Bionic photo-thermal nano-motor for targeted regulation of solid tumor matrix-immunosuppression microenvironment and preparation method and application of bionic photo-thermal nano-motor

Abstract

The invention discloses a bionic photo-thermal nano motor for targeted regulation and control of a solid tumor matrix-immunosuppression microenvironment and a preparation method and application thereof, and belongs to the technical field of biomedical materials.The preparation method comprises the steps that polydopamine nanoparticles and a K2PtCl4 aqueous solution are subjected to condensation reflux, and asymmetric PDA-Pt is prepared; and preparing a cancer-related fibroblast and breast cancer cell fusion membrane and coating the surface of PDA-Pt with the cancer-related fibroblast and breast cancer cell fusion membrane. The nano-motor realizes dual targeting of matrix cells and tumor cells through a surface modified fusion cell membrane, targeted ablation CAFs regulates and controls a tumor matrix microenvironment, a good tumor permeation effect is realized by cooperating with H2O2 responsive self-driving of the nano-motor, a mild photo-thermal-catalytic synergistic effect is formed by a photo-thermal effect of PDA and peroxidase-like activity of Pt nano-enzyme, and a good tumor permeation effect is achieved. And finally, the tumor immunosuppression microenvironment is remarkably improved, and the tumor immunotherapy effect is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical materials, and specifically relates to a biomimetic photothermal nanomotor for targeted regulation of a solid tumor matrix-immunosuppressive microenvironment, and a preparation method and application thereof. Background Art

[0002] Malignant tumors are among the most lethal diseases worldwide, and their treatment has long been hampered by the complex tumor microenvironment (TME). The dense extracellular matrix (ECM) and infiltrating immunosuppressive cells within solid tumors create a physical and biochemical barrier that not only impedes drug penetration but also weakens therapeutic efficacy through immune evasion. Cancer-associated fibroblasts (CAFs), core regulators of the tumor microenvironment, secrete excessive amounts of matrix components such as type I collagen and fibronectin, increasing the ECM stiffness from 1.13-1.83 kPa in normal tissue to 1.91-3.68 kPa in tumor regions. This change in mechanical stiffness not only creates a physical barrier that hinders drug penetration but also promotes tumor cell invasion and metastasis by activating integrin signaling pathways. Simultaneously, chemokines such as IL-6 and CCL2 secreted by CAFs continuously recruit myeloid-derived suppressor cells (MDSCs) and regulatory T cells (Tregs), creating an immunosuppressive microenvironment. Under these conditions, tumor cells produce large amounts of lactic acid through the Warburg effect, causing the local pH to drop below 6.5, further inhibiting T cell activity and inducing apoptosis. This physical rigidity, in synergistic with immunosuppressive signals, causes tumors to exhibit a "cold" immune phenotype, characterized by inefficient tumor antigen presentation, insufficient cytotoxic T lymphocyte (CTL) infiltration, and high expression of immune checkpoint molecules such as PD-L1, ultimately leading to immune escape and therapeutic resistance.

[0003] In recent years, innovative strategies such as photothermal therapy (PTT), nanomotor delivery systems, and biomimetic nanomaterials have provided new avenues for overcoming this bottleneck, but their clinical translation still faces multiple challenges. Photothermal therapy has become a research hotspot due to its non-invasive nature, spatiotemporal controllability, and potential for immune activation. However, the clinical application of traditional PTT is severely limited by dose-dependent thermal side effects: when treatment temperatures exceed 50°C, thermal damage caused by tumor vascular rupture can exacerbate necrosis of surrounding normal tissue and even lead to scarring. Furthermore, the physical barrier formed by the dense ECM severely hinders the deep penetration of photothermal agents. In 3D tumor spheroid models, passively diffused nanoparticles can only reach approximately 100 μm from the surface, leading to residual tumor margins and recurrence. Furthermore, the immune response induced by PTT alone is often neutralized by immunosuppressive cells in the TME. For example, Tregs suppress CTL function by secreting IL-10 and TGF-β, while MDSCs deplete local L-arginine through arginase-1, further impairing T cell proliferation. Therefore, how to achieve synergistic enhancement of mild photothermal therapy (≤45°C) and normalization of the immune microenvironment has become a key link in breaking through the limitations of PTT.

[0004] The emergence of nanomotor technology provides a new strategy for overcoming biological barriers. This type of system achieves autonomous movement through the drive of chemical energy, physical energy or biological energy, significantly improving the efficiency of drug delivery. Experiments have shown that in a 3D tumor sphere model, the penetration depth of autonomously moving nanoparticles can reach 3-5 times that of passive diffusion, and their cross-matrix migration ability is closely related to surface ligand modification and size. For example, red blood cell membrane-coated magnetic nanomotors are targeted and enriched in tumor areas through magnetic navigation, and combined with microenvironment-responsive drug release design, tumor vascular normalization can be achieved within 72 hours. However, existing nanomotors still face multiple challenges: the enzymatic activity of chemically driven motors is easily interfered with by proteases in the TME, the biosafety of physically driven systems has not been fully verified, and the long-term circulation characteristics of biohybrid motors still need to be optimized. In addition, how to achieve precise regulation of the motor movement direction and deep penetration path remains a difficult problem that needs to be overcome in this field. Summary of the Invention

[0005] To address these shortcomings, the present invention provides a biomimetic photothermal nanomotor for targeted regulation of the solid tumor stroma-immunosuppressive microenvironment, as well as its preparation method and application. This nanomotor, through its surface-modified fusion cell membrane, can achieve dual targeting of stromal and tumor cells, targeted ablation of CAFs to regulate the solid stromal microenvironment, and further synergizes with the nanomotor's H2O2-responsive self-propulsion to achieve excellent tumor penetration. Furthermore, the photothermal effect of PDA and the peroxidase-like activity of Pt nanozymes form a mild photothermal-catalytic synergistic effect, promoting immunogenic cell death. Ultimately, this improves the tumor immune microenvironment and significantly enhances the efficacy of tumor immunotherapy, effectively resolving the challenges of the prior art.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention to solve the technical problem is: A method for preparing a biomimetic photothermal nanomotor for targeted regulation of a solid tumor matrix-immunosuppressive microenvironment comprises the following steps: (1) PDA nanoparticles were dispersed in ultrapure water, and a K2PtCl4 aqueous solution was added thereto under stirring conditions, followed by condensation and reflux reaction in a water bath to prepare a PDA-Pt asymmetric nanophotothermal agent; (2) The CAFs cell membrane and breast cancer cell membrane were mixed and ultrasonically fused to obtain a fused membrane. The fused membrane, PDA-Pt nanophotothermal agent and ultrapure water were mixed and then ultrasonically treated in an ice water bath. The mixture was then passed through a polycarbonate membrane with gradually decreasing pore size in sequence and finally centrifuged to obtain a biomimetic photothermal nanomotor (PDA-Pt@MM).

[0007] Furthermore, the particle size of the PDA nanoparticles in step (1) is 80-200 nm. Furthermore, in step (1), the mass ratio of PDA nanoparticles to K2PtCl4 is 1:2-7.5. Furthermore, in step (1), the reflux reaction temperature is 75-85° C., and the reaction time is 20-30 h. Furthermore, in step (2), the mass ratio of CAFs cell membrane to breast cancer cell membrane is 1:1, and the ultrasonic fusion time is 8-15 min.

[0008] Furthermore, in step (2), the mass ratio of the PDA-Pt asymmetric nanophotothermal agent to the fusion membrane is 1:1-3, the water bath ultrasonic power is 160-200w, and the ultrasonic treatment time is 2-5min.

[0009] Furthermore, the pore sizes of the polycarbonate membrane in step (2) are 800 nm, 400 nm and 200 nm, respectively.

[0010] A bionic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment is prepared by the above method.

[0011] The application of the above-mentioned bionic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment in the preparation of drugs for treating solid tumors.

[0012] Furthermore, the above-mentioned bionic photothermal nanomotor is used to prepare drugs for treating various solid tumors appearing on the surface or inside organs, including one of skin cancer, malignant melanoma, nasopharyngeal cancer, esophageal cancer, gastric cancer, liver cancer, breast cancer, laryngeal cancer, thyroid cancer, tongue cancer, prostate cancer, penile cancer, testicular tumor, vaginal malignancy, and vulvar malignancy.

[0013] Furthermore, the method of use is intratumoral administration or intravenous injection.

[0014] The beneficial effects produced by the present invention are: 1. The PDA-Pt nanophotothermal agent prepared in the present invention has an asymmetric structure. During the synthesis process, the Pt nanoparticles are preferentially anchored on the active sites on the PDA surface, forming growth points of localized high-concentration Pt cations. Subsequently, the Pt 2+ Driven by the electrochemical potential, heterogeneous reduction growth continues along the surface of the deposited Pt particles, and the small-sized Pt crystals at the edge gradually dissolve and are absorbed by the adjacent large particles due to their higher surface energy, causing the latter to continue to increase in size, and eventually forming an asymmetric morphology with Pt enriched at one end. The size of the growing Pt nanozyme cluster is 2-10nm. This morphology can provide sufficient power at one end during the release process, promoting the increase in the penetration depth of the nanomotor in the tumor.

[0015] 2. The Pt nanozyme clusters in the biomimetic photothermal nanomotor of this invention catalyze the decomposition of H2O2 to produce O2 bubbles, driving the nanoparticles to penetrate the 3D tumor spheroids to a depth of 70 μm (a three-fold increase compared to conventional nanoparticles), significantly increasing drug concentration within the tumor. PDA generates mild photothermal energy under 808nm laser (1W / cm²) irradiation. Combined with the Pt nanozyme cluster's catalytic activity in H2O2 to generate hydroxyl radicals (·OH), this induces immunogenic cell death (ICD) while preventing the damage of normal tissue caused by high temperatures. Furthermore, the nanomotor disrupts the redox balance within tumor cells. Specifically, the nanomotor utilizes the peroxidase activity of the Pt nanozyme to generate oxidative hydroxyl radicals (·OH). Furthermore, the quinone groups on the PDA oxidize glutathione, a substance that maintains intracellular redox balance, further promoting intracellular oxidative stress. Combined with the mild photothermal activity, this leads to tumor cell death.

[0016] 3. The bionic photothermal nanomotor in the present invention achieves simultaneous targeting of tumor cells and stromal cells by fusing the 4T1 tumor cell membrane with the CAFs cell membrane (the targeting efficiency is 3 times higher than that of ordinary nanoparticles), reducing non-specific distribution. Therefore, the bionic photothermal nanomotor reshapes the solid tumor stromal microenvironment and immunosuppressive microenvironment through the dual "attack" of tumor cells and CAFs, thereby activating systemic anti-tumor immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 Schematic diagram of the synthesis and action mechanism of the biomimetic photothermal nanomotor in an embodiment of the present invention; Figure 2 The synthesis and characterization results of PDA-Pt@MM include: (A) Fluorescence co-localization results of 4T1M (DiO, green) and CAFM (DiI, red) (scale bar = 5 μm), (B) Fluorescence spectra of 4T1M co-labeled with DiI and DiD and fused with CAFM at different mass ratios, (C) Zeta potential and hydrated particle size of PDA-Pt@MM, (D) Transmission electron microscopy characterization of PDA-Pt and PDA-Pt@MM, (E) SDS-PAGE gel images of 4T1M, CAFM, MM, and PDA-Pt@MM, and (F) Changes in hydrated particle size of PDA-Pt@MM in PBS and culture medium over 7 days. Figure 3 Characterization results of the photothermal heating capability of PDA-Pt@MM. (A) Temperature rise curves of PDA-Pt@MM with different concentrations (808 nm, 1 W / cm 2 ), (B) Temperature rise curves of PDA-Pt@MM (200 μg / mL) at different laser power densities, (C) Photothermal temperature rise curves of PDA-Pt@MM, PDA-Pt and PDA at the same concentration, (D) Thermal imaging of PDA-Pt@MM and PDA-Pt under 808 nm laser irradiation, (E) Photothermal stability of PDA-Pt@MM, (F) Photothermal conversion efficiency of PDA-Pt@MM; Figure 4 Results of in vitro ·OH generation and glutathione scavenging by PDA-Pt@MM. (A) Michaelis-Menten curve of PDA-Pt@MM, (B) Peroxidase-like activity of PDA-Pt@MM enhanced by hyperthermia, (C) GSH consumption rate by PDA-Pt@MM, (D) GSH consumption efficiency of PDA, PDA-Pt, and PDA-Pt@MM at 25°C and 45°C. Figure 5Figure 3 shows the oxygen production performance and autonomous actuation function of PDA-Pt@MM. (A) Oxygen production of PDA-Pt@MM in vitro, (B) Diffusion coefficients of different nanoparticle components (PDA, Pt, PDA + Pt, and PDA-Pt@MM) in the presence of H2O2, (C) Particle size distribution of PDA-Pt@MM before and after H2O2 addition and after H2O2 depletion. Figure 6 Comparison of cellular uptake of different nanoparticle components. (A) Fluorescence images of 4T1 breast cancer cells uptake of different nanoparticles. (B) Flow cytometry analysis of 4T1 cell uptake of different nanoparticles. (C) Fluorescence images of CAFs uptake of different nanoparticles. (D) Flow cytometry analysis of CAFs uptake of different nanoparticles (cell nuclei: blue fluorescence; nanoparticles: red fluorescence). Figure 7 Results of deep tumor penetration of PDA-Pt@MM. (A) Stratified scanning image of H2O2-dependent tumor penetration of PDA-Pt@MM, (B) Quantitative image of the fluorescence distribution of PDA-Pt@MM with or without H2O2 added at 70 μm, (C) Comparison of fluorescence intensity in cross-sections of 3D tumor spheres at different depths. Figure 8 Figure 3. The in vitro mild photothermal synergistic antitumor effect of PDA-Pt@MM. Effects of different nanoparticles on (A) 4T1 cell and (B) CAF cell viability under conditions with or without illumination. (C) DCFH-DA assay for ROS levels in 4T1 cells under different treatment groups (green fluorescence: DCFH-DA). (D) Lipid peroxide probe assay for lipid peroxide levels in 4T1 cells under different treatment groups. (E) Fluorescence images of mitochondria in 4T1 cells under different treatment groups using a JC-1 mitochondrial membrane potential probe (red fluorescence: aggregates; green fluorescence: monomers). Figure 9 Figure 3. In vivo targeting and penetration results of PDA-Pt@MM. (A) In vivo fluorescence distribution of 4T1 tumor-bearing mice after intravenous injection of PDA@MM, PDA-Pt@M4T1, PDA-Pt@MCAF, and PDA-Pt@MM. (B) Fluorescence imaging of isolated tumors and major organs and (C) quantitative analysis. CLSM images of tumor sections from the PDA@MM, PDA-Pt@M4T1, PDA-Pt@MCAF, and PDA-Pt@MM treatment groups 48 hours after intravenous injection. Blood vessels were stained with anti-CD31-FITC. Figure 10Figure 3. PDA-Pt@MM in vivo anti-tumor effects and immunosuppressive microenvironment remodeling. (A) Schematic diagram of the in vivo anti-tumor treatment regimen for PDA-Pt@MM, (B) Tumor volume growth curves for each group of mice during treatment, (C) H&E staining of tumor tissue sections from each group of mice, detection and analysis of cytokines (D) CXCL1 and (E) CXCL12 in mouse serum, flow cytometry detection of MDSCs in tumor tissue (G) and statistical analysis (F), and flow cytometry detection of Tregs in tumor tissue (I) and statistical analysis (H). Figure 11 Results of PDA-Pt@MM remodeling the stromal microenvironment in vivo. (A) Analysis of extracellular matrix and cell density in mouse tumor sections after control + L and PDA-Pt@MM + L treatment (green: Collgen I; blue: cell nuclei). (B) α-SMA fluorescence levels in tumor sections (green: α-SMA; blue: cell nuclei). (C) Collgen I fluorescence levels in tumor sections (green: Collgen I; blue: cell nuclei). DETAILED DESCRIPTION

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. That is, the embodiments described are only part of the embodiments of the present invention, rather than all embodiments.

[0019] Therefore, the detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the invention as claimed, but is merely intended to represent selected embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative work are within the scope of protection of the present invention.

[0020] It should be noted that relational terms such as "first" and "second" are used only to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the terms "comprises," "comprising," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0021] The features and performance of the present invention are further described in detail below with reference to the embodiments and drawings.

[0022] Example 1 A biomimetic photothermal nanomotor for targeted regulation of a solid tumor matrix-immunosuppressive microenvironment, the preparation method of which comprises the following steps: (1) Dopamine solution was slowly added dropwise to a mixed solution consisting of Tris and anhydrous ethanol in a volume ratio of 5:1, stirred for 16 hours, and centrifuged at 13,000 rpm / min to obtain PDA nanoparticles; (2) PDA nanoparticles were added to ultrapure water to prepare a solution with a concentration of 0.5 mg / mL, and 10 mL was added to a round-bottom flask. Under slow stirring, 500 μL of K2PtCl4 solution (120 mM) was slowly added, and the reaction was condensed and refluxed at 80°C for 24 h to prepare a PDA-Pt asymmetric nanophotothermal agent. (3) Equal amounts of CAFs cell membranes and 4T1 cell membranes were mixed and ultrasonically fused at 37°C for 10 min to obtain fused membranes. The fused membranes (1 mg / mL, 400 μL), PDA-Pt asymmetric nanophotothermal agent solution (1 mg / mL, 200 μL) and 400 μL ultrapure water were blown and mixed, and then ultrasonically treated in an ice water bath with an ultrasonic power of 180 W for 3 min. The mixture was then passed through polycarbonate membranes with pore sizes of 800, 400 and 200 nm in sequence and finally centrifuged to obtain (PDA-Pt@MM).

[0023] Example 2 A biomimetic photothermal nanomotor for targeted regulation of a solid tumor matrix-immunosuppressive microenvironment, the preparation method of which comprises the following steps: (1) Dopamine solution was slowly added dropwise to a mixed solution consisting of Tris and anhydrous ethanol in a volume ratio of 5:1, stirred for 14 hours, and centrifuged at 13,000 rpm / min to obtain PDA nanoparticles; (2) PDA nanoparticles were added to ultrapure water to prepare a solution with a concentration of 0.5 mg / mL, and 10 mL was added to a round-bottom flask. Under slow stirring, 250 μL of K2PtCl4 solution (120 mM) was slowly added, and the reaction was condensed and refluxed at 80°C for 24 h to prepare a PDA-Pt asymmetric nanophotothermal agent. (3) Equal amounts of CAFs cell membranes and 4T1 cell membranes were mixed and ultrasonically fused at 37°C for 8 min to obtain fused membranes. The fused membranes (1 mg / mL, 400 μL), PDA-Pt asymmetric nanophotothermal agent solution (1 mg / mL, 200 μL) and 400 μL ultrapure water were blown and mixed, and then ultrasonically treated in an ice water bath with an ultrasonic power of 160 W for 5 min. The mixture was then passed through polycarbonate membranes with pore sizes of 800, 400 and 200 nm in sequence and finally centrifuged to obtain (PDA-Pt@MM).

[0024] Example 3 A biomimetic photothermal nanomotor for targeted regulation of a solid tumor matrix-immunosuppressive microenvironment, the preparation method of which comprises the following steps: (1) Dopamine solution was slowly added dropwise to a mixed solution consisting of Tris and anhydrous ethanol in a volume ratio of 5:1, stirred for 18 hours, and centrifuged at 13,000 rpm / min to obtain PDA nanoparticles; (2) PDA nanoparticles were added to ultrapure water to prepare a solution with a concentration of 0.5 mg / mL, and 10 mL was added to a round-bottom flask. Under slow stirring, 750 μL of K2PtCl4 solution (120 mM) was slowly added, and the reaction was condensed and refluxed at 80°C for 24 h to prepare a PDA-Pt asymmetric nanophotothermal agent. (3) Equal amounts of CAFs cell membranes and 4T1 cell membranes were mixed and ultrasonically fused at 37°C for 8-15 min to obtain a fused membrane. The fused membrane (1 mg / mL, 400 μL), PDA-Pt asymmetric nanophotothermal agent solution (1 mg / mL, 200 μL) and 400 μL ultrapure water were blown and mixed, and then ultrasonically treated in an ice water bath with an ultrasonic power of 200 w and an ultrasonic treatment time of 2 min. The mixture was then passed through polycarbonate membranes with pore sizes of 800, 400 and 200 nm in sequence and finally centrifuged to obtain (PDA-Pt@MM).

[0025] Test example Figure 1 Schematic diagram of the synthesis and action mechanism of the biomimetic photothermal nanomotor in the present invention; 1. Taking the biomimetic nanomotor prepared in Example 1 as an example, the following tests were performed on it: 1. Use fluorescence images and fluorescence spectra to detect cell membrane fusion. The results are shown in Figure 2 .in, Figure 2 A is the fluorescence image result, Figure 2 B is the fluorescence spectrum result. The results show that the 4T1 cell membrane and CAFs cell membrane were successfully fused by ultrasound.

[0026] 2. Use particle size analyzer and transmission electron microscope to characterize the morphology of nanosystem. The results are shown in Figure 2 .in, Figure 2 C is the particle size and potential result, Figure 2 D is the transmission electron microscopy result. The results show that the particle size of the PDA-Pt composite system has increased slightly, and further increased after the modified fusion membrane. Figure 2 D shows the asymmetric morphology of PDA-Pt, and an obvious core-shell structure can be seen after cell membrane modification.

[0027] 3. Use SDS-PAGE to detect the modification of cell membrane. The results are shown in Figure 2 .Depend on Figure 2 E shows that the fusion membrane (MM) has special bands of 4T1M and CAFM, and the membrane protein bands of PDA-Pt@MM are very similar to those of MM, proving the successful coating of the fusion membrane on the PDA-Pt surface.

[0028] 4. Use particle size analyzer to test the stability of biomimetic photothermal nanomotor. The results are shown in Figure 2 .Depend on Figure 2 F shows that the particle size of PDA-Pt@MM did not change significantly in PBS within 7 days and can be used for subsequent in vitro and in vivo experiments.

[0029] 2. Test the photothermal heating capability of the bionic nanomotor. The specific operation method is as follows: 1. Testing the heating performance of nanomotors at different concentrations PDA-Pt@MM was prepared into aqueous solutions with different concentrations using UP water, which were set to 50, 100, and 200 μg / mL, respectively. Water was used as a control group. Then, an 808 nm laser (laser power was set to 1.0 W / cm 2 ) Each solution was irradiated for 15 min, and the temperature change of the solution was recorded every 30 s.

[0030] like Figure 3 As shown in A, when the laser power is fixed at 1.0W / cm 2 When irradiated for 15 minutes, PDA-Pt@MM showed a concentration-dependent temperature rise behavior. It is worth noting that when the concentration of the nanophotothermal agent is 200 μg / mL, the maximum temperature difference (ΔT) can reach 22.8°C.

[0031] 2. Photothermal heating performance test of nano-photothermal agents at different powers A 200 μg / mL PDA-Pt@MM aqueous solution was used, and then the 808 nm laser was adjusted to different powers (0.5, 1.0, 1.5 W / cm 2) The solution was irradiated continuously for 15 min, and the temperature change of the solution was recorded every 30 s.

[0032] like Figure 3 As shown in Figure B, at the same concentration, the solution was irradiated with lasers of different powers, and PDA-Pt@MM also showed a power-dependent temperature rise effect.

[0033] 3. Photothermal heating performance test of each group of nanoparticles PDA-Pt@MM, PDA-Pt, and PDA were prepared into aqueous solutions with the same concentration (200 μg / mL) using UP water, and water was used as a control group. Subsequently, an 808 nm laser (laser power was set to 1.0 W / cm 2 ) Each solution was irradiated for 15 min, and the temperature change of the solution was recorded every 30 s.

[0034] like Figure 3 As shown in Figures C and D, PDA, PDA-Pt, and PDA-Pt@MM exhibit similar temperature increases, with PDA-Pt exhibiting a greater temperature increase than PDA. This is likely due to the photothermal conversion capabilities of Pt nanoparticles. Thermal images of PDA-Pt and PDA-Pt@MM under laser irradiation were recorded using an infrared thermal imager. Modification of the fusion membrane has no effect on the photothermal effect of PDA-Pt.

[0035] 4. Calculation of PDA-Pt@MM temperature stability and photothermal conversion efficiency A 200 μg / mL PDA-Pt@MM aqueous solution was used, and then a power of 1.0 W / cm 2 The aqueous solution was irradiated with an 808 nm laser for 10 minutes, and the temperature change of the solution was recorded every 30 seconds. After one irradiation, the laser was turned off, and the solution was allowed to cool down naturally, and the temperature change of the solution was recorded every 30 seconds. The recovery of the solution to a temperature close to the initial temperature was recorded as one cycle, and then the laser was continued to be used at the same power to perform the same operation as the previous cycle, and a total of four cycles were repeated.

[0036] like Figure 3 As shown in Figures E and F, PDA-Pt@MM maintains excellent photothermal conversion capabilities after several cycles of 808 nm laser irradiation, demonstrating its exceptional photothermal stability. Furthermore, under 808 nm laser irradiation, the photothermal conversion efficiency of PDA-Pt@MM reached 32.0%. These results demonstrate that PDA-Pt@MM is an excellent nanophotothermal agent.

[0037] 3. Test the ability of biomimetic nanomotors to disrupt oxidation balance. The specific test method is as follows: 1. Performance test of PDA-Pt@MM in generating hydroxyl radicals To evaluate the peroxidase-mimetic activity of PDA-Pt@MM, a chromogenic substrate, TMB, was used. First, TMB was prepared in DMSO at various concentrations (3, 2.0, 1.5, 1, 0.8, 0.4, 0.2, and 0.1). PBS (pH 5.5), PDA-Pt@MM, and H₂O₂ solutions were rapidly mixed and pipetted thoroughly. The mixture was then added to a cuvette and the absorbance was measured every 1 second.

[0038] like Figure 4 As shown in Figure A, H2O2 can be catalyzed by platinum nanozymes to decompose into ·OH, which in turn oxidizes 3,5',5,5'-tetramethylbenzidine (TMB) into blue ox-TMB, which has a characteristic absorbance at 652 nm. The reaction rate increases with increasing substrate concentration. Since increasing temperature can increase the rate of enzyme reaction, the enzyme activity of PDA-Pt@MM before and after laser application was further compared. Figure 4 As shown in Figure B, compared to the unexposed group, the group treated with 808 nm laser light exhibited a significant increase in absorbance. This is likely due to the local temperature increase triggered by the photothermal effect, which significantly increased the rate of the catalytic reaction. Therefore, PDA-Pt@MM promotes the generation of ·OH through photothermal treatment, which further enhances the photothermal therapeutic effect, demonstrating the potential of PDA-Pt@MM in synergistic photothermal-catalytic tumor therapy.

[0039] 2. Performance test of PDA-Pt@MM in scavenging glutathione (1) Detection of glutathione (GSH) consumption at different time points: GSH solution (1 mM) and PDA-Pt@MM (200 μg / mL) were mixed, and PBS solution alone was used as a control. At the preset time points (10-60 min, each time with an interval of 10 min), 100 μL of the reaction solution was centrifuged (12000 rpm, 10 min), and 50 μL of the supernatant was taken for color reaction with 50 μL of DTNB (20 mM). The absorbance of the test solution at 412 nm was quickly recorded using a microplate reader to obtain the GSH consumption.

[0040] like Figure 4As shown in Figure C, GSH consumption gradually increased with reaction time, exceeding 50% within 60 minutes. This is because the platinum nanozyme is in situ reduced by the PDA nanoparticles, oxidizing the catechol groups of PDA to quinone groups. On this basis, the quinone groups can be further reduced by the reducing substance GSH, leading to the consumption of endogenous GSH.

[0041] (2) Detection of the effect of different particles and different temperatures on GSH consumption: GSH solution (1 mM) and PDA-Pt@MM (200 μg / mL) were mixed, and PDA and PDA-Pt solutions of the same concentration were used as controls. The room temperature experimental group was placed in a dark environment overnight, while the elevated temperature experimental group was placed in a 45°C water bath overnight waiting for treatment. After the reaction was completed, 100 μL of the solution in each tube was further treated with 100 μL of DTNB (20 mM), and the absorbance of the mixed solution at 412 nm was recorded using a microplate reader to calculate the amount of GSH loss.

[0042] like Figure 4 As shown in Figure D, increasing the temperature can further promote GSH consumption. PDA-Pt and PDA-Pt@MM show stronger GSH consumption behavior than PDA alone. This may be because the reduced Pt promotes the presence of more quinone groups on the PDA surface. The above results fully demonstrate that PDA-Pt@MM can not only generate reactive oxygen species in cells, but also consume the intracellular reducing substance GSH to further disrupt the intracellular redox balance, showing the ability to effectively kill tumor cells.

[0043] 4. Taking the bionic nanomotor in Example 1 as an example, its oxygen production performance and autonomous driving function were tested by the following method: 1. PDA-Pt@MM oxygen production performance test To evaluate oxygen production performance, a dissolved oxygen meter was used. The meter was pre-opened and placed in zero oxygen calibration solution for zero calibration. Zero calibration was completed after the index stabilized. The oxygen electrode probe was placed on the surface of ultrapure water to calibrate the instrument to full scale. PBS (pH 6.5) was pre-sonicated to remove oxygen from the water. 10 mL of PBS buffer containing 2 mM H₂O₂ was added to a glass bottle. PDA-Pt and PDA-Pt@MM (200 μg / mL) were then added. A single H₂O₂ solution was used as a control. After sufficient dissolution, the bottle was sealed with plastic film. The oxygen electrode was inserted for measurement, and the dissolved oxygen concentration was recorded every 1 s for 240 s per sample.

[0044] like Figure 5As shown in Figure A, PDA-Pt@MM was placed in a PBS solution containing H2O2. The amount of oxygen released in the solution was measured using a dissolved oxygen instrument. The results showed that compared to the H2O2 solution alone, both PDA-Pt@MM and PDA-Pt reacted with H2O2 to release a large amount of oxygen, which provided a driving force for their movement. However, compared with PDA-Pt, the oxygen production capacity of PDA-Pt@MM was weakened, which may be due to the reduced contact and reaction of its surface cell membrane with H2O2.

[0045] 2. PDA-Pt@MM diffusion performance test Dynamic light scattering (DLS) test dishes were ultrasonically cleaned three times with ultrapure water and dried at room temperature before use. PDA, Pt, PDA + Pt, and PDA-Pt@MM were prepared to the same concentration using pure water and mixed with a 2 mM H₂O₂ solution. The mixture was immediately added to the test dish for testing. The diffusion coefficient was calculated using the Stokes-Einstein equation.

[0046] like Figure 5 As shown in Figure B, the diffusion coefficients of nano-PDA, Pt, and PDA + Pt mixed components did not increase in H2O2 solution, while the diffusion coefficient of PDA-Pt@MM increased significantly, which is obviously related to the asymmetric structure of PDA-Pt@MM. In addition, the particle size distribution of PDA-Pt@MM before and after incubation with H2O2 was studied (see Figure 4 C) The particle size distributions of the two groups, without H2O2 addition and after H2O2 depletion, were essentially the same. However, the particle size distribution of the group reacting with H2O2 decreased significantly, consistent with the Stokes-Einstein equation that fluid dynamics is inversely proportional to the diffusion coefficient. The particles then returned to their original size after H2O2 depletion. These results demonstrate that PDA-Pt@MM exhibits excellent H2O2-dependent self-propelled motion.

[0047] 5. Taking the biomimetic nanomotor of Example 1 as an example, its specific targeting ability was tested using the following method: At the cellular level, using Nile red-labeled PDA nanoparticles as a tracer, single-cell suspensions of tumor-associated fibroblasts (CAFs) and breast cancer 4T1 cells were prepared and seeded into 48-well plates. The cells were incubated in a humidified incubator for 12-16 hours until the cells adhered. The culture medium was aspirated from the plates, washed three times with PBS, and then RPMI 1640 (4T1 cells) or DMEM (CAFs) containing 200 μg / mL PDA-Pt@MM was added. At predetermined time points (2, 4, and 6 hours), any nanoparticles not internalized by the cells were discarded and washed three times with PBS. The nuclear dye Hoechst was added and incubated with the cells for 15 minutes in the dark. Internalization of PDA-Pt@MM by CAFs and 4T1 cells was observed using an inverted fluorescence microscope at different times.

[0048] like Figure 6 As shown in A, 4T1 tumor cells reacted with PDA-Pt without membrane component modification and with the CAFs membrane modified nanosystem (PDA-Pt@M CAF ) uptake was low. In contrast, the 4T1 membrane alone (PDA-Pt@M 4T1 There was no significant difference in the uptake of 4T1 tumor cells by the modified PDA-Pt, indicating that the fusion membrane retained the homologous targeting ability of the 4T1 membrane, enabling it to be targeted and taken up by 4T1 tumor cells. The results of flow cytometry showed the same pattern (see Figure 6 B). Similarly, if Figure 6 As shown in C and D, the PDA-Pt@MM and CAFs membrane-modified nanosystems exhibited similar cellular uptake behaviors, while the 4T1 cell membrane-modified nanosystem was less taken up by CAFs cells.

[0049] VI. Taking the biomimetic nanomotor of Example 1 as an example, its ability to penetrate 3D tumor spheres in vitro was tested using the following method: Using a conical-bottom 96-well plate that had been blocked with PF 127 overnight, 4T1 cells and CAFs were counted using a cell counter and a concentration of 2 × 10 4 Prepare a suspension of 4T1 and CAFs cells at a concentration of 100 μg / mL. Add 50 μL of the diluted cell suspension to each well, balance, and centrifuge in a microplate centrifuge (1000 rpm, 5 minutes). Add 50 μL of culture medium to each well to replenish nutrients. Continue culturing for two days before use.

[0050] like Figure 7As shown in A, in the absence of H2O2, PDA-Pt@MM showed stronger cell-targeting permeability than single membrane components. In the presence of H2O2, the permeability of PDA-Pt@MM was significantly improved, and the red fluorescence signal of each layer of the 3D tumor spheroid was much stronger than that of other control groups. In addition, Figure 7 Figures B and C compare the fluorescence intensity of the 3D tumor spheroid model at the interface 70 μm from the top in the presence or absence of H2O2 solution. It can be observed that the PDA-Pt@MM + H2O2 group significantly promoted the penetration of PDA-Pt@MM. These results indicate that PDA-Pt@MM, driven by the homologous targeting effect of the fused membrane and the oxygen production stimulated by H2O2, can effectively penetrate deep into the tumor and be efficiently taken up by deep-seated tumor cells.

[0051] VII. Taking the biomimetic nanomotor of Example 1 as an example, its mild photothermal synergistic catalytic anti-tumor properties in vitro were tested using the following method: 1. In vitro proliferation inhibition experiment of PDA-Pt@MM biomimetic photothermal nanomotor 4T1 cells in the logarithmic growth phase were seeded in 96-well plates (density of 1.0×10 4 The culture medium in the well plate was removed and 100 μL of RPMI 1640 (4T1 cells) or DMEM (CAFs) solution of each nanosystem was added and incubated at 37°C in a cell culture incubator for 6 h. After the cells were internalized, the culture medium containing the nanoparticles was discarded and washed three times with sterile PBS before adding fresh culture medium to the well plate. For the light treatment group, a laser with a wavelength of 808 nm (output power 1 W / cm 2 Cells were treated with irradiation (irradiation time 8 min) and cultured for an additional 24 h after illumination. After incubation, the culture medium containing the nanoparticle system was aspirated and the cells were gently rinsed three times with sterile PBS. 100 μL of pre-prepared CCK-8 assay solution was added to each well and incubated at 37°C for 1 h. The absorbance of each well at 450 nm was measured using a microplate reader, and the survival rate was calculated using the formula = (OD value of the experimental group - OD value of the blank group) - (OD value of the control group - OD value of the blank group).

[0052] like Figure 8As shown in A, under laser irradiation, PDA-Pt@MM has a significant cell killing effect compared to the mild photothermal therapy group (PDA@MM + L). This is mainly due to the fact that Pt nanoperoxidase produces hydroxyl radicals that destroy the intracellular redox balance, further promoting the killing effect of mild photothermal therapy. The tumor cell killing effect of PDA-Pt@MM + L is also 45.21% higher than that of the non-membrane modified nanophotothermal agent treatment group (PDA-Pt + L), indicating that the fusion membrane-mediated cell-targeted endocytosis significantly enhances its tumor cell killing effect. Similarly, PDA-Pt@MM + L also showed a similar killing effect on CAFs cells (see Figure 8 B). In summary, these results demonstrate that mild photothermal synergistic catalytic therapy brings about a powerful cell-killing effect, which, combined with the targeting effect of the fusion membrane, further enhances the anti-tumor efficacy.

[0053] 2. PDA-Pt@MM induces intracellular ROS production 4T1 cells in the logarithmic growth phase were seeded in 24-well plates (density 5×10 4 / well) and cultured for 24 h. Remove the old culture medium, wash three times with PBS buffer, and add cell culture medium containing PDA-Pt, PDA@MM, and PDA-Pt@MM ([PDA]=200 μg / mL) to each well according to the experimental group. The blank control is cultured with PBS and cultured at 37°C for another 4 h. After the culture time is up, the old culture medium in each well is aspirated, gently washed three times with sterile PBS and re-added with cell culture medium. For the laser treatment group, a laser with a wavelength of 808 nm (output power 1 W / cm 2 Treat cells with 1% dapoxetine (1% dapoxetine) and illuminate for 8 minutes. Continue incubating at constant temperature for 6 hours after illumination. Prepare DCFH-DA staining solution (1:1000 dilution in serum-free RPMI 1640 medium). Aspirate the old culture medium, wash three times with sterile PBS, then add DCFH-DA staining solution, ensuring that the stain completely covers the cells. Stain at 37°C in the dark for 20 minutes. Aspirate the staining solution, wash three times with sterile PBS, and stain with Hoechst for 15 minutes. Remove the staining solution and wash with PBS, then add culture medium. Finally, capture fluorescence images using an inverted fluorescence microscope.

[0054] like Figure 8 As shown in Figure C, PDA-Pt@MM exhibited less green fluorescence, proving that the efficiency of Pt nanozyme alone in generating ROS was low, while the ROS level in the PDA-Pt + L treatment group increased significantly, indicating that mild photothermal treatment enhanced the catalytic efficiency of Pt nanozyme. The strongest green fluorescence was observed in the PDA-Pt@MM + L treatment group, proving that PDA-Pt@MM + L can significantly increase the ROS content in cells.

[0055] 3. PDA-Pt@MM induces intracellular lipid oxidation 4T1 cells in the logarithmic growth phase were seeded in 24-well plates (density 5×10 4 / well) and cultured for 24 h. Remove the old culture medium, wash three times with PBS buffer, and add cell culture medium containing PDA-Pt, PDA@MM, and PDA-Pt@MM ([PDA]=200 μg / mL) to each well according to the experimental group. The blank control is cultured with PBS and cultured at 37°C for another 4 h. After the culture time is up, the old culture medium in each well is aspirated, gently washed three times with sterile PBS and re-added with cell culture medium. For the laser treatment group, a laser with a wavelength of 808 nm (output power 1 W / cm 2 The cells were treated with 5 μM BODIPY probe (10 μM PBS, 8 min of light exposure) and then incubated at constant temperature for 6 h after the illumination period. The culture medium was aspirated and 5 μM BODIPY probe was added. After complete contact of the cells with the dye solution, the cells were incubated at 37°C for 30 min. After staining, the cells were washed three times with sterile PBS, 1 mL of PBS was added, and images were taken using an inverted fluorescence microscope.

[0056] The cell membrane is mainly composed of lipids containing unsaturated fatty acids, in which the double bonds of unsaturated fatty acids are easily attacked by ROS, causing chain free radical reactions and leading to lipid oxidation and degradation. 665 / 676 ) were used to detect lipid peroxidation accumulation in 4T1 cells. Figure 8 As shown in D, compared with the control group, strong red fluorescence appeared in the 4T1 cells in the PDA-Pt@MM + L treatment group, indicating that there was a large accumulation of lipid peroxidation. The above results show that mild photothermal treatment can promote PDA-Pt@MM to produce ·OH, destroy the redox balance in the cells, lead to the destruction of cell mitochondrial function, and trigger lipid peroxidation on the cell membrane, further enhancing the anti-tumor effect.

[0057] 4. PDA-Pt@MM induces intracellular mitochondrial damage 4T1 cells in the logarithmic growth phase were seeded in 24-well plates (density 5×10 4 / well) and cultured for 24 h. Remove the old culture medium, wash three times with PBS buffer, and add cell culture medium containing PDA-Pt, PDA@MM, and PDA-Pt@MM ([PDA]=200 μg / mL) to each well according to the experimental group. The blank control is cultured with PBS and cultured at 37°C for another 4 h. After the culture time is up, the old culture medium in each well is aspirated, rinsed three times with sterile PBS, and then the cell culture medium is added again. For the laser treatment group, a laser with a wavelength of 808 nm (output power 1 W / cm 2 Treat the cells with 1 mL of fresh culture medium and 1 mL of JC-1 staining buffer (for 8 minutes). Continue incubating at constant temperature for 12 hours after the illumination period. Remove the old culture medium and add 2 mL of a premix (1 mL of fresh culture medium and 1 mL of JC-1 staining working solution). Once the cells are fully exposed to the stain, incubate at constant temperature for 20 minutes in the dark. After staining, wash twice with preheated JC-1 staining buffer and then add 1 mL of cell culture medium. Immediately image the cells using an inverted fluorescence microscope.

[0058] It is well known that mitochondria are essential organelles in cells and play an important role in energy metabolism and cell apoptosis. Excessive ROS can induce mitochondrial dysfunction, leading to mitochondrial membrane potential depolarization and activating apoptosis pathways associated with mitochondrial damage. JC-1 is a membrane potential-dependent dye. When the mitochondrial transmembrane potential is intact, it forms aggregates with red fluorescence in the mitochondrial matrix. When mitochondrial function is damaged, the membrane potential decreases and the probe produces green fluorescence in the form of monomers. Figure 8 As shown in Figure E, in the PBS and PBS + L control groups, 4T1 cells exhibited significant red fluorescence from JC-1 aggregates, significantly higher than the green fluorescence from monomers, indicating that the mitochondria of 4T1 cells were in homeostasis. In contrast, the red fluorescence in the PDA-Pt + L and PDA-Pt@MM groups weakened, while the green fluorescence increased, indicating that some mitochondria were damaged. This is clearly related to the higher intracellular ROS levels in these two groups. Most notably, the red fluorescence in the PDA-Pt@MM + L treatment group almost completely disappeared, while the green fluorescence intensity of the monomers increased significantly, indicating that the large amount of ROS caused an imbalance in the mitochondrial membrane potential of 4T1 cells. Therefore, the heat generated by PDA-Pt@MM combined with light can enhance intracellular ROS levels, thereby disrupting mitochondrial function.

[0059] 8. Taking the biomimetic nanomotor of Example 1 as an example, its in vivo targeting and tumor penetration effects were tested using the following methods: Balb / c mice were provided by the Jicui Biological Animal Experiment Center. All mice were female, 4-6 weeks old, and weighed 18-22 g.

[0060] 4T1 cells and CAFs were digested and centrifuged, resuspended in physiological saline and counted. 100 μL of mixed cell suspension (containing 5×10 5 4T1 and 2.5×10 5 CAFs) were injected into the right lower fourth mammary pad of mice to establish orthotopic stroma-rich 4T1 tumors.

[0061] A unilateral in situ matrix-rich 4T1 tumor model was constructed and randomly divided into four groups, with 3 mice in each group. PDA@MM, PDA-Pt@M labeled with Cy5.5 fluorescent probe, and PDA-Pt@M were injected into the tail vein respectively. 4T1 、PDA-Pt@M CAF and PDA-Pt@MM ([PDA-Cy5.5] = 75 mg / kg). In vivo fluorescence imaging of mice was performed at pre-set time points, 2, 4, 8, 24, and 48 hours after injection. At the final time point (48 hours), mice were sacrificed by cervical dislocation, and tumor tissue and major organs (heart, liver, spleen, lung, and kidney) were removed for ex vivo organ fluorescence imaging. Fluorescence images were quantitatively analyzed using Living Image® 4.4 software.

[0062] like Figure 9 As shown in A, during the entire drug administration process, the strongest fluorescence signals were observed in the PDA@MM and PDA-Pt@MM treatment groups, indicating that the modification of the fusion membrane enables the nanophotothermal agent to accumulate in the tumor site, promoting more nanomedicines to enter the tumor site. 4T1 It also showed strong tumor targeting accumulation, which was due to the higher proportion of tumor cells in the tumor tissue; while the CAFs single membrane modified group PDA-Pt@M CAF The results showed that the tumor targeting ability of the drug was relatively low, which was related to the relatively low proportion of CAFs cells in the tumor tissue. It is worth noting that the fluorescence signal in the tumor of each group reached the highest value at 8 hours and gradually decreased after 24 hours, indicating that 8 hours is the optimal time point for phototherapy. In addition, fluorescence imaging of the dissected tumors and major organs was performed 48 hours after drug injection (see Figure 9 B) and compare the fluorescence intensity of tumors and other organs (see Figure 9 C), it can be observed that the two groups modified with fusion membrane (PDA@MM and PDA-Pt@MM) showed relatively high intratumor fluorescence intensity, indicating that the fusion membrane caused the photothermal agent to accumulate at a higher level in the tumor site through homologous targeting, which is conducive to the subsequent anti-tumor effect.

[0063] 48 hours after administration, the ex vivo tumors were dissected out, fixed with paraformaldehyde, and then sectioned in paraffin. The tumor sections were stained with anti-CD31-FITC, a vascular endothelial marker. Figure 9As shown in D, the red fluorescence around the blood vessels in the PDA@MM treated group is weak, indicating that although some PDA@MM can reach the tumor through the EPR effect, since PDA@MM itself does not have the driving ability, it is limited to passive accumulation, and a large number of nanoparticles are trapped in the vascular system. In contrast, the fluorescence intensity around the blood vessels of the other groups containing Pt nanozymes is significantly stronger. Although the single membrane modified group (PDA-Pt@M 4T1 、PDA-Pt@M CAF ) accumulates less in tumor tissue, but due to its autonomous driving ability, it is able to achieve deeper tumor penetration. Notably, PDA-Pt@MM combines the dual targeting and autonomous driving capabilities of the fusion membrane, with its red fluorescence being significantly strongest around blood vessels, indicating that PDA-Pt@MM achieves targeted deep tumor penetration.

[0064] IX. Taking the biomimetic nanomotor of Example 1 as an example, its ability to fight tumors and reshape the immunosuppressive microenvironment in vivo was tested using the following methods: 4T1 cells and CAFs were digested and centrifuged, resuspended in physiological saline and counted. 100 μL of mixed cell suspension (containing 5×10 5 4T1 and 2.5×10 5 CAFs) were injected into the fourth lower right mammary pad of mice to establish orthotopic stroma-rich 4T1 tumors. Three days after orthotopic tumor establishment, 100 μL of mixed cell suspension (containing 1×10 5 4T1 and 5×10 4 CAFs) were injected into the lower left fourth mammary pad of mice to establish orthotopic contralateral stroma-rich 4T1 tumors.

[0065] A bilateral in situ matrix-rich 4T1 tumor model was constructed and randomly divided into 6 groups, with 6 mice in each group. The drugs were administered intravenously, and the primary tumor site was irradiated with laser irradiation 8 hours after administration. The blank group was intravenously injected with 100 μL normal saline. The other groups were: G1: Control + L group (Saline injection and photothermal therapy group); G2: PDA@MM + L group (mild photothermal therapy group); G3: PDA-Pt@MM (nanozyme catalytic therapy group); G4: PDA-Pt@M 4T1 + L (mild photothermal synergistic catalytic therapy group targeting tumor cells); G5: PDA-Pt@M CAF + L (mild photothermal synergistic catalytic treatment group targeting stromal cells); G6: PDA-Pt@MM + L (dual-targeted mild photothermal synergistic catalytic treatment group); The tumor size in two dimensions was measured with a vernier caliper every other day, and the weight of the tumor-bearing mice was checked. The second administration and laser irradiation were performed on the third day, and the third administration and laser irradiation were performed on the fifth day. Figure 11 A). Tumor volume calculation formula is V = WL 2 = / 2 (W represents the long diameter of the tumor, L represents the short diameter of the tumor). On day 18, the mice were sacrificed, and the tumor masses were isolated and weighed. Tumor tissues were collected, embedded in paraffin, and then sectioned.

[0066] like Figure 11 As shown in B, although mild photothermal therapy alone (G2) can cause partial thermal damage to the tumor, it does not affect the overall trend of tumor growth; the Pt nanozyme treatment group alone (G3) has a weak tumor inhibition effect, which may be due to the fact that the peroxidase activity of the Pt nanozyme cannot produce enough ·OH at lower temperatures. In addition, the targeted mild photothermal synergistic catalytic treatment mediated by CAFs single membrane modified PDA-Pt (G5) also showed a poor tumor inhibition effect, which may be due to the limited proportion of stromal cells in the tumor; at the same time, tumor targeting mediated by 4T1 single membrane modified PDA-Pt (G4) (G5) has an enhanced inhibitory effect on in situ tumor growth. It is worth noting that under laser irradiation conditions, the PDA-Pt@MM treatment group (G6) showed a significant inhibitory effect, which may be mainly attributed to the enhanced tumor inhibition effect of the dual targeting mediated by the fusion membrane. The tumor tissues of the G1-G6 groups were further stained with H&E, and the results are shown in Figure 2. Figure 11 C, Compared with the negative control group, the PDA-Pt@MM + L-treated group showed significant histopathological damage, demonstrating that PDA-Pt@MM + L can lead to tumor ablation through enhanced tissue penetration and dual targeting effects.

[0067] CAFs, as core stromal cells within the TME, not only secrete ECM but also release CXCL1 / CCL2 (recruiting neutrophils / MDSCs) and CXCL12 / CCL5 (recruiting Tregs), establishing an immunosuppressive network. Therefore, ELISA assays were used to assess the levels of cytokines associated with maintaining an immunosuppressive microenvironment in the blood of mice treated with different therapies. Compared with the combined treatment group targeting 4T1 (G4), the synergistic treatments (G5 and G6) targeting CAFs and dual cell-targeting significantly reduced serum CXCL1 and CXCL12 levels, demonstrating that PDA-Pt@MM can target and kill CAFs and reduce the levels of cytokines they secrete. Flow cytometry was then used to analyze the immunosuppressive MDSCs and Tregs associated with these two cytokines. The results showed that the proportion of MDSCs in tumor tissue was approximately 33.6% in the untreated group (G1). However, after dual-targeting synergistic treatment, the MDSC level in the PDA-Pt@MM + L group (G6) was significantly reduced to 6.48%, demonstrating its ability to improve the tumor's immunosuppressive microenvironment. Notably, a comparison of the 4T1-targeted therapy group (25.8 ± 0.7%, G4) and the CAFs-targeted therapy group (16.8 ± 2.6%, G5) revealed that CAFs depletion significantly reduced the infiltration of MDSCs within the tumor, further validating the aforementioned findings. Therefore, depleting CAFs in solid tumors can significantly improve the tumor immune microenvironment, further promoting a robust anti-tumor immune response and transforming immune "cold" tumors into immune "hot" tumors. Furthermore, analysis of the proportion of Treg cells in tumor tissue revealed that the PDA-Pt@MM + L treatment group (G6) had the lowest Treg ratio of all treatment groups (5.21%). This is consistent with the previously reported cytokine trends associated with CAFs depletion. PDA-Pt@MM demonstrated the strongest clearance, thereby reducing Treg infiltration into tumor tissue. In addition, it can be seen that in the synergistic treatment group (G5) that targets CAFs alone, its Treg ratio was significantly downregulated (13.27%) compared with the synergistic treatment group (G4) that targets 4T1 alone, proving that CAFs are a key factor in maintaining the tumor immunosuppressive microenvironment, and targeted depletion of CAFs can further promote the tumor killing rate.

[0068] 10. In the eighth item, the tumor targeting effect of the nanosystem was evaluated by detecting the fluorescence intensity of the nanosystem in mouse tumors. The results showed that the tumor targeting ability of PDA-Pt@MM was significantly increased compared with the single membrane targeting group. However, PDA-Pt@MM still faces many obstacles from the extracellular matrix components, which limits the large influx and deep penetration of PDA-Pt@MM in solid tumors. Since PDA-Pt@MM can reshape the extracellular matrix by targeting CAFs cells, the reshaping of the extracellular matrix was further detected by tissue fluorescence sectioning. Figure 11 A, Compared with the control group Control + L, the content of Collgen I, the main component of ECM, was significantly reduced in PDA-Pt@MM treated with light, and the tumor tissue became vacuolated and loose, indicating that the physical barrier of solid tumors can be improved after treatment with PDA-Pt@MM treated with light. The level of α-SMA, a marker of CAFs, was also detected. Figure 11 As shown in Figure B, the CAFs-targeted PDA-Pt group (G5) and the dual-targeted PDA-Pt group (G6) significantly reduced the level of CAFs in the tumor, proving that PDA-Pt can kill CAFs through homologous targeting of the cell membrane and reduce its level in tumor tissue. The level of type I collagen in each treatment group was further tested (see Figure 11 C), it can be seen that the collagen level in the final group decreased significantly, proving that PDA-Pt@MM can reduce the level of extracellular matrix components in solid tumors by targeted depletion of CAFs, reduce the complex matrix components of solid tumors, and promote the penetration of the nanosystem.

Claims

1. A method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment, characterized in that: The following steps are involved: (1) PDA nanoparticles were dispersed in ultrapure water, and a K2PtCl4 aqueous solution was added thereto under stirring conditions, followed by condensation and reflux reaction in a water bath to prepare a PDA-Pt asymmetric nanophotothermal agent; (2) The CAFs cell membrane and the breast cancer cell membrane were mixed and ultrasonically fused to prepare a fused membrane. The fused membrane, the PDA-Pt nanophotothermal agent and ultrapure water were mixed and then ultrasonically treated in an ice water bath. The mixture was then passed through a polycarbonate membrane with gradually decreasing pore sizes in sequence and finally centrifuged to prepare.

2. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: PDA nanoparticles were prepared by the following method: dopamine solution was added dropwise to a mixed solution of Tris and anhydrous ethanol in a volume ratio of 4-6:1, stirred for 14-18 hours, and centrifuged to obtain PDA nanoparticles.

3. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: The particle size of the PDA nanoparticles in step (1) is 80-200 nm.

4. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: The mass ratio of PDA nanoparticles to K2PtCl4 is 1:2-7.5, the reflux reaction temperature is 75-85°C, and the reaction time is 20-30h.

5. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: In step (2), the mass ratio of CAFs cell membrane to breast cancer cell membrane is 1:1, and the ultrasonic fusion time is 8-15 min.

6. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: In step (2), the mass ratio of the PDA-Pt asymmetric nanophotothermal agent to the fusion membrane is 1:1-3, the water bath ultrasonic power is 160-200 W, and the ultrasonic treatment time is 2-5 min.

7. The method for preparing a biomimetic photothermal nanomotor for targeted regulation of solid tumor matrix-immunosuppressive microenvironment according to claim 1, characterized in that: The pore sizes of the polycarbonate membrane in step (2) are 800 nm, 400 nm, and 200 nm, respectively.

8. A biomimetic photothermal nanomotor that targets and regulates the solid tumor matrix-immunosuppressive microenvironment, characterized in that: Prepared by the method according to any one of claims 1 to 7.

9. Use of the biomimetic photothermal nanomotor for targeted regulation of the solid tumor stroma-immunosuppressive microenvironment as claimed in claim 8 in the preparation of drugs for treating solid tumors.

10. The use according to claim 9, characterized in that The method of use is intratumoral administration or intravenous injection.

Images