the adsorption of water-soluble or water-insoluble membrane antigens alone, the MnBP@pGluCP nanocomplexes that simultaneously captured water-insoluble and water-soluble antigens showed the highest SIINFEKL peptide level, confirming that their cross-presentation efficiency was significantly improved. More importantly, the nanocomplexes that captured multiple types of antigens were superior to single antigen nanocomplexes in terms of simultaneously inducing DC maturation and enhancing antigen presentation ability ( Figure 6 b, de)

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

[0065] 1. Immune effects of nanomaterials based on choline phosphate-cell membrane synergy to enhance antigen capture in primary tumor models

[0066] On day 0, 5×10 4T1 tumor cells (purchased from Guangzhou First Military Region Hospital) were injected subcutaneously. 5 cells / mouse) to the right hind flank of BALB / c female mice (4-6 weeks, 18-20 g, purchased from Beijing Huafukang Biotechnology Co., Ltd.) and wait until they grow to 100 mm 3 Follow-up experiments were performed around 2 pm.

[0067] On days 7, 11, and 15, 100 μL of saline, MnBP, and MnBP@pGluCP (100 ug / mouse) were administered. The power was 1.5 W / cm 2 The mice were irradiated with 808 nm laser for 5 min. After 21 days of treatment, the mice were killed and the spleen and lymph were collected for subsequent experiments.

[0068] The spleen was ground into a single cell suspension of spleen cells, and the spleen cells and tumor cells were inoculated in a 96-well plate at a ratio of 25:1. The cells were cultured for 24 hours, and the cell status was observed under an inverted fluorescence microscope. The activity of tumor cells was detected by CCK-8. In addition, the spleen and lymph were ground into single cell suspensions, and the spleen cells and lymphocytes (1×10 6 The cells were stained with fluorescent dye-labeled antibodies (APC-anti-CD3, PerCP-Cy5.5-anti-CD8a, FITC-anti-CD4) for 30 min and detected by flow cytometry.

[0069] The experimental results are as follows Figure 7 As shown, 4T1 tumor cells were subcutaneously inoculated on the right posterior back on day 0 and allowed to grow to 100 mm 3 Around 20 days, mice received intratumoral injections of the drug 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 showed a certain degree of tumor killing, but the tumor killing efficiency of the MnBP@pGluCP+L group was significantly higher than that of the other groups, providing strong evidence for the antigen-specific CTLs response ( Figure 7 bc). In addition, CD8 + The results of T cell detection were as follows Figure 7 As shown in dg, compared with the saline and MnBP+L groups, the MnBP@pGluCP+L group showed the highest CD8 + T cell infiltration. Among them, CD8 +The T cell infiltration level was significantly higher than that in the MnBP+L group, which benefited from its excellent antigen capture and presentation ability.

[0070] These results indicate that nanomaterials with enhanced antigen capture function based on choline phosphate-cell membrane synergistic effects kill tumor cells under photothermal action, capture tumor-associated antigens during tumor cell death through the "chassis" of the pGluCP-enhanced material, recruit more DCs, and significantly enhance the vaccine effect.

[0071] 2. Antitumor effect of nanomaterials based on choline phosphate-cell membrane interaction to enhance antigen capture in a remote tumor model

[0072] To further investigate the antitumor effects of orthotopic tumor vaccine preparations on distal tumor mouse models, a bilateral tumor model was established. 4T1 cells (5×10 5 / mouse) (primary tumor), and 4 days later, 4T1 cells (5×10 5 When the right tumor of the mouse reached about 50 mm 3 At 4 pm, the mice were randomly divided into groups (n=5) and treated with the same anti-tumor treatment as in the primary tumor model. The tumor volume and body weight of the mice were measured every 2 days (also once on the day of inoculation). Tumor volume = (width 2 × length) / 2.

[0073] The results are as follows Figure 8 As shown, 4T1 tumor cells were subcutaneously inoculated on the right posterior flank of mice on day 0 and on the left posterior flank on day 4. When the tumor on the right side of the mouse reached about 50 mm 3 The drug was injected into the tumor once every 2 days, and laser irradiation was performed the next day for a total of three times ( Figure 8 a). The tumor growth curve and image results showed that compared with the saline group, the MnBP+L group could significantly inhibit the growth of primary 4T1 tumors and also had a significant inhibitory effect on the growth of distal tumors ( Figure 8 b, ef), while the MnBP@pGluCP+L group had a more significant effect in inhibiting tumors and reducing tumor weight ( Figure 8 c, gh). At the same time, the body weight of mice did not change significantly during the treatment period, indicating that the drug has good biocompatibility ( Figure 8 d).

[0074] In summary, reference Figure 1(b), it can be seen that the present invention, by adsorbing the modified manganese-mineralized black phosphorus nanomaterials through the bioinspired polymer pGluCP, retains the excellent biocompatibility and photothermal properties of BP on the one hand, while pGluCP plays the role of a "chassis" for reinforcing materials, captures tumor-associated antigens, and can simultaneously capture water-soluble antigens and water-insoluble membrane antigens and form pathogen-like micro-nanoparticles, which are helpful for antigen delivery and presentation of APCs, and provide a feasible tool for the major challenge of capturing water-insoluble membrane antigens. MnBP and pGluCP work synergistically to achieve personalized transformation of autologous tumors into vaccine production and delivery.

[0075] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A functional nanomaterial based on choline phosphate-cell membrane interaction, characterized in that: The functional nanoparticles are prepared from manganese-mineralized black phosphorus nanosheets MnBP and polyglutamic acid-choline phosphate pGluCP.

2. The functional nanomaterial according to claim 1, characterized in that: The mass ratio of the MnBP to the pGluCP is 1:6-24.

3. The functional nanomaterial according to claim 1 or 2, characterized in that: The preparation method comprises the steps of: (1) Preparation of black phosphorus nanosheets; The blocky black phosphorus crystals are dispersed in N-methylpyrrolidone, and ultrasonically treated in an ice bath, and then the suspension is centrifuged, washed, and the supernatant is collected to obtain a 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 a black phosphorus nanosheet suspension II, NH3·H2O was added, and the mixture was stirred at 40°C. Mn 2+ The solution was stirred in the dark, centrifuged, and washed to obtain manganese-mineralized black phosphorus nanosheets; (3) The manganese-mineralized black phosphorus nanosheet suspension and the polyglutamic acid-choline phosphate solution are mixed and stirred to obtain the functional nanomaterial based on choline phosphate-cell membrane interaction.

4. The functional nanomaterial according to claim 3, characterized in that: In the step (1), N-methylpyrrolidone: black phosphorus = 1 mL: 1 mg.

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

6. The functional nanomaterial according to claim 1, characterized in that: In the step (2), the concentration of black phosphorus in the black phosphorus nanosheet suspension II is 50 μg / mL, calculated based on the original amount of black phosphorus, and the mass percentage concentration of the added NH3·H2O is 1.9-2.0%; the volume of the added NH3·H2O is 0.1% of the volume of the black phosphorus nanosheet suspension II.

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

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

9. The functional nanomaterial according to claim 1, characterized in that: In the steps (2) and (3), the washing is to wash the precipitate until the supernatant is clear and transparent.

10. Use of the nanomaterial according to any one of claims 1 to 9 in the preparation of antigen capture functional materials and in in situ tumor vaccines.

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