T cell mimicking nanoparticles A-AuNPs@M and their application in photothermal-immune combined therapy of tumor

Abstract

This invention provides T-cell biomimetic nanoparticles A-AuNPs@M and their application in photothermal-immunotherapy for tumors. Prepared by coating A-AuNPs with a T-cell membrane, this invention provides their application in tumor treatment, belonging to the field of nanomaterials technology. The biomimetic nanoparticles provided by this invention possess excellent near-infrared II photothermal conversion properties; the localized high temperature generated by PTT induces ICDs to recruit CTLs to the tumor site; and they can activate the FasL protein-dependent tumor cell apoptosis pathway using T-cell membrane surface proteins. The PD-1 protein on their surface endows the biomimetic nanoparticles with the function of an immune checkpoint inhibitor, enhancing the ability to activate adaptive immune responses. This biomimetic nanoparticle-mediated photothermal-immunotherapy not only eliminates the primary tumor but also generates memory T cells, effectively preventing tumor recurrence and metastasis, providing a possibility for effective clinical cancer treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanomaterials technology, and in particular relates to a T-cell biomimetic nanoparticle A-AuNPs@M and its application in photothermal-immunotherapy for tumors. Background Technology

[0002] Tumor cells promote tumorigenesis through immune escape mechanisms that evade immune surveillance or prevent the activation of immune responses. Undoubtedly, fully activating the human immune system is crucial for combating cancer recurrence and metastasis. However, in the context of tumor-infiltrating lymphocyte depletion and a complex immunosuppressive environment with diverse immunosuppressive factors, single-modality immunotherapy often results in low response efficiency and severe cytokine release syndrome (CRS). To generate a durable adaptive immune response against cancer, multimodal treatment approaches based on immunotherapy combined with chemotherapy, photothermal therapy, photodynamic therapy, or radiotherapy have been designed.

[0003] To achieve combined therapy, researchers utilize nanomaterials as assembly platforms to integrate multiple elements and enhance anti-tumor effects. It has been reported that nanomaterial-mediated photothermal therapy (PTT) reduces the density of tumor tissue, increases blood perfusion, facilitates the accumulation of therapeutic drugs, and promotes the recruitment of endogenous immune cells at the tumor site, thus creating a positive feedback loop that transforms "cold" tumors into "hot" tumors. Furthermore, photothermal-induced tumor immunogenic cell death (ICD) can recruit CD8+ CTLs and activate the immune system. However, due to the immunosuppressive effect of tumor-infiltrating lymphocytes (TME), the immune response is limited and lacks durable immunity. Therefore, how to rapidly eliminate the primary tumor, increase the recruitment of tumor-infiltrating lymphocytes (TILs), and generate memory T cells to suppress distant untreated tumors, effectively preventing tumor recurrence, is currently a hot research topic. Summary of the Invention

[0004] This invention provides a T-cell biomimetic nanoparticle, A-AuNPs@M, and its application in photothermal-immunotherapy of tumors. This nanoparticle possesses both excellent photothermal conversion properties and the ability to activate adaptive immune responses. This biomimetic nanoparticle-mediated photothermal-immunotherapy not only eliminates the primary tumor but also increases the recruitment of tumor-infiltrating lymphocytes (TILs) and generates memory T cells to suppress distant untreated tumors, effectively preventing tumor recurrence and providing a possibility for effective clinical treatment of cancer.

[0005] To achieve the above objectives, the present invention provides a T-cell biomimetic nanoparticle A-AuNPs@M, which is prepared by coating the surface of aggregated gold nanoparticles A-AuNPs with a T-cell membrane.

[0006] Preferably, the A-AuNPs@M nanoparticles have an average hydrodynamic diameter of approximately 202.4 nm and a PDI of 0.159.

[0007] Preferably, the A-AuNPs@M is prepared by the following method:

[0008] Add 20-1000 μL of 20 mg / mL to A-AuNPs nanoparticles -1 The PAH was reacted at room temperature for 10-60 minutes.

[0009] Centrifuge at 5000-12000 rpm for 5-20 min and collect the product;

[0010] The prepared T cell membrane was thoroughly mixed with A-AuNPs nanoparticles and sonicated in an ice bath at a frequency of 10kHz-40kHz for 20-60 minutes.

[0011] Centrifuge at 0-4℃ and 12000-16000g for 15-50 minutes to remove uncoated T cell membranes and obtain T cell biomimetic nanoparticles A-AuNPs@M.

[0012] Preferably, the weight ratio of the added A-AuNPs nanoparticles to T cell membrane proteins is 1:1.5-1:3.

[0013] This invention also provides the application of T-cell biomimetic nanoparticles A-AuNPs@M as photothermal conversion materials in the preparation of drugs for treating cancer.

[0014] This invention also provides an application of T-cell biomimetic nanoparticles A-AuNPs@M based on photothermal-immunotherapy in improving cancer cell killing ability or prolonging survival rate.

[0015] As a preferred option, T-cell biomimetic nanoparticles A-AuNPs@M, at a concentration of 0-200 μg / mL, were used with an NIR-II laser at a speed of 1.0 W / cm². 2 A laser with high power can kill cancer cells if it is continuously irradiated for 10 minutes.

[0016] This invention also provides an anticancer material that can generate immune memory, prepared using the T-cell biomimetic nanoparticles A-AuNPs@M described in any of the above technical solutions, and prepared using them as the main active ingredient.

[0017] The present invention also provides a PD-L1 blocker, which is prepared by using the T-cell biomimetic nanoparticles A-AuNPs@M as described in any of the above technical solutions, and is prepared with A-AuNPs@M as the main active ingredient.

[0018] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0019] The T-cell biomimetic nanoparticles A-AuNPs@M provided by this invention possess both excellent photothermal conversion properties and the ability to activate adaptive immune responses. Specifically, with the adjuvant effect of photothermal therapy, they can achieve rapid and complete ablation of subcutaneous solid tumors; under the action of immunotherapy, they can activate the adaptive immune response in mice, generating effector memory T cells and preventing tumor recurrence and metastasis. Compared with T cells, the biomimetic nanoparticles are smaller in size and can more easily bind to PD-L1 on the surface of tumor cells via PD-1 on the T cell membrane, effectively reducing the inhibition of CTL immune activity by tumor cells through immune checkpoints, preventing CTL exhaustion, and enhancing the CTL immune response against tumor cells. In addition, similar to T cells, the biomimetic nanoparticles target tumors through proteins on the T cell membrane, enhancing accumulation at the tumor site and inducing FasL-dependent apoptosis of cancer cells. The results show that the photothermal-immunotherapy mediated by biomimetic nanoparticles not only eliminates the primary tumor, but also increases the recruitment of tumor-infiltrating lymphocytes (TILs) and generates memory T cells to suppress distant untreated tumors. It can stimulate strong anti-tumor immunity and long-term immune memory, effectively prevent tumor recurrence, and provide a possibility for effective clinical treatment of cancer. Attached Figure Description

[0020] Figure 1 The diagram illustrates the preparation process of T cell membrane biomimetic nanoparticles A-AuNPs@M provided in this embodiment of the invention and their application in tumor NIR-II photothermal-immunotherapy.

[0021] Figure 2 For the characterization analysis of A-AuNPs provided in the embodiments of the present invention, (a) TEM image of the prepared ultra-small AuNCs, scale bar: 2nm; (b) TEM image of the prepared A-AuNPs nanoparticles; (c) TEM image of the prepared A-AuNPs nanoparticles.

[0022] Figure 3 Characterization analysis of A-AuNPs@M provided in the embodiments of the present invention: (a) TEM image of A-AuNPs@M nanoparticles; (b) dynamic light scattering of A-AuNPs nanoparticles and A-AuNPs@M nanoparticles.

[0023] Figure 4The photothermal performance analysis of A-AuNPs@M provided in the embodiments of the present invention includes: (a) UV-Vis-NIR absorption spectra of A-AuNPs@M nanoparticles at different concentrations; (b) temperature curves of 200 μg / mL A-AuNPs@M nanoparticles at different laser power densities; and (c) laser power density of 1.0 W / cm² for A-AuNPs@M nanoparticles at different concentrations. 2 The corresponding temperature curves; (d) A-AuNPs@M nanoparticles at different concentrations (0-200 μg / mL) with a laser power density of 1.0 W / cm². 2 Thermal infrared images lasting 10 minutes;

[0024] Figure 5 Photothermal performance analysis of A-AuNPs@M provided in embodiments of the present invention, (a) at different powers (0.5, 0.8 and 1.0 W / cm²). 2 (a) The photothermal properties of 200 μg / mL A-AuNPs@M nanoparticles were investigated under the action of 1064 nm laser light on chicken breast muscles of different thicknesses (2, 5, and 10 mm); (b) The photothermal properties of 200 μg / mL A-AuNPs@M nanoparticles were investigated under the action of 1064 nm laser light on chicken breast muscles of different thicknesses (2, 5, and 10 mm); 2 Under different pectoral muscle thicknesses (2, 5, and 10 mm), the penetration capabilities of 1064 nm and 808 nm lasers were compared at different laser power densities.

[0025] Figure 6 Photothermal performance analysis of A-AuNPs@M provided in the embodiments of the present invention, (a) using a power density of 1.0 W / cm² 2 (a) Time-temperature graph of 200 μg / mL A-AuNPs@M solution after irradiation with NIRII laser for 600 s and then stopping the laser irradiation; (b) Curve of cooling time versus the negative natural logarithm of temperature driving force obtained from the cooling stage; (c) Photothermal stability of A-AuNPs@M nanoparticles after four laser on / off cycles.

[0026] Figure 7 For the analysis of the tumor cell targeting ability provided in the embodiments of the present invention, (a) SDS-PAGE protein analysis of T cell lysates, T cell membranes and A-AuNPs@M by Coomassie blue staining; (b) flow cytometry analysis of the expression of FasL and PD-1 on T cells, A-AuNPs@M treated with trypsin and A-AuNPs@M.

[0027] Figure 8Representative confocal fluorescence microscopy images of 4T1 tumor cells treated with DiD-labeled nanoparticles, such as tr-A-AuNPs@M, Pb-A-AuNPs@M, and A-AuNPs@M, provided for embodiments of the present invention. This indicates that AuNPs@M interacts with tumor cells through PD-1 / PD-L1 binding; cell nuclei are labeled with DAPI (blue). Scale bar: 25 μm.

[0028] Figure 9 Western blot analysis of caspase-8 expression proteins after treatment of 4T1 cells with PBS, tr-A-AuNPs@M, Fb-A-AuNPs@M or A-AuNPs@M, as provided in the embodiments of the present invention;

[0029] Figure 10 The relative survival rates of LO2 cells treated with different concentrations of A-AuNPs@M nanoparticles provided in this embodiment of the invention;

[0030] Figure 11 For the analysis of the combined treatment results at the cellular level provided in the embodiments of the present invention, (a) the relative survival rate of 4T1 cells treated with different nanoparticles. Wherein 1 represents PBS, 2 represents tr-A-AuNPs, 3 represents Fb-A-AuNPs, 4 represents A-AuNPs@M, 5 represents A-AuNPs+laser, and 6 represents A-AuNPs@M+laser; (b) flow cytometry analysis of apoptosis in 4T1 cells; (c) CLSM images of representative 4T1 tumor cells treated with Calcein (green) / PI (red) and PBS, tr-A-AuNPs, Fb-A-AuNPs, or A-AuNPs@M under infrared light irradiation or without irradiation; scale bar: 100 μm;

[0031] Figure 12 For the biological distribution at the mouse level provided in this embodiment of the invention, (a) in vivo infrared fluorescence imaging of 4T1 tumor-bearing mice injected via tail vein with tr-A-AuNPs, Fb-A-AuNPs, or A-AuNPs@M nanoparticles at 0h, 2h, 4h, 8h, 12h, 24h, and 48h; (b) images of the heart, liver, spleen, lung, kidney, and tumor of 4T1 tumor-bearing mice injected via tail vein with tr-A-AuNPs, Fb-A-AuNPs, or A-AuNPs@M nanoparticles 24 hours later; and (c) near-infrared fluorescence imaging of in vivo metabolism of 4T1 tumor-bearing mice injected via tail vein with A-AuNPs@M nanoparticles.

[0032] Figure 13For the in vivo photothermal efficiency at the mouse level provided in this embodiment of the invention, (a) infrared images of different groups of tumor-bearing mice; (b) temperature changes at the tumor site within 5 minutes of laser irradiation;

[0033] Figure 14 For the analysis of the combined treatment results of the mouse model provided in the embodiments of the present invention, (a) digital photos of mice on day 0 and day 14 after treatment; (b) digital photos of mouse tumors after day 14 after treatment; (c) tumor weight; (d) tumor growth curves of mice under different treatments; (e) average weight change of mice during treatment; (f) survival curves of 4T1 tumor-bearing mice after different treatments (n=5).

[0034] Figure 15 Fourteen days after treatment as provided in this embodiment of the invention, H&E staining was performed on the major organs (heart, liver, spleen, lung, and kidney) of 4T1 tumor-bearing mice; scale bar: 100 μm;

[0035] Figure 16 For the analysis of tumor recurrence and metastasis results after combined treatment provided in the embodiments of the present invention, (a) tumor growth curves of mice in all groups after treatment; (b) tumor growth curves of mice in the PBS group after treatment; (c) tumor growth curves of mice in the tr-A-AuNPs@M group after treatment; (d) tumor growth curves of mice in the A-AuNPs@M group after treatment.

[0036] Figure 17 Analysis of tumor recurrence and metastasis results after combined treatment provided in the embodiments of the present invention: (a) Tumor growth curves of mice in the A-AuNPs+laser and A-AuNPs@M+laser groups; (b) Tumor growth curves of mice in the A-AuNPs+laser group; (c) Tumor growth curves of mice in the A-AuNPs@M+laser group.

[0037] Figure 18 For the analysis of the results of the combined treatment provided in the embodiments of the present invention, the following were performed: (a) flow cytometry analysis of the killing ability of effector memory T cells; (b) CCK-8 analysis of the killing ability of effector memory T cells (1 represents CTL extracted from normal mice, 2 represents CTL extracted from the spleen of PBS tumor-bearing mice, and 3 represents CTL extracted from the spleen of mice in the A-AuNPs@M+laser group); (c) flow cytometry analysis of effector memory T cells. Detailed Implementation

[0038] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0039] Example 1: Preparation of A-AuNPs@M

[0040] 1.1 Culture EL4 cells and extract cell membranes

[0041] The EL4 cell line stably expresses a variety of plasma membrane proteins similar to those of T cells, therefore this study used T cell membranes derived from the EL4 cell line.

[0042] EL4 cells were passaged in basal DMEM medium containing 10% bovine serum (FBS) and 1% antibiotic (PS, penicillin-streptomycin). After centrifugation and washing, the cell pellet was collected. Following the instructions of Beyotime's membrane protein extraction kit, the cells in the above solution were repeatedly disrupted using a freeze-thaw cycle. The mixture was then centrifuged at 4°C and 700g for 10 min. Finally, the supernatant was centrifuged at 4°C and 15000g for 30 min to precipitate cell membrane fragments. The collected membrane fragments were stored at -80°C for subsequent experiments.

[0043] 1.2 Preparation of A-AuNPs

[0044] According to previous reports, at room temperature, 5 mL of 20 mM HAuCl4 and 1.5 mL of 100 mM GSH were mixed separately, and 43.5 mL of DMSO was added. The mixture was heated to 70 °C in an oil bath with gentle stirring at 500 rpm for 24 h to obtain ultra-small gold nanoclusters (AuNCs). 960 μL of 20 mM reduced sodium ascorbate was added dropwise to 1 mL of the AuNCs solution. After standing at room temperature for 30 min, a flocculent gray-black precipitate was obtained. This precipitate was washed and centrifuged to remove residual DMSO, yielding aggregated gold nanoparticles (A-AuNPs).

[0045] 1.3 Preparation of A-AuNPs@M

[0046] Add an appropriate amount of PAH (20 mg·mL⁻¹) to A-AuNPs nanoparticles -1The mixture was reacted at room temperature for 30 minutes. Then, it was centrifuged at 8000 rpm for 5 minutes, and the product was collected. The prepared T-cell membrane was thoroughly mixed with A-AuNPs nanoparticles at a weight ratio of 1:2 (A-AuNPs nanoparticles to membrane protein), and sonicated on ice for 30 minutes at low frequency and low sonication power. Then, it was centrifuged at 14000g for 20 minutes at 4°C to remove the uncoated T-cell membrane.

[0047] Characterization analysis of A-AuNPs@M in Example 2

[0048] Ultrasmall gold nanoclusters (AuNCs) were synthesized using an organic system based on DMSO solution and glutathione and HAuCl4. Transmission electron microscopy (TEM) imaging showed that the average particle size of the AuNCs was approximately 2 nm. Figure 2 (As shown in a). The reducing properties of sodium ascorbate further induce the aggregation of small-sized AuNCs, forming large-sized aggregated A-AuNPs. TEM clearly shows that the nanoparticles have a particle size of approximately 200 nm and are spherical nanoparticles with uneven surfaces. Figure 2 (as shown in b); SEM images show that A-AuNPs have a spherical morphology that is uniform in size and well-dispersed. Figure 2 (as shown in c).

[0049] To enhance tumor targeting and biocompatibility, A-AuNPs were encapsulated on the cell membranes of EL4 cells to obtain gold-inspired biomimetic nanomaterials encapsulated in T cell membranes. This reduced the blood clearance (ABC) phenomenon and increased the in vivo circulation time of the nanocarrier, enabling targeted delivery to tumor tissues and active accumulation and retention. To ensure successful encapsulation of A-AuNPs nanoparticles on T cell membranes, we validated the encapsulation using TEM imaging and dynamic light scattering (DLS). The TEM images clearly showed... Figure 3 As shown in (a), a clear and thin film coats the nanocore; furthermore, the average hydrodynamic diameter of the A-AuNPs nanoparticles, measured using a Zetasizer Nano ZS90, is approximately 185.5 nm (PDI = 0.111), while the average hydrodynamic diameter of the A-AuNPs@M nanoparticles is approximately 202.4 nm (PDI = 0.159). Figure 3 As shown in b), when coated with a cell membrane, the average hydrodynamic diameter of A-AuNPs@M nanoparticles is slightly larger than that of A-AuNPs nanoparticles without T cell membrane, indicating that the cell membrane can successfully coat the nanoparticles.

[0050] Example 3: Photothermal Performance Analysis of A-AuNPs@M

[0051] A-AuNPs@M nanoparticles of different concentrations exhibit broad absorption in the NIR-II region (1000-1350 nm). Figure 4 As shown in a), it exhibits great photothermal conversion potential under NIR-II laser irradiation. To investigate the suitable laser power density, a laser power density of 0.5 W / cm² was used. 2 1.0W / cm 2 and 1.5W / cm 2 After irradiating A-AuNPs@M nanoparticles of the same concentration with a NIR-II laser (1064 nm) for 10 minutes, the temperature of the A-AuNPs@M nanoparticles increased by 22.9℃, 42.3℃, and 50.4℃, respectively. Figure 4 (As shown in b, 4c, and 4d), this is sufficient to kill cancer cells through photothermal therapy. However, considering that the local temperature generated by the laser exceeds 50°C, which can destroy the tumor but may also burn surrounding normal tissue, the most suitable laser power density is selected as 1.0 W / cm². 2 Therefore, at a laser power density of 1.0 W / cm², 2 Under the given conditions, A-AuNPs@M nanoparticles of different concentrations were irradiated with NIR-II laser for 10 minutes. The results showed that the temperature increase was closely related to the irradiation time and the concentration of A-AuNPs@M nanoparticles.

[0052] Example 4: Tissue Penetration of A-AuNPs@M

[0053] Since tumors have a certain depth and thickness of invasion, in order to explore whether A-AuNPs@M can induce high temperature at an ideal tissue depth, aqueous solutions of A-AuNPs@M nanoparticles were used to coat chicken breast meat with thicknesses of 2, 5, and 10 mm, respectively, and then heated with 0.5 W / cm² water. 2 0.8W / cm 2 and 1.0W / cm 2 Irradiation with NIR-II laser for 10 minutes at different power densities. Figure 5 As shown in figure a, A-AuNPs@M nanoparticles can convert light energy into heat, raising the solution temperature to 4-32℃, even with a power density below 1.0 W / cm³. 2 It can also achieve satisfactory photothermal performance. Furthermore, it uses a power density of 1.0 W / cm². 2 Chicken breasts containing the same concentration of A-AuNPs@M nanoparticles were irradiated with NIR-I and NIR-II near-infrared lasers. The results showed that, with increasing chicken breast thickness, the temperature change under NIR-II laser irradiation was greater than that under NIR-I irradiation. Figure 5 (As shown in b), this is because the NIR-II laser has a deeper penetration ability into tissues.

[0054] according to Figure 6 The photothermal conversion efficiency of A-AuNPs@M nanoparticles under NIR-II laser irradiation was calculated to be 51.1% (a-6c). Furthermore, after four consecutive laser irradiations with a power density of 1.0 W / cm², the efficiency was further improved. 2 The A-AuNPs@M nanoparticles remained stable after irradiation with a NIR-II laser switch, with no significant temperature decay observed in each cycle and ΔT remaining essentially constant. This phenomenon indicates that A-AuNPs@M nanoparticles are durable and stable photothermal conversion materials. Therefore, A-AuNPs@M nanoparticles irradiated with NIR-II lasers exhibit high tissue penetration depth and high maximum permissible optical power, along with excellent photothermal stability and other outstanding photothermal conversion properties, suggesting their potential as highly reliable photothermal conversion materials for cancer treatment.

[0055] Example 5: Ability of A-AuNPs@M to target tumor cells

[0056] 5.1 Characterization of A-AuNPs@M membrane proteins

[0057] Membrane protein profiles were studied using sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The concentrations of the tested substances—prepared T-cell lysates, extracted T-cell membrane proteins, and A-AuNPs@M protein—were determined using the BCA protein assay. The lysate was mixed with NuPAGE·LDS sample buffer, and then 15 μg of protein was added to a 10% (w / v) SDS polyacrylamide gel. After electrophoresis, the gel was immediately stained with Coomassie blue and then washed to observe the protein bands.

[0058] The preservation of membrane proteins on A-AuNPs@M nanoparticles was verified using Coomassie blue staining and flow cytometry. Therefore, T cell lysates, T cell membranes, and A-AuNPs@M nanoparticles were stained with Coomassie blue and analyzed by SDS-PAGE for protein content. Figure 7 As shown in a, proteins on the T cell membrane are highly preserved on A-AuNPs@M.

[0059] To further verify the high preservation of FasL and PD-1 proteins on the surface of A-AuNPs@M nanoparticles, flow cytometry analysis was performed using fluorescein-conjugated antibodies against FasL and PD-1. 200 μg of A-AuNPs@M was adsorbed onto 500 μL of microspheres at room temperature for 15 min. The A-AuNPs@M beads were then stained with fluorescein-conjugated APC anti-mouse CD279 (PD-1) or PE anti-mouse CD178.1 (FasL), respectively. The mixture was then washed twice. After washing, the stained mixture was analyzed by flow cytometry.

[0060] As a result, flow cytometry analysis showed high levels of FasL and PD-1 proteins on A-AuNPs@M nanoparticles, while the levels of both were very low on trypsin-treated T cell membrane-encapsulated nanoparticles (tr-A-AuNPs@M). Figure 7 (as shown in b). In summary, A-AuNPs@M containing T cell membrane proteins has the potential to serve as biomimetic nanoparticles for T cells.

[0061] 5.2A-AuNPs@M's ability to target tumor cells

[0062] Programmed death ligand-1 (PD-L1) is highly expressed on the surface of tumor cells and can interact with programmed death receptor (PD-1) expressed on the surface of immune T cells. Therefore, we investigated whether AuNPs@M nanoparticles and tumor cells can interact through PD-1 / PD-L1 binding.

[0063] Specifically, PD-1 on A-AuNPs@M was blocked with an anti-PD-1 antibody to obtain antiPD-1Ab-treated A-AuNPs@M (Pb-A-AuNPs@M). A-AuNPs@M were then treated with trypsin to obtain tr-A-AuNPs@M. Approximately 2 × 10⁻⁶ 5 4T1 cells were in a solution containing 20 ng / mL of recombinant mouse protein. -1 Cells were cultured in IFN-γ medium for 24 hours to upregulate PD-L1 expression on cancer cells. After washing three times with PBS, the cells were fixed in 10% neutral buffered formalin for 10 minutes and blocked with 1% BSA for 1 hour. Cells were then stained with 200 μg / mL of DiD (a lipophilic carbonyl cyanine dye with a long lipophilic hydrocarbon chain that diffuses laterally across the cell membrane to stain the entire cell membrane, emitting stable far-infrared fluorescence). -1tr-A-AuNPs@M, Pb-A-AuNPs@M, and A-AuNPs@M were incubated in PBS at 4°C for 60 s. After incubation, the cells were washed three times with PBS, and the nuclei were sealed with a mounting solution containing DAPI. The fluorescence distribution of DAPI (excitation / emission = 364 nm / 454 nm) and DID (excitation / emission = 644 nm / 665 nm) cells was detected using a confocal laser scanning microscope (C2, Nikon, Japan).

[0064] like Figure 8 Confocal imaging data showed that, compared to the group treated with other nanoparticles (tr-A-AuNPs@M and Pd-1 blocked Pd-A-AuNPs@M), the A-AuNPs@M treatment group exhibited significant red fluorescence in the DID-labeled nanoparticles, indicating the binding of A-AuNPs@M to cancer cells. In the group of nanoparticles digesting proteins on the membrane with trypsin, almost no fluorescence was observed indicating the binding of nanoparticles to cancer cells through protein-protein interactions. When PD-1 protein on the membrane was blocked with an anti-PD-1 antibody, the resulting Pd-A-AuNPs@M hardly bound to cancer cells, suggesting that the interaction between A-AuNPs@M and cancer cells mainly occurs through PD-1 / PD-L1 binding. In summary, these results indicate that A-AuNPs@M coating T cell membranes possesses the ability to target tumors through protein adhesion, and that A-AuNPs@M can serve as both an anti-cancer immunomodulator and a PD-L1 blocker.

[0065] 5.3A-AuNPs@M FasL-Fas Mechanism

[0066] 4T1 cells were loaded at approximately 1 × 10⁴ cells per well. 5 Cells were cultured in 6-well plates for 12 hours, and the culture medium contained 20 ng / mL of recombinant mouse protein that can upregulate Fas expression in cancer cells. -1IFN-γ was used to mimic the in vivo TME. 4T1 cells were then washed three times and incubated at 37°C for 48 h with a blank control, tr-A-AuNPs@M, Fb-A-AuNPs@M, or A-AuNPs@M, respectively. Cells, including the supernatant, were collected, centrifuged, washed, and lysed with cell lysis buffer. The cells were then centrifuged at 10000g for 10 min at 4°C, and the supernatant was transferred to clean centrifuge tubes to obtain protein samples. Protein concentration was determined using the BCA protein assay. 15 μg of protein was then added to a 10% (w / v) SDS-polyacrylamide gel for electrophoresis. After electrophoresis, the gel was transferred to a membrane. The membrane was then blocked with primary antibody (Caspase 8 antibody) and secondary antibody. Finally, ECL imaging agent was prepared. The PVDF membrane, drained of water, was removed with tweezers, the ECL luminescent agent was poured on, and the reaction was allowed to proceed for 2 min. Imaging was then performed using a chemiluminescence imaging system.

[0067] Depend on Figure 9 The experimental results showed that, compared with other treatment groups, the A-AuNPs@M group increased the activation of caspase-8, a key downstream component of the Fas / FasL signaling pathway, and significantly inhibited cancer cell proliferation.

[0068] Example 6: Cytotoxicity of A-AuNPs@M

[0069] The in vitro toxicity of different concentrations of A-AuNPs@M solution to normal LO2 cells was detected using a cell counting kit (CCK-8). LO2 cells were counted at approximately 2 × 10⁶ cells per well. 4 Cells were cultured in 24-well plates for 12 hours, with the culture medium containing 20 ng / mL of recombinant mouse cells. -1 The IFN-γ was used to mimic the in vivo TME. LO2 cells were then washed three times and mixed with a blank control group, and at concentrations of 25, 50, 100, and 200 μg / mL, respectively. -1 The A-AuNPs@M solution was incubated in a cell culture incubator for 48 h. Potential cytotoxicity was then assessed using a CCK-8 assay. Absorbance was measured at 450 nm using a microplate reader, and the relative number of viable cells was expressed as a percentage of the number of viable cells in the untreated group at 0 h.

[0070] like Figure 10 As shown, even when the concentration used to treat LO2 cells was increased to 200 μg / mL, cell viability was hardly affected, indicating that the synthesized A-AuNPs@M nanocomposite material has good biocompatibility.

[0071] Example 7: Combined therapy of A-AuNPs@M at the cellular level

[0072] Next, CCK-8 and flow cytometry analyses were performed to understand the synergistic anti-cancer effect of A-AuNPs@M nanoparticles on 4T1 cells in photothermal immunotherapy. Figure 11 As shown in Figure a, the cell survival rate of 4T1 cells treated with A-AuNPs@M after NIR-II laser irradiation was significantly lower than that of the untreated control group or the control group treated with other nanoparticles (including tr-A-AuNPs@M and Fb-A-AuNPs@M). This indicates that the FasL protein retained on the T cell membrane of the nanoparticles, as confirmed by Western blotting experiments, induces cancer cell apoptosis through the Fas / FasL signaling pathway. When 4T1 cells incubated with A-AuNPs or A-AuNPs@M were irradiated with a 1064nm laser, the cell survival rate dropped sharply to 30% and approximately 22%, respectively, indicating that the photothermal effect significantly promoted the cell-killing ability. Compared with laser-irradiated A-AuNPs, A-AuNPs@M had a stronger cell-killing ability, which is due to the immune effect of the coated T cell membrane, indicating that the strategy of combining photothermal and immunotherapy can improve cell-killing ability. The apoptosis rate was highest in the photothermal immunotherapy group (62.12% ± 0.74% = 62.86%), significantly higher than that in the photothermal therapy alone group (41.44% ± 1.05% = 45.49%) and the immunotherapy alone group (24.15% ± 0.90% = 25.05%). Figure 11 b). Therefore, the results of flow cytometry were consistent with cell viability, indicating that the combination therapy was superior to the single therapy. After staining with calcein-acetoxymethyl ester (Calcein-AM) and propidium iodide (PI) to show live and dead cells, confocal fluorescence imaging was used to further verify the killing ability of cells treated under different conditions. As shown in the figure, the combination therapy group showed the strongest red fluorescence ( Figure 11 c) indicates that almost all cells are still in the late apoptotic stage, which also confirms the conclusions drawn from CCK-8 and flow cytometry experiments.

[0073] Example 8: Biodistribution and in vivo photothermal efficiency of A-AuNPs@M in mice.

[0074] Inspired by the in vitro cytotoxic activity of A-AuNPs@M nanoparticles, we investigated their in vivo therapeutic efficiency. First, various DiD-labeled nanoparticles (DiD-tr-A-AuNPs@M, DiD-Pb-A-AuNPs@M, and DiD-A-AuNPs@M) were injected intravenously into Balb / c mice carrying subcutaneous 4T1 tumors, and the biodistribution of the A-AuNPs@M nanoparticles was investigated. We used an in vivo imaging system (IVIS) to track the fluorescence of DiD to study the distribution of the labeled nanoparticles. In our design, the T cell membranes used to encapsulate the nanoparticles were derived from the EL4 cell line because the EL4 cell line expresses multiple plasma membrane proteins. The T cell membranes on the nanoparticles can avoid nanoparticle clearance mediated by the mononuclear phagocyte system, thereby improving the circulation time and tumor targeting of the nanoparticles. In particular, the PD-1 protein on the nanoparticle coating membrane plays a dual role, including binding to cancer cells through adhesion and blocking the PD-L1 immunosuppressive checkpoint. Similarly, the FasL protein also enhanced the binding of nanoparticles to cancer cells. As expected, fluorescence appeared earliest and remained strongest in mouse tumors treated with DiD-A-AuNPs@M within 48 hours post-injection, indicating that most DiD-A-AuNPs@M accumulated at the tumor site compared to DiD-Pb-A-AuNPs@M and DiD-tr-A-AuNPs@M. This is highly consistent with observations made by CLSM in experiments exploring the interaction between nanoparticles and cancer cells. Figure 12 a). 48 hours after injection, major organs and tumor tissue were removed for ex vivo imaging. Figure 12 (b) It was readily observed that in the two groups of mice injected with DiD-Pb-A-AuNPs@M and DiD-tr-A-AuNPs@M, respectively, most nanoparticles accumulated in the reticuloendothelial system, including the liver and spleen, while in the group treated with DiD-A-AuNPs@M, most nanoparticles accumulated in the tumors. Furthermore, we investigated the ability to remove the injected nanoparticles via in vivo imaging. Figure 12 As shown in Figure c, the fluorescence signal of DiD-labeled A-AuNPs@M at the tumor site reached its maximum on the second day and gradually decreased to normal levels after 5 days, indicating that the designed nanoparticles can be excreted from the body. These results demonstrate that the side effects of our nanoparticles are negligible at the administered dose. Therefore, modifying nanoparticles with T-cell membranes can significantly enhance their tumor-targeting ability and provide an important prerequisite for synergistic therapy using this nanoplatform.

[0075] When the nanoparticles accumulated 24 hours after injection, we locally irradiated the tumor site with a 1064nm laser (1W / cm2) for 5 minutes, and simultaneously recorded temperature changes using an infrared thermometer. Compared with the PBS group, the temperature of the tumor site in mice receiving AuNPs@M and A-AuNPs plus NIRII laser irradiation increased by 15℃ and 22℃, respectively. Figure 13 (a) and (b) demonstrate that the designed nanoparticles possess excellent photothermal generation capabilities. Compared to the single photothermal therapy group (mice treated with A-AuNPs), the combined therapy group (mice treated with AuNPs@M) showed a higher temperature increase at the tumor site, indicating that AuNPs@M promotes in vivo drug delivery to the tumor and achieves greater accumulation and longer retention time. These nanoparticles can generate high temperatures under NIRII laser irradiation to meet the requirements of tumor thermal ablation, and AuNPs@M exhibits good biocompatibility.

[0076] Due to the continuous accumulation of tumors and the favorable NIR-II photothermal effect, we hypothesized that the A-AuNPs@M nanoparticles might produce a significant synergistic NIR-II photothermal immunotherapy effect, and further evaluated its anti-tumor activity in vivo. An orthotopic tumor model was established in female BALB / c mice via lateral subcutaneous injection of 4T1 cells. After tumor formation, tumor-bearing mice were randomly divided into 5 groups (n=5 per group): PBS group, tr-A-AuNPs@M group, A-AuNPs@M group, A-AuNPs+laser group, and A-AuNPs@M+laser group. Tumor-bearing mice were first injected via tail vein with PBS, tr-A-AuNPs@M, A-AuNPs, and A-AuNPs@M, once every 2 days. Among them, the A-AuNPs+laser group and the A-AuNPs@M+laser group were treated with NIRII laser with a power density of 1.0W / cm2 for 5 minutes after injection 24 hours later.

[0077] Tumor volume was measured every two days in mice receiving different treatments, and photographs of the mice were recorded at the start and end of treatment. Figure 14 a). After treatment, mouse tumors were collected, photographed, and weighed. Figure 14 b, c). For example Figure 14As shown in Figure d, mice in the A-AuNPs+laser group or the AuNPs@M+laser group completely eliminated tumors, exhibiting the best tumor growth inhibition, indicating that PTT alone and combination therapy can effectively eliminate tumors. Tumor growth inhibition in the AuNPs@M group was also better than in the PBS group and the tr-AuNPs@M group. However, during treatment, the tumors in mice treated with tr-AuNPs@M monotonically increased, similar to the blank control group. In the single immunotherapy group, mice treated with AuNPs@M without laser irradiation showed effective tumor growth inhibition in 40% of the mice, even eliminating the tumors, while 60% of the mice showed gradual tumor growth at a slower rate. Mice receiving single PTT (A-AuNPs+laser group) showed significant differences from those receiving combination therapy (AuNPs@M+laser group). In the PTT alone group, tumor volume initially increased and then decreased. In the combination therapy group, tumor volume monotonically decreased, and the tumors were eliminated earlier. Therefore, in the comprehensive comparison, synergistic photothermal-immunotherapy showed the best anti-tumor effect. We also analyzed the biocompatibility and biosafety of AuNPs@M in vivo. During treatment, there was no significant change in body weight in any of the mice. Figure 14 e). Furthermore, the photothermal-immunotherapy combination significantly prolonged their survival rate ( Figure 14 f).

[0078] In addition, such as Figure 15 H&E staining of the sections of the major organs (heart, liver, spleen, lung, and kidney) shown in the figures revealed no obvious signs of inflammation or cell death, indicating that the nanoparticles are non-toxic.

[0079] Example 9: Analysis of Tumor Recurrence and Metastasis After Combined Treatment

[0080] To verify whether nanoparticles can effectively inhibit tumor recurrence, mice carrying tumors underwent surgical resection after treatment. Approximately 15 days later, the mice were observed for recurrence after different treatments, and the relative tumor volume was measured during the observation period. The experimental results showed that the PBS group and the tr-A-AuNPs@M group were ineffective in preventing tumor recurrence, with 100% tumor recurrence in the mice. Figure 16 (b, c). While the single-immunotherapy group (A-AuNPs@M) can suppress tumor recurrence or metastasis by inducing host immunity, the individual variability of immunotherapy is significant due to the weak immunogenicity of the tumor itself and the immunosuppressive tumor microenvironment. Therefore, it cannot eradicate the tumor initially, resulting in a 50% recurrence rate. Figure 16d). The single photothermal group (A-AuNPs+laser) can efficiently kill in situ tumors through thermal ablation, and the dead tumor cells can effectively inhibit tumor recurrence through PTT-induced immunogenic cell death (ICD). The photothermal-immune group (A-AuNPs@M+laser) rapidly ablates in situ tumors through the heat generated by PTT while simultaneously activating the host immune system, achieving the highest anti-tumor effect and a strong immune effect. Figure 16 a) No tumors were detected in any of the mice.

[0081] To verify whether the treatment group could effectively inhibit tumor metastasis, after 15 days of observation, mice without tumor recurrence, namely the photothermal group and the photothermal-immune group, were treated with homologous 4T1 cell suspension (approximately 1 × 10⁻⁶ cells per mouse). 7 (A-AuNPs + laser) cells were subcutaneously injected into mice on the side opposite the primary tumor. The volume of the re-inoculated tumor was measured every 3 days. The results show that while the single photothermal group (A-AuNPs + laser) can effectively kill tumors in situ and induce ICD, its effectiveness in preventing tumor metastasis is limited due to the difficulty in controlling the temperature during treatment. Figure 17 b). The photothermal-immune group (A-AuNPs@M+laser) showed a 100% metastasis-free result, indicating that it can enhance host immunity and induce long-term immune memory to prevent tumor metastasis. Figure 17 c). The above results indicate that ( Figure 17 a) Our synergistic strategy successfully activated the systemic immune response, effectively preventing tumor recurrence and inhibiting distant metastasis.

[0082] Example 10: Analysis of memory T cell production after combined treatment

[0083] The ability to form memories is crucial for the adaptive immune system to prevent reinfection. Memory T cells reactivate their remembered antiviral methods upon the next antigen invasion, destroying target cells again and releasing the antigens from them. To better understand relapse and distant metastasis, we further inoculated mice on day 45 after combination therapy with 4T1 cells to form distant tumors, and then extracted spleen cells (a type of T lymphocyte). Spleen cells from tumor-bearing mice (treated with PBS) served as isotype controls, while spleen cells from the control group were obtained from healthy mice. All extracted cells were digested into single-cell suspensions. Spleen cells from different groups of mice were mixed with 4T1 cancer cells in the exponential growth phase and incubated for 36 hours. Cell viability of the cancer cells was assessed using a CCK-8 assay. Figure 18 As shown in b, after incubation with extracted T cells from mice treated with the same therapy, the viability of cancer cells was significantly lower than that of cancer cells co-incubated with the same type of cells. Figure 18As shown in figure a, the percentage of apoptotic cancer cells analyzed by flow cytometry was consistent with the percentage of dead cancer cells detected by CCK-8 assay, which means that the combination therapy generated CD8+CTLs and enhanced the ability to kill cancer cells.

[0084] It is well known that immune memory can effectively inhibit tumor growth and generate a long-term immune response. To investigate whether organisms induce immune memory, mice 30 days after self-elimination of tumors in a tumor metastasis model were used to analyze central memory T (TCM) cells and effector memory T (TEM) cells in the spleen using flow cytometry. It is known that TCM cells can rapidly proliferate upon initiation of antigen stimulation, while TEM cells (CD3+, CD8+, CD62L, -CD44+) can provide a robust protective immune memory response by producing IFN-γ, IL-4 cytokines, and perforin. Notably, the percentage of TEM cells observed in mice receiving combination therapy was significantly higher than in mice undergoing surgery. Figure 18 c) This demonstrates that the synergistic strategy of photothermal immunotherapy induces strong immune memory, meaning that photothermal-immunotherapy combined therapy can produce significant synergistic therapeutic effects. A schematic diagram of the entire process is shown below. Figure 1 As shown.

Claims

1. T cell biomimetic nanoparticles A-AuNPs@M, characterized in that, It was prepared by coating the surface of aggregated gold nanoparticles A-AuNPs with T cell membranes; The A-AuNPs@M nanoparticles have an average hydrodynamic diameter of 202.4 nm and a PDI of 0.

159. The aggregated gold nanoparticles A-AuNPs were prepared by the following method: At room temperature, 5 mL of 20 mM HAuCl4 and 1.5 mL of 100 mM GSH were mixed separately, and 43.5 mL of DMSO was added. The mixture was heated to 70 °C in an oil bath with gentle stirring at 500 rpm and reacted for 24 h to obtain ultra-small gold nanoclusters AuNCs. 960 μL of 20 mM reduced sodium ascorbate was added dropwise to 1 mL of AuNCs solution. After standing at room temperature for 30 min, a flocculent gray-black precipitate was obtained. The precipitate was washed and centrifuged to remove residual DMSO, yielding aggregated gold nanoparticles.

2. The T-cell biomimetic nanoparticles A-AuNPs@M according to claim 1, characterized in that, The A-AuNPs@M were prepared by the following method: Add 20-1000 μL of 20 mg / mL to A-AuNPs nanoparticles -1 The PAH was reacted at room temperature for 10-60 minutes. Centrifuge at 5000-12000 rpm for 5-20 min and collect the product; The prepared T cell membrane was thoroughly mixed with A-AuNPs nanoparticles and sonicated in an ice bath at a frequency of 10 kHz-40 kHz for 20-60 min. Centrifuge at 0-4℃ and 12000-16000 g for 15-50 minutes to remove uncoated T cell membranes and obtain T cell biomimetic nanoparticles A-AuNPs@M.

3. The T-cell biomimetic nanoparticles A-AuNPs@M according to claim 2, characterized in that, The weight ratio of the added A-AuNPs nanoparticles to T cell membrane proteins is 1:0.5-1:

3.

4. The use of the T-cell biomimetic nanoparticles A-AuNPs@M as a photothermal conversion material in the preparation of drugs for treating cancer, according to any one of claims 1-3.

5. The use of the T-cell biomimetic nanoparticles A-AuNPs@M according to any one of claims 1-3 in the preparation of products that enhance the killing power of cancer cells based on photothermal-immunotherapy combination therapy.

6. The application according to claim 4 or 5, characterized in that, T-cell biomimetic nanoparticles A-AuNPs@M, at a concentration of 100-200 μg / mL, were used with a NIR-II laser at a frequency of 1.0 W / cm². 2 A laser with high power can kill cancer cells if it is continuously irradiated for 10 minutes.

7. An anticancer material capable of generating immune memory, characterized in that, The T-cell biomimetic nanoparticles A-AuNPs@M as described in any one of claims 1-3 were used as the main active ingredient.

8. A PD-L1 blocker, characterized in that, The T-cell biomimetic nanoparticles A-AuNPs@M as described in any one of claims 1-3 were used as the main active ingredient.

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