Efficient membrane-rupturing tumor immunogenic cell death biomimetic nanovesicles, preparation method and application thereof

Abstract

The present application relates to the technical field of biological medicine, and particularly relates to a tumor immunogenic death biomimetic nanovesicle with high membrane breaking efficiency, a preparation method and application thereof.The tumor immunogenic death biomimetic nanovesicle with high membrane breaking efficiency comprises a tumor cell membrane and a photothermal reagent; the photothermal reagent is compound A or compound B.The biomimetic nanovesicle provided by the present application has a killing rate of more than 95% on various tumor cells, can be prepared into ICMs for different tumors by flexibly matching cell membranes of different cancer types, supports individualized treatment and combined immunotherapy, and provides an innovative scheme with high efficiency, wide spectrum and low toxicity for solid tumor treatment through synergistic action of targeted delivery and photothermal-induced cell membrane destruction, and has high clinical application potential.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a biomimetic nanovesicle for tumor immunogenic death with high efficiency of membrane permeation, its preparation method and application. Background Technology

[0002] In the field of cancer treatment, biomimetic nanotechnology provides an ideal carrier platform for nanomedicines by mimicking the properties of biological membranes. This novel carrier not only improves the stability and blood circulation time of nanomedicines in vivo, but also significantly enhances their targeting ability to tumor tissues through homologous targeting, thereby increasing the efficiency of drug accumulation at the tumor site. Furthermore, the design of biomimetic nanoparticles fully considers biocompatibility and biodegradability, reducing potential immune responses and toxicity risks. Through these innovations, biomimetic nanotechnology not only improves therapeutic efficacy but also brings greater safety and tolerability to patients.

[0003] Photothermal therapy, as an important means of cancer treatment, has demonstrated immense potential. In this therapy, carefully designed photothermal agents can accumulate at specific locations within tumor cells, enhancing the efficacy of photothermal therapy. When irradiated by an external near-infrared light source, these nanoparticles efficiently convert light energy into heat energy, precisely heating the tumor area without damaging surrounding normal tissue, leading to cancer cell death. Notably, in addition to the traditional method of destroying cancer cells by generating localized high temperatures, photothermal therapy can also destroy cancer cells by targeting the cell membrane, inducing physical changes in the cell membrane. This method avoids the traditional cell membrane damage pathway of pyroptosis, which relies on the expression level of the Gasdermin protein, providing a more direct and effective cell-killing strategy. Therefore, cell membrane-targeted photothermal therapy not only improves the precision of cancer treatment but also broadens its clinical applications.

[0004] To address the limitations of existing cancer treatments, this invention aims to explore a novel approach combining biomimetic nanotechnology and photothermal therapy. First, we obtained tumor cell membranes and, by varying the ratio of cell membrane to IR813, screened for an optimal drug loading ratio to prepare a simple biomimetic nanovesicle loaded with a photothermal reagent. The near-infrared fluorescence and photothermal properties of these nanovesicles were then evaluated. Second, this invention evaluated the homology targeting and immune escape properties of the nanovesicles, confirming their highly efficient and broad-spectrum anti-tumor effect when combined with near-infrared laser. Finally, we analyzed the potential mechanism by which these nanovesicles, combined with near-infrared laser, kill tumor cells, verifying that the cell death induced by these nanovesicles is a highly efficient immunogenic death. This research not only promises to provide new perspectives and tools for cancer treatment but also aims to address challenges in current treatment regimens, such as poor selectivity and significant side effects. Summary of the Invention

[0005] The purpose of this invention is to develop a nanovesicle loaded with a photothermal reagent on a cell membrane. The photothermal reagent is delivered to the cell membrane through the homologous targeting and membrane fusion functions of the cell membrane. After being irradiated with a near-infrared laser, the photothermal effect on the cell membrane is used to achieve efficient destruction of the cell membrane, thereby effectively exerting an anti-tumor effect.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a highly efficient membrane-permeable biomimetic nanovesicle for tumor immunogenic death, comprising a tumor cell membrane and a photothermal reagent; The method for preparing the tumor cell membrane includes the following steps: a) digesting the tumor cells with trypsin and then washing them; b) resuspending the washed cells in a hypotonic lysis buffer and sonicating them; c) centrifuging to remove the cell nuclei, collecting the supernatant and centrifuging again to collect the cell membrane, and washing it; the hypotonic lysis buffer is a mixture of 18~22mM Tris-HCl; 8~12mM KCl; 1.8~2.2mM MgCl2 and 1× protease inhibitor.

[0007] Preferably, the photothermal reagent can be compound A.

[0008] Formula 1

[0009] Wherein, R: methyl, ethyl, propyl or butyl; including p-toluenesulfonate, perchlorate, chloride, bromide or iodide; Or compound B

[0010] Formula 2

[0011] Wherein, R: methyl, ethyl, propyl or butyl; including p-toluenesulfonate, perchlorate, chloride, bromide or iodide.

[0012] Preferably, the photothermal reagent is IR813, IR780, or IR775.

[0013] Preferably, the final concentration of the tumor cell membrane is 150-250 μg / ml; and the final concentration of the photothermal reagent is 450-550 μg / ml.

[0014] Preferably, the ultrasonic treatment in step b) has a frequency of 20~40kHz, a power of 100~200W, and a treatment time of 3~5min.

[0015] Preferably, the tumor cells in step a) are derived from breast cancer, glioma, lung cancer, or colorectal cancer.

[0016] This invention provides a method for preparing the aforementioned nanovesicles, which involves mixing and stirring tumor cell membranes and photothermal reagents, washing with water, and then extruding the mixture.

[0017] Preferably, the extrusion method is as follows: extruding 8 to 12 times using a liposome extruder, wherein the pore size of the polycarbonate membrane in the liposome extruder is 700 to 900 nm.

[0018] This invention provides the application of the aforementioned nanovesicles in the preparation of therapeutic antitumor drugs.

[0019] Preferably, the antitumor drug is a breast cancer treatment drug, a glioma treatment drug, a lung cancer treatment drug, or a colorectal cancer treatment drug.

[0020] This invention provides an antitumor drug comprising the aforementioned nanovesicles; the nanovesicles are used in conjunction with near-infrared light.

[0021] Preferably, the near-infrared light has a wavelength of 808nm, a power density of 0.9~1.1W / cm², and an irradiation time of 4~6min.

[0022] Preferably, the innovation of the treatment scheme provided by the present invention is reflected in the following aspects: synergistic effect of targeted delivery and photothermal destruction: utilizing the homologous targeting of tumor cell membranes to locate photothermal reagents to tumor cell membranes, avoiding the dependence of traditional photothermal therapy on intracellular targets; cell membrane damage mechanism: directly destroying tumor cell membranes to induce immunogenic death, while reducing damage to normal cells; applicability of near-infrared absorbing photothermal molecules: applicable to positively charged photothermal molecules with near-infrared absorption.

[0023] Preferably, the present invention can be applied not only to the treatment of solid tumors, including but not limited to breast cancer, glioma, lung cancer, melanoma, liver cancer, and head and neck cancer; but also to combination therapy, using it in combination with chemotherapy drugs (such as paclitaxel, docetaxel, cyclophosphamide, gemcitabine, etc.) or immune checkpoint inhibitors (such as anti-PD-1 antibodies, PD-L1, CTLA-4, etc.) to synergistically enhance the anti-tumor effect.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1) Targeted cell membrane delivery: Through the homologous targeting and membrane fusion mechanism of tumor cell membrane (CM), photothermal reagents (including but not limited to IR813, IR780, IR775 and other similar photothermal reagents) are directly delivered to the surface of homologous tumor cell membranes, so as to achieve precise positioning of photothermal reagents on tumor cell membranes.

[0025] 2) Photothermal Damage and Immunogenic Cell Death: After near-infrared laser irradiation, the photothermal reagent releases energy locally on the tumor cell membrane, directly disrupting cell membrane integrity (e.g., SYTOX staining shows a significant increase in inner membrane permeability 10 min after irradiation), triggering immunogenic cell death (ICD), releasing immunogenic substances such as ATP, HMGB1, and Calreticulin, and activating the anti-tumor immune response. Experiments show that this method has a killing rate of ≥95% on various tumor cells and extremely low toxicity to normal cells.

[0026] 3) Broad Applicability: Cell membrane-derived materials can be flexibly matched to different cancer types (such as breast cancer, glioma, lung cancer, etc., and are also applicable to other types of cancer). By preparing ICMs from different tumor sources, "customized" treatment can be achieved, supporting personalized therapy and combined immunotherapy. This technology provides an innovative, efficient, broad-spectrum, and low-toxicity solution for solid tumor treatment through the synergistic effect of targeted delivery and photothermal-induced cell membrane disruption. Attached Figure Description

[0027] Figure 1 The results show the physicochemical properties of ICM (nanovesicles).

[0028] Where A: Optical images and yields of ICMs with different feed ratios

[0029] B: Zeta potential and drug loading efficiency of ICMs with different feed ratios

[0030] C: Potentiometric spectra of IR813, CM and ICM

[0031] D: Transmission electron microscopy image of ICM

[0032] E: UV-Vis-NIR absorption spectra of IR813, CM (cell membrane), and ICM.

[0033] F: Fluorescence absorption spectra of IR813, CM and ICM

[0034] G: ICM photothermal imaging image

[0035] H: SDS-PAGE images of 4T1 cells (breast cancer cells), CM, and ICM.

[0036] I: Western blot images of 4T1 cells, CM and ICM.

[0037] Figure 2 To investigate the uptake of ICM by 4T1 tumor cells and the homologous targeting and immune evasion effects of ICM.

[0038] Among them, A: CLSM images of 4T1 cells incubated with ICM for different times (bar=20μm).

[0039] B: Quantitative flow cytometry fluorescence intensity graphs of 4T1 cells incubated with ICM for different time periods (n=3)

[0040] C: CLSM images (bar=20μm) of 4T1 cells incubated with ICM (RBC), ICM (GL261), and ICM (4T1) for 2 h.

[0041] D: Flow cytometry fluorescence intensity quantification of 4T1 cells incubated with ICM (RBC), ICM (GL261), and ICM (4T1) for 2 h (n=3)

[0042] E: CLSM image of RAW264.7 cells incubated with ICM or IR813 for 2 h (bar=20μm)

[0043] F: Quantitative flow cytometry fluorescence intensity of RAW264.7 cells incubated with ICM or IR813 for 2 h (n=3).

[0044] Figure 3 To investigate the therapeutic effects of ICM combined with near-infrared laser on tumor cells and on various types of tumor cells.

[0045] Among them, A: Representative bright-field morphology images (bar=20μm) of 4T1 cells after incubation with different concentrations of ICM for 2 h and irradiation with near-infrared laser for 3 h.

[0046] B: Bar graph of cell death in 4T1 cells after incubation with different concentrations of ICM for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0047] C: A bar graph of cell death in GL261 cells after incubation with 5 μg / ml ICM (GL261) for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0048] D: A bar graph of cell death in EMT-6 cells after incubation with 5 μg / ml ICM (EMT-6) for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0049] E: A bar graph of cell death in A549 cells after incubation with 5 μg / ml ICM (A549) for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0050] F: A bar graph of cell death in MC38 cells after incubation with 5 μg / ml ICM (MC38) for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0051] G: A bar graph of cell death in HCT116 cells after incubation with 5 μg / ml ICM (HCT116) for 2 h, followed by near-infrared laser irradiation for 3 h (n=3).

[0052] H: Cell viability statistics

[0053] I: Representative bright-field morphology images (bar=20μm) of 4T1 and MCF-10A cells after incubation with 5μg / ml ICM (4T1) for 2 h followed by near-infrared laser irradiation for 3 h.

[0054] J: Bar graphs of cell death (n=3) of 4T1 and MCF-10A cells after incubation with 5 μg / ml ICM (4T1) for 2 h and irradiation with near-infrared laser for 3 h.

[0055] Figure 4 To investigate the distribution of ICM in cells and its damage to the cell membrane after combined NIR irradiation.

[0056] A: Fluorescence image of 4T1 cells colocalized with the cell membrane after incubation with 5 μg / ml ICM for 2 h (bar=5μm).

[0057] B: Analysis diagram of colocalization of 4T1 cells with cell membrane after incubation with 5 μg / ml ICM for 2 h.

[0058] C: 4T1 cells incubated with 5 μg / ml ICM (4T1) for 2 h, followed by near-infrared laser irradiation, and the resulting cell membrane damage fluorescence image within 1 h (bar=20μm).

[0059] D: Fluorescence signal changes of 4T1 cells after incubation with 5 μg / ml ICM (4T1) for 2 h, followed by near-infrared laser irradiation, within 1 h, indicating cell membrane damage (n=3).

[0060] E: AFM image of cell membrane damage (bar=1μm) of 4T1 cells after incubation with 5μg / ml ICM (4T1) for 2 h followed by near-infrared laser irradiation for 1 h.

[0061] F: AFM image of cell membrane damage (bar=20μm) of 4T1 cells after incubation with 5μg / ml ICM (4T1) for 2 h followed by near-infrared laser irradiation for 1 h.

[0062] Figure 5 To investigate the exposure of tumor cells to immunogenic substances by ICM combined with near-infrared laser.

[0063] Among them, A: The amount of ATP released by 4T1 cells after incubation with 5 μg / ml ICM for 2 h and irradiation with near-infrared laser for 3 h was detected (n=3).

[0064] B: Western blot analysis results of 4T1 cells incubated with 5 μg / ml ICM for 2 h, followed by near-infrared laser irradiation for 3 h, and HMGB1 in the cell culture medium, with GADPH as the standard control.

[0065] C: Western blot analysis of Calreticulin on the cell membrane of 4T1 cells after incubation with 5 μg / ml ICM for 2 h followed by near-infrared laser irradiation for 3 h, with ATPA1 as the standard control.

[0066] D: Immunofluorescence image of HMGB1 cells in 4T1 cells after incubation with 5 μg / ml ICM for 2 h, followed by near-infrared laser irradiation for 3 h (bar=20μm).

[0067] E: Immunofluorescence image of Calreticulin in 4T1 cells after incubation with 5 μg / ml ICM for 2 h followed by near-infrared laser irradiation for 3 h (bar=20μm). Detailed Implementation

[0068] The technical solutions provided by this invention will be described in detail below with reference to experimental examples, but they should not be construed as limiting the scope of protection of this invention.

[0069] In the following methods, the electrophoresis conditions are 250 V for 30 min. In the following methods, the membrane transfer conditions are 200 mA for 30 min. In the following methods, the TBST washing conditions are 100 rpm for 10 min.

[0070] Example 1

[0071] A highly efficient tumor immunogenic death biomimetic nanovesicle that permeates the cell membrane comprises a tumor cell membrane at a final concentration of 150 μg / ml (based on membrane proteins, the same below) and a photothermal reagent at a final concentration of 450 μg / ml; the photothermal reagent is IR813. The method for preparing the tumor cell membrane includes the following steps: a) The tumor cells were digested with pancreatic enzymes and then washed three times with PBS; the tumor cells were derived from colorectal cancer.

[0072] b) Resuspend the washed cells in hypotonic lysis buffer and sonicate (4°C, 20kHz, 100W, 3min). c) Centrifuge (3000 g, 5 min, 4°C) to remove cell nuclei, collect the supernatant and centrifuge again (20000 g, 30 min, 4°C) to collect cell membranes. After washing three times with ultrapure water, cell membranes (CM) are obtained. The hypotonic lysis buffer is 20 mM Tris-HCl; 10 mM KCl; 2 mM MgCl2; 1× protease inhibitor mixture; pH=7.4.

[0073] The method for preparing the nanovesicles (ICM) involves mixing and stirring tumor cell membranes and photothermal reagents (stirring speed 1400 rpm, overnight, temperature 4℃), washing with water (centrifugation speed 20000 g, time 30 min, temperature 4℃), and then extruding them 8 times using a liposome extruder. The polycarbonate membrane in the liposome extruder has a pore size of 700 nm.

[0074] Example 2

[0075] A biomimetic nanovesicle for tumor immunogenic death with high efficiency of membrane permeation includes tumor cell membrane at a final concentration of 250 μg / ml and a photothermal reagent at a final concentration of 550 μg / ml; the photothermal reagent is IR775. The method for preparing the tumor cell membrane includes the following steps: a) The tumor cells were digested with trypsin and then washed three times with PBS; the tumor cells were from lung cancer. b) Resuspend the washed cells in hypotonic lysis buffer and sonicate (4°C, 40kHz, 200W, 5min). c) Centrifuge (3000 g, 5 min, 4°C) to remove cell nuclei, collect the supernatant and centrifuge again (20000 g, 30 min, 4°C) to collect cell membranes. After washing three times with ultrapure water, cell membranes (CM) are obtained. The hypotonic lysis buffer is 20 mM Tris-HCl; 10 mM KCl; 2 mM MgCl2; 1× protease inhibitor mixture; pH=7.4.

[0076] The method for preparing the nanovesicles (ICM) involves mixing and stirring tumor cell membranes and photothermal reagents (stirring speed 1400 rpm, overnight, temperature 4℃), washing with water (centrifugation speed 20000 g, time 30 min, temperature 4℃), and then extruding 12 times using a liposome extruder. The polycarbonate membrane in the liposome extruder has a pore size of 900 nm.

[0077] Example 3

[0078] A highly efficient tumor immunogenic death biomimetic nanovesicle that can perforate cell membranes comprises a tumor cell membrane at a final concentration of 200 μg / ml and a photothermal reagent at a final concentration of 500 μg / ml; the photothermal reagent is IR780. The method for preparing the tumor cell membrane includes the following steps: a) The tumor cells were digested with trypsin and then washed three times with PBS; the tumor cells were from breast cancer. b) Resuspend the washed cells in hypotonic lysis buffer and sonicate (4°C, 30kHz, 150W, 4min). c) Centrifuge (3000 g, 5 min, 4°C) to remove cell nuclei, collect the supernatant and centrifuge again (20000 g, 30 min, 4°C) to collect cell membranes. After washing three times with ultrapure water, cell membranes (CM) are obtained. The hypotonic lysis buffer is 20 mM Tris-HCl; 10 mM KCl; 2 mM MgCl2; 1× protease inhibitor mixture; pH=7.4.

[0079] The method for preparing the nanovesicles (ICM) involves mixing and stirring the tumor cell membrane and photothermal reagent (stirring speed of 1400 rpm, overnight, temperature of 4℃), washing with water (centrifugation speed of 20000 g, time of 30 min, temperature of 4℃), and then extruding 10 times using a liposome extruder. The polycarbonate membrane in the liposome extruder has a pore size of 800 nm.

[0080] Experimental Example 1

[0081] This invention characterized the prepared ICM using multiple methods, including transmission electron microscopy, DLS detection, UV-Vis-NIR spectroscopy, Fourier transform infrared spectroscopy, fluorescence spectroscopy, and near-infrared photothermal imaging. These methods demonstrated the retention of the properties of IR813. Sodium hexadecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot characterization were also performed, demonstrating the complete retention of the CM properties. Figure 1 ).

[0082] In the SDS-PAGE image, the bands of ICM and CM are similar, indicating that the characteristic proteins of the original membrane were preserved after drug loading during the preparation of ICM (this is the basis for achieving "immune escape" or "targeting"); it also proves that extrusion / ultrasound did not break down the membrane proteins and that the drug loading process did not introduce serum proteins or other impurities. The WB results specifically characterized the retention of some proteins with homologous targeting functions (CD44, E-cadherin) and immune escape functions (CD47).

[0083] Experiment Example 2

[0084] This invention uses confocal microscopy and flow cytometry to investigate the effective uptake time of ICM by tumor cells. 5 μg / ml of ICM was incubated in 4T1 tumor cells for 0 h, 0.5 h, 1 h, 2 h, and 4 h. After washing with PBS, the nuclei were labeled with DAPI and observed using CLSM. ICM was then digested with trypsin and analyzed by flow cytometry. Confocal images showed no significant difference in IR813 fluorescence intensity between 2 and 4 hours of incubation; flow cytometry measurements of the average fluorescence intensity of cells also showed no difference between 2 and 4 hours, indicating that IR813 uptake by cells was essentially saturated after 2 hours of incubation.

[0085] The results showed that the red fluorescence signal reached its maximum value after 2 hours of incubation, with no significant difference from that after 4 hours of incubation, indicating that ICM can be effectively taken up by tumor cells after two hours of incubation.

[0086] This invention uses confocal microscopy and flow cytometry to investigate the homologous targeting function of ICM on tumor cells and its immune escape effect on macrophages. ICM from different sources, including ICM derived from breast cancer cells (4T1), ICM derived from glioma cells (GL261), and ICM derived from erythrocytes (RBC), were collected and incubated with 4T1 breast cancer cells for 2 hours. After washing with PBS, they were observed under a confocal microscope. After digestion with trypsin, flow cytometry analysis was performed. The results showed that the red fluorescence of the ICM (4T1) group was the brightest, and the flow cytometry results were consistent. This indicates that homologous cell membranes are better taken up by tumor cells. IR813 and ICM were co-incubated with macrophages for 2 hours, washed with PBS, and observed under a confocal microscope. After digestion with trypsin, flow cytometry analysis was performed. The results showed that compared with free drug, the amount of ICM taken up by macrophages was significantly reduced, indicating that the cell membrane endows IR813 with immune escape function. Figure 2 ).

[0087] Experimental Example 3

[0088] This invention uses the CCK8 cell proliferation assay kit to detect the therapeutic effect of ICM combined with near-infrared laser irradiation in 4T1 tumor cells. Different concentrations of ICM (1 μg / ml, 2 μg / ml, 3 μg / ml, 4 μg / ml, 5 μg / ml) were incubated with 4T1 tumor cells for 2 h. After washing with PBS, the cells were irradiated with a near-infrared laser at a power of 1 W / cm². 2The incubation time was 5 min. After another 3 h of incubation, CCK8 reagent was added, and the results were analyzed using an ELISA reader. The results showed that the cell death rate of 4T1 breast cancer cells gradually increased with the increase of ICM concentration. When the concentration was 5 μg / ml, the cell death rate was greater than 95%, indicating that ICM combined with near-infrared laser irradiation can effectively kill tumor cells and has a significant anti-tumor effect.

[0089] This invention uses the CCK8 cell proliferation assay kit to detect the therapeutic effect of ICM (derived from treated tumor cells) combined with near-infrared laser in various tumor cell types. Different ICMs (5 μg / ml) were incubated with GL261, EMT-6, 4T1, MCF-7, A549, MC38, and HCT116 tumor cells for 2 h. After washing with PBS, the cells were irradiated with a near-infrared laser at a power of 1 W / cm². 2 The incubation time was 5 minutes. After another 3 hours of incubation, CCK8 detection reagent was added, and the results were analyzed using an ELISA reader. The results showed that at a concentration of 5 μg / ml, the mortality rate of tumor cells in each group was greater than 95%, indicating that the present invention has a good therapeutic effect on various tumor cells. Figure 3 ).

[0090] Experiment Example 4

[0091] This invention uses laser confocal microscopy to observe the distribution of ICM on tumor cell membranes. 5 μg / ml of ICM was incubated with 4T1 tumor cells for 2 h. After washing with PBS, the cell membrane was labeled with DIO, and the cell nuclei were labeled with DAPI. The results were observed using laser confocal fluorescence microscopy. The results showed that ICM and DIO overlapped well and could be distributed on the tumor cell membrane, indicating that this invention has a certain degree of cell membrane targeting.

[0092] This invention uses SYTOX to detect the damaging effect of ICM combined with near-infrared laser on the cell membrane of 4T1 tumor cells. 4T1 tumor cells were incubated with 5 μg / ml ICM for 2 h, washed with PBS, and then irradiated with a near-infrared laser at a power of 1 W / cm². 2 The exposure time was 5 minutes. Then, SYTOX (2.5 μM) was added, and green fluorescence images were acquired over the next hour using a laser confocal microscope (images taken every 5 minutes), or changes in green fluorescence were read over the next hour using a microplate reader (read every 5 minutes). The results showed that green fluorescence signals appeared 20 minutes after exposure, indicating cell membrane damage. Extensive green fluorescence enhancement occurred within 0.5 hours, and almost all cells showed green fluorescence signals within 1 hour, indicating that tumor cell membranes could be effectively damaged within 1 hour of exposure. This demonstrates that the present invention can effectively damage tumor cell membranes.

[0093] in, Figure 4D is a quantitative detection of SYTOX cellular uptake and also a detection of cell membrane damage; Figure 4 E and F in the diagram show the morphological imaging and height profile analysis results of atomic force microscopy (AFM), which are used to compare the nanoscale structural changes on the surface of cell membranes or biomembranes under two treatment conditions. After ICM + NIR treatment, significant local structural damage or reconstruction occurred on the membrane surface, resulting in the formation of local "pits" or "holes" on the membrane surface, suggesting possible photothermal induced membrane damage.

[0094] Experimental Example 5

[0095] This invention uses immunofluorescence staining and an ATP assay kit to detect the release of immunogenic substances from 4T1 tumor cells induced by ICM combined with near-infrared laser. 4T1 tumor cells were incubated with 5 μg / ml ICM for 2 h, washed with PBS, and then irradiated with a near-infrared laser at a power of 1 W / cm². 2 The incubation time was 5 min. After incubation for another 3 h, the cell supernatant was collected, centrifuged at 300 g for 5 min, and 20 μl of supernatant was taken. 80 μl of ATP detection working solution was added, and chemiluminescence was detected using a microplate reader. For HMGB1 detection, cells were fixed with 4% paraformaldehyde, blocked with blocking buffer, permeabilized with 0.1% Triton-X 100 for 10 min, anti-HMGB1 was added, and the cells were incubated overnight at 4℃. After washing three times with PBS, FITC-conjugate anti-Rabbit was added, and the cells were incubated at room temperature for 2 h. After washing three times with PBS, the cell nuclei were labeled with DAPI, and images were acquired using CLSM. For Calreticulin detection, cells were fixed with 4% paraformaldehyde, blocked with blocking buffer, anti-Calreticulin was added, and the cells were incubated overnight at 4℃. After washing three times with PBS, FITC-conjugate anti-Rabbit was added, and the cells were incubated at room temperature for 2 h. After washing three times with PBS, the cell nuclei were labeled with DAPI, and images were acquired using CLSM. The results showed that after ICM combined with near-infrared laser treatment, cells released a large amount of ATP, HMGB1 in the cells was also released from the cell nucleus, and Calreticulin on the endoplasmic reticulum was also exposed, indicating that the cell death induced by the present invention is a strong immunogenic death.

[0096] This invention relates to Western blot detection of the release of immunogenic substances from 4T1 tumor cells induced by ICM combined with near-infrared laser. 4T1 tumor cells were incubated with 5 μg / ml ICM for 2 h, washed with PBS, and then irradiated with a near-infrared laser at a power of 1 W / cm². 2The incubation time was 5 min. After incubation for another 3 h, the cell supernatant was collected, centrifuged at 300 g for 5 min, and the supernatant was used for HMGB1 detection. Cells were collected separately. For HMGB1 detection, cells were lysed with RIPA lysis buffer, centrifuged at 12000 rpm for 5 min at 4℃, the supernatant was collected, and after BCA protein quantification, diluted to an appropriate concentration with protein loading buffer, heated at 98℃ for 5 min, electrophoresed, transferred to a membrane, washed 3 times with TBST, blocked with rapid blocking buffer for 10 min, washed 3 times with TBST, added anti-HMGB1 antibody and incubated at room temperature for 1 h, washed 3 times with TBST, added HRP-conjugate anti-Rabbit antibody and incubated at room temperature for 1 h, washed 3 times with TBST, and then added ECL luminescence detection reagent. Protein band images were acquired on a Bio-Rad gel imaging system. For Calreticulin detection, cells were used to extract cell membranes, and the obtained cell membranes were then subjected to the above operations for Western blot experiments. Figure 5 ).

[0097] in: Figure 5 A: The amount of ATP released in the cell supernatant after different treatments. The release of ATP (a type of DAMPS) was significantly increased after ICM + NIR treatment. Figure 5 B and E: Different methods were used to detect the release of HMGB1 (a type of DAMPS). Western blotting detected HMGB1 in cell supernatant and cells, respectively. The results showed a decrease in HMGB1 in cells and an increase in HMGB1 in the supernatant, indicating that HMGB1 was released extracellularly. Immunofluorescence detection of the remaining HMGB1 in cells also showed a decrease, indicating effective release of HMGB1. Figure 5 C and D: Different methods were used to detect CRT exposure (a type of DAMPS). Western blotting was used to detect the CRT content in the cell membrane under different treatment conditions. After ICM + NIR treatment, the CRT content in the cell membrane increased significantly, and immunofluorescence showed the same experimental results, indicating effective CRT exposure. It can be concluded that ICM + NIR treatment can effectively release DAMPS, which is an immunogenic cell death process.

[0098] In summary, this invention introduces a nanovesicle (ICM) based on a tumor cell membrane loaded with a photothermal reagent. This technology utilizes the homologous targeting and membrane fusion mechanism of the tumor cell membrane (CM) to precisely deliver photothermal reagents (such as IR813, IR780, and IR775) to the surface of homologous tumor cell membranes, achieving highly efficient localization of the tumor cell membrane. Under near-infrared laser irradiation, the photothermal reagent locally releases energy, disrupting the integrity of the tumor cell membrane (SYTOX staining shows a significant increase in membrane permeability 10 minutes after irradiation), triggering immunogenic cell death (ICD), and releasing AT. Immunogenic substances such as P, HMGB1, and Calreticulin activate anti-tumor immune responses. Experiments show that this method has a killing rate of ≥95% against various tumor cells and extremely low toxicity to normal cells. In addition, by flexibly matching cell membrane sources from different cancer types (such as breast cancer, glioma, lung cancer, etc.), ICMs targeting different tumors can be prepared, supporting personalized treatment and combined immunotherapy. This technology provides an innovative, efficient, broad-spectrum, and low-toxicity solution for solid tumor treatment through the synergistic effect of targeted delivery and photothermal-induced cell membrane disruption, and has high potential for clinical application.

[0099] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A biomimetic nanovesicle for tumor immunogenic death with high efficiency of membrane perforation, characterized in that, Includes tumor cell membranes and a photothermal reagent; the photothermal reagent may be compound A. Formula 1 Wherein, R: methyl, ethyl, propyl or butyl; including p-toluenesulfonate, perchlorate, chloride, bromide or iodide; Or compound B Formula 2 Wherein, R: methyl, ethyl, propyl or butyl; including p-toluenesulfonate, perchlorate, chloride, bromide or iodide; The method for preparing the tumor cell membrane includes the following steps: a) digesting the tumor cells with trypsin and then washing them; b) resuspending the washed cells in a hypotonic lysis buffer and sonicating them; c) centrifuging to remove the cell nuclei, collecting the supernatant and centrifuging again to collect the cell membrane, and washing it; the hypotonic lysis buffer is a mixture of 18~22mM Tris-HCl; 8~12mM KCl; 1.8~2.2mM MgCl2 and 1× protease inhibitor.

2. The nanovesicles according to claim 1, characterized in that, The final concentration of the tumor cell membrane is 150-250 μg / ml; the final concentration of the photothermal reagent is 450-550 μg / ml; and the photothermal reagent is IR813, IR780, or IR775.

3. The nanovesicles according to claim 1, characterized in that, Step b) describes an ultrasonic treatment with a frequency of 20-40 kHz, a power of 100-200 W, and a treatment time of 3-5 min.

4. The nanovesicles according to claim 1, characterized in that, The tumor cells described in step a) are from breast cancer, glioma, lung cancer, or colorectal cancer.

5. The method for preparing nanovesicles according to any one of claims 1 to 4, characterized in that, Mix the tumor cell membrane and photothermal reagent, wash with water, and then squeeze out.

6. The preparation method according to claim 5, characterized in that, The extrusion method is as follows: extruding 8 to 12 times using a liposome extruder, wherein the pore size of the polycarbonate membrane in the liposome extruder is 700 to 900 nm.

7. The use of the nanovesicles according to any one of claims 1 to 4 in the preparation of antitumor drugs.

8. The application according to claim 7, characterized in that, The anti-tumor drugs mentioned are drugs for treating breast cancer, glioma, lung cancer, and colorectal cancer.

9. An antitumor drug, characterized in that, Includes the nanovesicles according to any one of claims 1 to 4; the nanovesicles are used in conjunction with near-infrared light.

10. The medicament according to claim 9, characterized in that, The near-infrared light has a wavelength of 808nm, a power density of 0.9~1.1W / cm², and an irradiation time of 4~6min.

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