Macrophage membrane-coated pH-responsive bionic nanoparticles as well as preparation method and application thereof

Abstract

The invention relates to the field of biological medicines and nano materials, in particular to a macrophage membrane-coated pH responsive bionic nanoparticle which can target tumors and is used for combination of photothermal therapy and immunotherapy as well as a preparation method and application of the macrophage membrane-coated pH responsive bionic nanoparticle. The bionic nano-drug delivery system disclosed by the invention can specifically target tumor cells and tissues, prolong the residence time of the drug in the bladder, improve the anti-tumor curative effect, enhance systemic immune activation, reduce adverse reactions and effectively inhibit tumor metastasis and relapse, and shows good clinical application potential.

Description

TECHNICAL FIELD

[0001] The present application relates to the fields of biological medicine and nanomaterials, in particular, a macrophage membrane-coated nanoparticle with pH responsiveness, tumor targeting and for photothermal therapy combined with immunotherapy and its preparation method and application. BACKGROUND

[0002] Bladder cancer is one of the most common malignant tumors worldwide, and its high recurrence and metastasis rates are the main challenges in clinical treatment. In recent years, immunotherapy, especially immune checkpoint blockade (such as anti-PD-L1 antibody), has shown great potential in tumor treatment. However, the immunosuppression of the tumor microenvironment and the low efficiency of drug delivery limit its efficacy. Photothermal therapy (PTT) is a local treatment method that uses photothermal agents to generate heat under near-infrared laser irradiation to kill tumor cells. In addition to direct killing of tumors, PTT can induce immunogenic cell death (ICD) in tumor cells, releasing tumor-associated antigens and damage-associated molecular patterns (DAMPs), thereby activating the body's anti-tumor immune response, equivalent to an "in situ tumor vaccine". However, single PTT is difficult to effectively inhibit tumor metastasis and recurrence. Nanomedicine delivery systems can accumulate at tumor sites through the enhanced permeability and retention (EPR) effect, but the passive targeting efficiency is limited. In recent years, cell membrane biomimetic coating technology has provided a new strategy for active targeting of nanomedicines. Among them, macrophages have the ability to naturally migrate to tumor sites, and the proteins expressed on their cell membranes, such as integrins (such as α4, β1), can effectively mediate the targeting of nanoparticles to tumor tissues. Therefore, developing a nanoplatform that can effectively combine PTT with immunotherapy and has the ability of active targeting and controlled release is of great significance for improving the treatment effect of bladder cancer and inhibiting recurrence. SUMMARY

[0003] The present application aims to overcome the shortcomings of the prior art and provide a macrophage membrane-coated pH-responsive biomimetic nanoparticle with good biocompatibility, tumor targeting, pH-responsive drug release, and photothermal-immune synergistic therapy function, as well as its preparation method and application in the preparation of bladder cancer treatment drugs.

[0004] In the first aspect of the present application, a macrophage membrane-coated pH-responsive biomimetic nanoparticle (HSS-PCPDTBT / anti-PDL1@MM) is provided. The biomimetic nanoparticle has a core-shell structure of "drug-loaded core-shell". The core is a polymer nanoparticle loaded with a photothermal agent and an immune checkpoint inhibitor, and the polymer is HA-SD-SA containing Se-Se bonds. The shell is a macrophage membrane. The photothermal agent is a semiconductor particle PCPDTBT, and the immune checkpoint inhibitor is an anti-PD-L1 antibody.

[0005] Furthermore, the core is composed of nanoparticles formed by the self-assembly of polymer long chains HA-SD-SA, loaded with a photothermal agent (semiconductor particles PCPDTBT) and an immune checkpoint inhibitor (anti-PD-L1 antibody, anti-PDL1).

[0006] Furthermore, the long-chain polymer HA-SD-SA is synthesized from hyaluronic acid (HA), selenocysteine ​​(SD), and stearic acid (SA) via an amidation reaction. The Se-Se bonds endow the nanoparticles with pH responsiveness, rapidly breaking down in the slightly acidic environment of tumors (e.g., pH 4.5-6.5), thereby accelerating the release of drugs from within.

[0007] Furthermore, the macrophage membrane is derived from RAW264.7 cells.

[0008] A second aspect of the present invention provides a method for preparing macrophage membrane-coated pH-responsive biomimetic nanoparticles (HSS-PCPDTBT / anti-PDL1@MM) as described above, comprising the following steps:

[0009] Step S1: Synthesize the polymer HA-SD-SA containing Se-Se bonds;

[0010] Step S2, Preparation of nanoparticle core (HSS-PCPDTBT / anti-PDL1): The polymer obtained in step S1 is used to load PCPDTBT and anti-PD-L1 antibody through self-assembly to form nanoparticle core;

[0011] Step S3: Extract macrophage membranes (MM);

[0012] Step S4: Preparation of biomimetic nanoparticles (HSS-PCPDTBT / anti-PDL1@MM): The nanoparticle core and the macrophage membrane are fused together by co-extrusion and ultrasonic treatment to obtain biomimetic nanoparticles.

[0013] Furthermore, step S1 includes the following steps:

[0014] 1) Stearic acid (SA) was dissolved in anhydrous DMSO, and the carboxyl group was activated by adding condensing agent EDC. Then, selenocysteine ​​(SD) was added to react and the SD-SA conjugate was obtained.

[0015] 2) Dissolve hyaluronic acid (HA) in MES buffer, activate the carboxyl group with EDC, and then slowly add DMSO solution of SD-SA to react; after the reaction is complete, precipitate with acetone, wash, and vacuum dry to obtain HA-SD-SA polymer.

[0016] Further, step S2 involves dispersing the polymer HA-SD-SA obtained in step S1 in water, adding PCPDTBT and anti-PD-L1 antibody, and forming a drug-loaded nanoparticle core through hydrophobic-hydrophobic interactions and self-assembly.

[0017] Furthermore, the method for extracting macrophage membranes (MM) in step S3 is as follows: RAW264.7 macrophages are cultured, and after the cells are collected, they are broken up using hypotonic lysis buffer and repeated freeze-thaw cycles. Cell membrane fragments are then purified by differential centrifugation.

[0018] Furthermore, step S4 involves mixing the nanoparticle core obtained in step S2 with the macrophage membrane obtained in step S3, repeatedly extruding the mixture using a micro extruder, and then subjecting it to ultrasonic treatment to ensure that the cell membrane completely coats the surface of the nanoparticles, thereby obtaining the target product.

[0019] In an embodiment of the present invention, the method for preparing the macrophage membrane-coated pH-responsive biomimetic nanoparticles includes the following steps:

[0020] 1) The polymer HA-SD-SA obtained in S1 was dispersed in water, and PCPDTBT and anti-PDL1 antibody were added to form HSS-PCPDTBT / anti-PDL1 nanoparticles (NPs) through self-assembly.

[0021] 2) Mix the NPs with the macrophage membrane extracted from S2, and repeatedly squeeze at least 20 times using a micro extruder (through 400nm and 200nm polycarbonate membranes). Then sonicate in a bath sonicator (40 kHz, 100 W) for 2 minutes to complete the membrane coating, thus obtaining the biomimetic nanoparticles HSS-PCPDTBT / anti-PDL1@MM.

[0022] A third aspect of the present invention provides the use of macrophage membrane-coated pH-responsive biomimetic nanoparticles (HSS-PCPDTBT / anti-PDL1@MM) as described above in the preparation of bladder cancer therapeutic agents.

[0023] In a fourth aspect, the present invention provides the application of macrophage membrane-coated pH-responsive biomimetic nanoparticles (HSS-PCPDTBT / anti-PDL1@MM) as described above in combination with near-infrared laser in the preparation of bladder cancer therapeutic drugs.

[0024] Furthermore, the drug is a combined treatment drug for photothermal therapy and immunotherapy for bladder cancer.

[0025] Furthermore, the immunotherapy described is immune checkpoint blockade therapy.

[0026] Furthermore, the drug can inhibit tumor recurrence and induce immune memory.

[0027] Its mechanisms of action include:

[0028] Targeted delivery and long-lasting retention: Macrophage membranes endow nanoparticles with the ability to actively target tumors and prolong their circulation time in vivo.

[0029] pH-responsive drug release: In the slightly acidic environment of the tumor, polymer chains break down, precisely releasing PCPDTBT and anti-PDL1.

[0030] Photothermal-immune synergistic therapy:

[0031] 1) Under irradiation with 808 nm near-infrared laser, PCPDTBT generates local high heat, which directly kills tumor cells (photothermal therapy).

[0032] 2) Photothermal effects induce immunogenic death (ICD) in tumor cells, releasing signals such as CRT, HMGB1, and ATP, which activate dendritic cells (DCs).

[0033] 3) The synergistic release of anti-PDL1 relieves immunosuppression, promotes the infiltration and function of cytotoxic T cells (CTLs), and inhibits regulatory T cells (Tregs), thereby stimulating a strong systemic anti-tumor immune response, effectively inhibiting the growth of primary tumors and preventing tumor recurrence.

[0034] Compared with the prior art, the advantages of the present invention are as follows:

[0035] 1. This invention provides a novel strategy for treating bladder cancer using biomimetic nanoparticles coated with macrophage membranes, combined with photothermal therapy and immunotherapy. Semiconductor particles PCPDTBT and the immune checkpoint inhibitor anti-PDL1 are loaded into polymer particles via a long polymer chain HA-SD-SA. These polymer particles are pH-responsive, enhancing drug release at the tumor site. RAW264.7 macrophage cell membranes are extracted and coated with HA-SD-SA to form HSS-PCPDTBT / anti-PDL1@MM nanoparticles. This invention innovatively integrates photothermal therapy and immune checkpoint blockade therapy into a single nanoplatform, achieving a synergistic anti-tumor effect greater than the sum of its parts (1+1>2). This invention utilizes the natural targeting properties of macrophage membranes to achieve active targeted drug delivery; and utilizes pH-responsive polymers containing Se-Se bonds to achieve intelligent controlled drug release at the tumor site, improving efficacy and reducing side effects. Both the core materials (HA, SA) and the outer shell (autologous cell membrane) exhibit good biocompatibility and safety, with in vitro and in vivo toxicity studies demonstrating high safety.

[0036] 2. Both in vivo and in vitro experiments have confirmed that the biomimetic nanomedicine delivery system (HSS-PCPDTBT / anti-PDL1@MM) for tumor treatment of the present invention can specifically target tumor cells and tissues, prolong the retention time of drugs in the bladder, improve anti-tumor efficacy, enhance systemic immune activation, and reduce adverse reactions.

[0037] 3. The combined application of photothermal therapy and immune checkpoint blockade therapy can not only treat local tumors, but also produce a "distant effect" by activating the body's immune system, effectively inhibiting tumor metastasis and recurrence, showing good clinical application potential. Attached Figure Description

[0038] Figure 1 Schematic diagram of the preparation process and characterization of HSS-PCPDTBT / anti-PDL1@MM nanoparticles.

[0039] (A) Schematic diagram of the preparation process of HSS-PCPDTBT / anti-PDL1. (B) Representative TEM image. Scale bar: 500 nm. (C) Hydrolysis rate of HSS-PCPDTBT / anti-PDL1 at different pH values ​​(pH 4.7, pH 6.5, pH 7.4). (D) In ​​vitro drug release curve. (E) Schematic diagram of the preparation of MM-NPs by extrusion. (F) Representative TEM image of HSS-PCPDTBT / anti-PDL1@MM. Scale bar: 50 nm. Inset: Magnified TEM image of a single MM-NP. Scale bar: 50 nm. (G) Zeta potential of NPs and MM-NPs analyzed by DLS. (H) Particle size of NPs and MM-NPs analyzed by DLS. (I) Characteristic protein bands of NPs, macrophage membrane-derived vesicles, and MM-NPs. (J) Quantitative analysis of α4 and β4 gray integral values. All experiments were repeated three times (n = 3). P ≤ 0.05, P ≤ 0.01, P ≤ 0.001.

[0040] Figure 2 Biocompatibility and in vitro targeting of nanoparticles were validated.

[0041] (A) Live / dead staining images of different material groups (green: live cells, red: dead cells, scale bar: 100 µm). (B) Quantitative analysis of cell viability of different material groups using the CCK-8 assay. (C) CLSM images showing the targeting of HSS-PCPDTBT / anti-PDL1@MM to MB49 cells (green: FITC, blue: DAPI, scale bar: 20 µm). All experiments were repeated three times (n = 3). P ≤ 0.05, P ≤ 0.01, P ≤ 0.001.

[0042] Figure 3 The photothermal conversion efficiency and in vitro immunogenic cell death induction ability of HSS-PCPDTBT / anti-PDL1@MM nanoparticles.

[0043] (A) Temperature rise curves of different concentrations of PCPDTBT under 808 nm laser irradiation. (B) Quantitative analysis of cell viability after laser irradiation using the CCK-8 assay. (C) Representative flow cytometry images of CRT expression levels. (D) Quantitative analysis of CRT expression levels. (E) Ratio of extracellular ATP concentration to intracellular ATP concentration. P ≤ 0.05, P ≤0.01, P ≤ 0.001.

[0044] Figure 4 The immune activation effect of HSS-PCPDTBT / anti-PDL1@MM nanoparticles on dendritic cells (BMDCs) in vitro.

[0045] (A) Schematic diagram of the Transwell system. (B) Flow cytometry analysis of the proportion of mature BMDCs after different treatments in the Transwell co-culture system. (C) Quantification of BMDC maturation level in the Transwell system experiment (n = 5). (DE) Cytokine content of IFN-γ and TNF-α in the supernatant of BMDCs after different treatments in the Transwell co-culture system (n = 5). P < 0.05, P < 0.01, P < 0.001.

[0046] Figure 5 In vivo targeting and distribution of HSS-PCPDTBT / anti-PDL1@MM nanoparticles.

[0047] (A) Typical in vivo fluorescence images at 6, 12, 24 and 48 hours after tail vein injection. (B) Fluorescence imaging of HSS-PCPDTBT / anti-PDL1@MM in various organs and tumor sites 48 hours after injection.

[0048] Figure 6 The immune activation effect of nanoparticles in vivo (DC maturity).

[0049] (A) Schematic diagram of in vivo anti-tumor immune mechanism assessment; (B) Mouse body temperature rise curve; (C) Maturation level of dendritic cells (DCs) in the tumor draining lymph nodes of mice on day 3 after treatment (n=3). P < 0.05, P < 0.01, P < 0.001.

[0050] Figure 7 The immune activation effect of nanoparticles in vivo (CTL / Treg cell ratio).

[0051] (A) The ratio of cytotoxic T cells to regulatory T cells in the tumor-draining lymph nodes of mice; (B) The ratio of cytotoxic T cells to regulatory T cells in the spleen of mice; (C) Quantitative results of the ratio of CTLs to Tregs in the tumor-draining lymph nodes; (D) Quantitative results of the ratio of CTLs to Tregs in the spleen. P < 0.05, P < 0.01, P < 0.001.

[0052] Figure 8 The immune activation effect of nanoparticles in vivo (IFN-γ secretion).

[0053] (A) Representative scatter plot of CD25+FOXP3+ T cells (regulatory T cells, Tregs) in tumor-bearing spleen; (B) Representative flow cytometry atlas of IFN-γ expression in CD4+ T cells and CD8+ T cells; (C) Proportion of regulatory T cells (Tregs) in tumor-bearing spleen; (D) Percentage of IFN-γ secreted by CD4+ T cells and CD8+ T cells. P < 0.05, P < 0.01, P < 0.001.

[0054] Figure 9 In vivo antitumor efficacy of HSS-PCPDTBT / anti-PDL1@MM nanoparticles combined with photothermal therapy.

[0055] (A) Schematic diagram of anti-tumor effect and tumor re-attack experiment in B16-OVA tumor-bearing mouse model; (B) Volume growth curve of primary tumor; (C) Tumor weight measurement results at the end of treatment; (D) Body weight change curve of mice in different treatment groups; (E) In vivo imaging of mice in different treatment groups; (F) Survival rate of mice under different treatment regimens throughout the experiment. P < 0.05 P < 0.01, P < 0.001.

[0056] Figure 10 Evaluation of the anti-tumor recurrence effect and immune memory of nanoparticles.

[0057] (A) Schematic diagram of immune memory assessment and anti-tumor metastasis experiment; (B) Tumor volume change curve after secondary tumor attack; (C) Representative density map of CD44+CD62L- (effective memory T cells, TEM) and CD44+CD62L+ (central memory T cells, TCM) in CD3+CD4+ and CD3+CD8+ T cells; (D) Quantitative analysis of the ratio of TEM to TCM cells in CD3+CD4+ and CD3+CD8+ T cells. P < 0.01, P < 0.001.

[0058] Figure 11 Infrared and ultraviolet characterization of HSS-PCPDTBT / anti-PDL1 nanoparticles.

[0059] Figure 12 Photothermal conversion efficiency of nanoparticles at different PCPDTBT concentrations.

[0060] Figure 13 Representative live / dead staining images of MB49 cells under laser irradiation.

[0061] Figure 14 Flow cytometry analysis of HSS-PCPDTBT / anti-PDL1@MM-targeted MB49 tumor cells.

[0062] Figure 15 Changes in tumor volume in mice after treatment with different materials.

[0063] Figure 16 Representative H&E staining images of major organs after treatment with different materials. Detailed Implementation

[0064] The specific implementation methods provided by the present invention will be described in detail below with reference to the embodiments.

[0065] The reagents used in the following examples—hyaluronic acid, stearic acid, selenocysteine ​​dihydrochloride, dimethyl sulfoxide, hydrogen peroxide, and N-hydroxysuccinimide—were purchased from Shanghai Sinopharm Group. Anti-mouse CD3 (FITC-labeled), anti-mouse CD4 (APC-labeled), anti-mouse CD8 (Percp / Cyanine 5.5-labeled), anti-mouse CD25 (PE-labeled), CCK-8 assay kits, and ROS detection kits—were purchased from Beyotime Biotechnology Co., Ltd. All other reagents and chemicals were of analytical grade.

[0066] Data processing: Each experiment was repeated at least three times. Data processing and statistical analysis were performed using GraphPad Prism 8.0 software. Results are expressed as mean ± standard deviation. Unpaired two-tailed t-tests were used to determine statistical significance between the two groups. A p-value < 0.05 was considered statistically significant.

[0067] Example 1: Synthesis of polymer HA-SD-SA

[0068] 1) Dissolve 300 mg of stearic acid (SA) in 30 mL of anhydrous DMSO. Then, add 300 mg of EDC dissolved in 6 mL of DMSO to the SA solution and activate at room temperature for 30 minutes. Next, add 200 mg of SD dissolved in 10 mL of DMSO to the activated SA solution and react at room temperature for 6 hours. Dialyze and lyophilize the solution to obtain SD-SA.

[0069] 2) Dissolve 100 mg of hyaluronic acid (HA) in 5 mL of MES buffer. Then, add 100 mg of EDC dissolved in 0.5 mL of MES buffer to the HA solution and activate by stirring at room temperature for 1 hour. Next, slowly add 100 mg of SD-SA dissolved in 30 mL of DMSO to the activated HA solution and react at room temperature for 8 hours. After 8 hours of reaction, precipitate with acetone and wash three times. Then, vacuum dry the reactants for 30 minutes. Subsequently, disperse the powder in distilled water, dialyze the solution, and lyophilize to obtain the final product HA-SD-SA.

[0070] Example 2: Extraction of macrophage membranes (MM)

[0071] RAW264.7 cells were cultured at 2.0 × 10⁻⁶. 7Cells were suspended at a density of 0.25 mM / mL in ice-cold TM buffer (pH 7.4; 10 mM Tris + 1 mM MgCl2) and lysed at least 30 times using a microextruder. Then, 1 M sucrose was added to the cell homogenate to a final sucrose concentration of 0.25 M, and the mixture was centrifuged at 2000 × g for 10 minutes at 4 °C. The supernatant was collected and centrifuged again at 3000 × g for 30 minutes at 4 °C. The precipitate was the cell membrane. The membrane was purified by washing twice with TM buffer containing 0.25 M sucrose.

[0072] Example 3: Preparation of HSS-PCPDTBT / anti-PDL1@MM nanoparticles

[0073] 1) The HA-SD-SA polymer obtained in Example 1 was dispersed in water, and PCPDTBT and anti-PDL1 antibody were added (the molar mass ratio of the three was 10:1:1) to form HSS-PCPDTBT / anti-PDL1 nanoparticles (NPs) through self-assembly.

[0074] 2) Mix the NPs with the macrophage membrane extracted in Example 2 at a molar ratio of 1:1, and repeatedly extrude them at least 20 times using a micro extruder (through 400 nm and 200 nm polycarbonate membranes). Then, sonicate them in a bath sonicator (40 kHz, 100 W) for 2 minutes to complete the membrane coating, thus obtaining HSS-PCPDTBT / anti-PDL1@MM nanoparticles.

[0075] Example 4: Preparation and characterization of HSS-PCPDTBT / anti-PDL1@MM nanoparticles

[0076] The synthesized nanoparticles were resuspended in PBS buffer solutions at different pH values ​​(pH 4.7, pH 6.5, pH 7.4), and their particle size and dispersion were measured using a nanoparticle size analyzer. The in vitro thermogenic properties of the nanomicelles were evaluated using an infrared imaging system. One mL of the nanoparticle aqueous solution was placed in a cuvette and irradiated sequentially with an 808 nm laser (power 2 W / cm²) for 5 minutes. The temperature change of each solution under near-infrared laser irradiation was recorded every 0.5 minutes using a near-infrared thermal imaging camera. One mL of PBS was irradiated with the laser under the same conditions as a negative control.

[0077] like Figure 1 As shown in Figure A, a long-chain polymer HA-SA-SD was synthesized via a simple amidation reaction. Semiconductor particles and anti-PDL1 antibody were encapsulated through hydrophobic-hydrophobic interactions to form self-assembled nanoparticles. Figure 1 B). Due to the presence of Se-Se bonds in the polymer molecules, these nanoparticles exhibit pH-responsive properties. For example... Figure 1As shown in Figure C, HSS-PCPDTBT / anti-PDL1 NPs were dissolved in buffer solutions at different pH values. The results showed that the transmittance of the solution decreased significantly in the buffer solution at pH 4.7, indicating extensive hydrolysis of the long polymer chains. We also investigated drug release from HSS-PCPDTBT / anti-PDL1 NPs at different pH values. Figure 1 As shown in Figure D, drug release reached 82.5% at pH 4.5, while it was only 21.3% at pH 7.2, demonstrating the excellent pH responsiveness of the nanoparticles.

[0078] To synthesize MM-NPs, macrophage membranes derived from the mouse macrophage cell line (RAW264.7 cells) were coated onto ROS-responsive NPs using an extrusion method. Figure 1 E). For example Figure 1 As shown in Figure F, transmission electron microscopy revealed that MM-NPs exhibit a spherical core-shell structure, with each NP encapsulated by a single cell membrane. Dynamic light scattering analysis indicated that the diameter of the nanoparticles increased from ~123 nm to ~156 nm after cell membrane encapsulation. Figure 1 G). The zeta potential of MM-NPs is smaller than that of free NPs ( Figure 1 The H value, consistent with the zeta potential on the macrophage surface, indicates successful macrophage membrane incorporation. Western blot analysis confirmed the presence of key membrane antigens, such as α4, β1, and CD47, on both the macrophage membrane and the surface of MM-NPs, indicating the comparability of the macrophage membrane on MM-NPs with that on macrophages. Figure 1 (I-1J). In addition, due to antigens on the macrophage membrane, macrophage membrane-coated NPs can effectively target tumor sites.

[0079] Example 5: In vitro cytotoxicity and targeting

[0080] MB49 cells in logarithmic growth phase were digested with 0.25% trypsin at a concentration of 1×10⁻⁶. 5 Cells were seeded at a density of 1:1 in 24-well plates. The cells were incubated in a humidified incubator at 37°C and 5% CO2 for 24 hours. Then, FITC-labeled nanomedicine was added to each well of the 24-well plate, and the plates were incubated in the dark for 2–24 hours. At each time point, 500 µL of ready-to-use DAPI staining solution was added to each well of the 24-well plate to stain the cell nuclei for 10 minutes, followed by washing three times with PBS. Finally, coverslips containing cells were inverted onto slides, sealed with an anti-fluorescence quencher, and the cellular uptake of the nanomedicine was observed and photographed under an inverted fluorescence microscope.

[0081] Good biocompatibility is essential for nanomaterials to function in vivo. To evaluate the biotoxicity of HSS-PCPDTBT / anti-PDL1@MM nanoparticles, they were co-cultured with MB49 cells for 24 and 48 hours. Figure 2 As shown in Figure A, the live / dead staining results indicated high cell viability, with no significant cell death observed. The CCK-8 assay further quantified cell viability, showing that cell viability exceeded 95% in all groups. Figure 2 B) indicates that the designed material has excellent biocompatibility.

[0082] After co-culturing HSS-PCPDTBT / anti-PDL1@MM nanoparticles with MB49 cells, their targeting ability was observed using laser confocal microscopy. Figure 2 C and Figure 14 As shown, the green fluorescence gradually increases over time and mainly accumulates in the cytoplasm, indicating that HSS-PCPDTBT / anti-PDL1@MM nanoparticles can target and enter tumor cells.

[0083] Example 6: In vitro photothermal conversion capacity and induction of immunogenic cell death

[0084] Numerous studies have reported that photothermal therapy (PTT) can induce anti-tumor immune responses by triggering immunogenic cell death. Following PTT treatment, necrotic or apoptotic cells release tumor-associated antigens (TAAs) and express damage-associated molecular patterns. DAMPs bind to corresponding receptors or ligands on dendritic cells, activating the differentiation of immature dendritic cells (DCs) into mature phenotypes. Mature DCs further engulf TAAs and present them to T cells. We further evaluated the photothermal conversion capability of HSS-PCPDTBT / anti-PDL1@MM nanoparticles and their ability to induce ICD in tumor cells.

[0085] like Figure 3 As shown in Figure A, the heating capacity of the nanoparticles increases with increasing semiconductor particle concentration. At a PCPDTBT concentration of 1 mg / mL, the nanoparticles raised the temperature by over 32°C within 5 minutes, sufficient to kill tumor cells. Figure 3 B). Therefore, we assessed the secretion of CRT and ATP. (e.g.) Figure 3 As shown in C-3E, HSS-PCPDTBT / anti-PDL1@MM significantly increased the expression of CRT and ATP, inducing higher levels of ICD.

[0086] Example 7: Immune activation effect on BMDCs

[0087] The immune activation effect of nanomicelles on BMDCs under near-infrared laser irradiation was investigated using a 12-well Transwell co-culture system. First, ID8 cells were cultured to the logarithmic growth phase, digested into single-cell suspensions with trypsin, and then cultured at 1×10⁻⁶ wells. 5 Cells were seeded at a density of 2.5 × 10⁶ cells / well in the upper chamber of a Transwell co-culture system. The mixture was gently stirred, and nanomicelles were added to the upper chamber. Incubation was continued for 2 hours. BMDCs cultured to day 10 were collected and seeded at 2.5 × 10⁶ cells / well. 5 Cells were seeded at a density per well in the lower chamber of a 12-well Transwell co-culture system and co-cultured with MB49 cells in the upper chamber, which had been treated with near-infrared laser for 12 hours. Suspended DCs in the lower chamber were collected, centrifuged at 1200 rpm for 5 minutes, and the expression of relevant cytokines in the supernatant was detected. The expression of immune cell-related markers in the cell pellet was detected by flow cytometry.

[0088] Flow cytometry: After near-infrared irradiation, mice in each group were sacrificed, and spleens, inguinal lymph nodes, and axillary lymph nodes were harvested to obtain cell suspensions. Anti-mouse CD3 (FITC-labeled), anti-mouse CD4 (APC-labeled), anti-mouse CD8 (Percp / Cyanine 5.5-labeled), and anti-mouse CD25 (PE-labeled) staining was performed as needed. The cell pellet was resuspended in 200 µL PBS, and the proportions of various immune cells in the spleen and lymph nodes were analyzed using flow cytometry.

[0089] Cytokine detection: Tumor tissue was collected on day 3 post-injection and homogenized in cold PBS supplemented with digestive enzymes to prepare a single-cell suspension. The levels of IFN-γ and TNF-α were detected using an ELISA kit according to the kit instructions (all purchased from Invitrogen).

[0090] Studies have shown that PTT can induce ICD, releasing TAAs and DAMPs, thereby stimulating an immune response. Immune adjuvants can enhance this immune activation. This study confirms that HSS-PCPDTBT / anti-PDL1@MM nanoparticles can induce ICD in MB49 cells under near-infrared laser irradiation. Therefore, we further investigated whether PTT mediated by HSS-PCPDTBT / anti-PDL1@MM nanoparticles, combined with the effect of immune adjuvants, can enhance the immune activation of BMDCs using the Transwell co-culture system.

[0091] like Figure 4 As shown in Figure A, the immunomodulatory effects of each material treatment group were analyzed using an in vitro Transwell assay. Compared with the control group, the proportions of CD86+, CD80+, and CD40+ were significantly increased after laser treatment. Figure 4(B and 4C). In addition, the immune activation effect of HSS-PCPDTBT / anti-PDL1@MM + Laser is superior to that of HSS-PCPDTBT / anti-PDL1 + Laser, which is attributed to the enhanced targeting brought about by macrophage membrane coating.

[0092] Furthermore, we assessed the expression of IFN-γ and TNF-α using ELISA. Figure 4 As shown in Figure D, HSS-PCPDTBT / anti-PDL1@MM nanoparticles significantly increased the levels of these cytokines in the supernatant of BMDCs. Specifically, IFN-γ increased by 7.3-fold and TNF-α increased by 8.4-fold compared to the control group. These results indicate that HSS-PCPDTBT / anti-PDL1@MM nanoparticles, under near-infrared laser irradiation, can synergistically enhance PTT and anti-PDL1-induced immune activation, thereby effectively activating BMDCs and laying the foundation for in vivo anti-tumor effects.

[0093] Example 8: In vivo targeting and distribution of HSS-PCPDTBT / anti-PDL1@MM + Laser nanoparticles

[0094] An orthotopic bladder tumor model was established in C57BL / 6 mice by injecting luciferase-labeled MB49-Luc cells into the bladder. When the tumor volume reached 50 mm³, FITC-labeled nanoparticles were injected. Mice were anesthetized with isoflurane at 2, 12, 24, and 48 hours post-injection, and near-infrared fluorescence imaging was performed using a small animal in vivo imaging system (excitation wavelength 745 nm, emission wavelength 840 nm). Forty-eight hours post-injection, mice were sacrificed, and tumor tissue and major organs (heart, liver, spleen, lungs, and kidneys) were isolated for in vivo near-infrared fluorescence imaging to observe the distribution of the nanomedicine in vivo.

[0095] To further verify whether HSS-PCPDTBT / anti-PDL1@MM nanoparticles can specifically target bladder tumors in vivo, in vivo imaging was used to observe the localization of the nanoparticles after tail vein injection. Figure 5As shown in Figure A, strong fluorescence signals were observed in the liver and tumor sites within 2 hours after injection of HSS-PCPDTBT / anti-PDL1 and HSS-PCPDTBT / anti-PDL1@MM nanoparticles. Over time, 12 hours after injection of HSS-PCPDTBT / anti-PDL1@MM nanoparticles, significant fluorescence signals were observed at the tumor sites. Although HSS-PCPDTBT / anti-PDL1 also accumulated at the tumor sites, its near-infrared fluorescence intensity was significantly weaker than that of HSS-PCPDTBT / anti-PDL1@MM nanoparticles. Furthermore, HSS-PCPDTBT / anti-PDL1 nanoparticles showed strong accumulation outside the tumor area. Forty-eight hours after injection, strong near-infrared fluorescence signals were still observed at the tumor sites in mice injected with HSS-PCPDTBT / anti-PDL1@MM nanoparticles, indicating that they have specific targeting ability for subcutaneous ID8 tumors and remain within the tumor for a long time.

[0096] To further investigate the in vivo distribution of nanomedicines, mice were sacrificed 48 hours after administration, and tumors and major organs (heart, liver, spleen, lungs, and kidneys) were isolated for in vitro near-infrared fluorescence imaging. Figure 5 As shown in Figure B, the nanomedicine is mainly metabolized by the liver, with some distribution in the spleen, lungs, and kidneys. Furthermore, strong fluorescence signals were observed at the tumor site, indicating that the HSS-PCPDTBT / anti-PDL1@MM nanoparticles can actively target bladder tumors in vivo and remain within the tumor tissue for an extended period.

[0097] Example 9: In vivo immune activation of HSS-PCPDTBT / anti-PDL1@MM

[0098] like Figure 6 As shown in Figure A, the expression of immune activation-related factors in mice on day three after treatment was analyzed to determine whether the immune system was activated. First, we evaluated the photothermal effects of each group of materials in vivo. For example... Figure 6 As shown in Figure B, PCPDTBT could raise the temperature to 55°C within 5 minutes, sufficient to kill tumor cells and induce ICD. We further evaluated the maturation of DCs in the spleen of mice on day 3 post-treatment. The results are as follows... Figure 6As shown in Figure C, compared with the control group, the HSS-PCPDTBT@MM +Laser group only slightly increased the proportion of mature DCs. In contrast, the HSS-PCPDTBT / anti-PDL1 and HSS-PCPDTBT / anti-PDL1@MM nanoparticle treatment groups significantly promoted the maturation of DCs in TDLNs, indicating that effective in vivo photothermal therapy and immune activation can induce an anti-tumor immune response. Notably, the maturation rate in the HSS-PCPDTBT / anti-PDL1@MM nanoparticle group was significantly higher than that in the HSS-PCPDTBT / anti-PDL1 group. This suggests that HSS-PCPDTBT / anti-PDL1@MM +Laser can effectively stimulate the body to produce an anti-tumor immune response.

[0099] Example 10: Systemic immune activation of HSS-PCPDTBT / anti-PDL1@MM

[0100] Cytotoxic T cells can directly kill tumor cells, while helper T cells play an important role in regulating adaptive immunity, and regulatory T cells can suppress anti-tumor immune responses. We dissected TDLNs and spleens of mice in each group, ground them into single-cell suspensions, and analyzed the ratio of cytotoxic T cells to Treg cells to further analyze the systemic anti-tumor immune response. Figure 7 As shown in Figure A, compared with the control group, the photothermal therapy alone increased the proportion of TDLNs and Treg cells in the spleen, which may explain the rapid relapse of tumor-bearing mice after PTT therapy alone. Anti-PD-L1, an FDA-approved immune checkpoint inhibitor for cancer treatment, can downregulate the proportion of TDLNs and Treg cells in the spleen. Through the immunosuppressive effect of anti-PD-L1, the HSS-PCPDTBT / anti-PDL1@MM + Laser treatment group significantly reduced the proportion of TDLNs and Treg cells in the spleen. These results indicate that the HSS-PCPDTBT / anti-PDL1@MM photothermal therapy combined with an immune checkpoint inhibitor can significantly promote CTL infiltration in tumor tissue and reduce Treg cell infiltration. Furthermore, it can stimulate the body to produce a systemic anti-tumor immune response. In conclusion, the combined application can effectively stimulate the body to produce an anti-tumor immune response.

[0101] Regulatory T cells are a special subset of CD4+ helper T cells that play a crucial role in suppressing immune cell function and inhibiting anti-tumor immune responses. For example... Figure 8As shown in A and 8C, the percentage of Tregs in splenic CD4+ T cells was 6.5-fold higher in the control group than in the HSS-PCPDTBT / anti-PDL1@MM + Laser group. Therefore, HSS-PCPDTBT / anti-PDL1@MM + Laser significantly reduces tumor-associated immunosuppression.

[0102] IFN-γ is a cytokine secreted by activated CD8+ T cells and CD4+ Th1 cells, playing a crucial role in tumor immunotherapy. IFN-γ can directly exert cytotoxic effects on tumor cells, enhance MHC-I expression, and increase the sensitivity of tumor cells to CD8+ T cell-mediated lysis. We quantified the number of IFN-γ-producing CD4+ T and CD8+ T cells using ELISA and flow cytometry. Figure 8 As shown in B and 8D, the proportion of CD4+ T and CD8+ T cells producing IFN-γ was significantly higher in the combination therapy group than in other groups. Furthermore, HSS-PCPDTBT / anti-PDL1@MM+Laser significantly increased IFN-γ production compared to HSS-PCPDTBT / anti-PDL1+Laser. These results indicate that HSS-PCPDTBT / anti-PDL1@MM+Laser can activate antigen-specific CD8+ CTLs and CD4+ Th1 cells.

[0103] Example 11: Evaluation of in vivo antitumor effect

[0104] An orthotopic bladder tumor model was established using luciferase-labeled MB49 cells. When the tumor volume reached 50-100 mm³, tumor-bearing mice of uniform size were randomly divided into 6 groups of 6 mice each. Mice were injected via tail vein with PBS, HSS-anti-PDL1@MM, PCPDTBT / anti-PDL1@MM, HSS-PCPDTBT@MM, HSS-anti-PDL1@MM, and HSS-PCPDTBT / anti-PDL1@MM. Twenty-four hours after injection, mice were anesthetized with isoflurane and the tumor site was irradiated with an 808 nm laser (2 W / cm²) for 5 minutes. The length and width of the tumor were measured every 3 days, and the tumor volume was calculated. On day 6 after near-infrared laser irradiation, 3 mice were randomly selected from each treatment group, sacrificed, and the tumor tissue was dissected. The tissue was fixed in 4% paraformaldehyde for 24 hours, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. The remaining mice were observed, and the tumor volume was recorded.

[0105] like Figure 9 As shown in Figure A, we further analyzed the changes in tumor volume in each treatment group to assess its efficacy. Figure 9B shows that, compared with the control group, the tumor inhibition effect was not significant in any group that did not receive laser irradiation; however, after laser treatment, tumor growth was significantly inhibited. Among them, the HSS-PCPDTBT / anti-PDL1@MM + Laser group exhibited the strongest tumor inhibition effect. Tumors were removed and weighed on day 28, showing (…). Figure 9 C), the combined treatment group (HSS-PCPDTBT / anti-PDL1@MM +Laser) had the lightest tumor weight. Furthermore, the body weight of mice in all groups did not change significantly during treatment, indicating that the combined treatment regimen did not produce significant toxic side effects in mice. Figure 9 D and Figure 16 ).

[0106] Further in vivo imaging in mice showed that the HSS-PCPDTBT / anti-PDL1@MM + Laser group had the lowest fluorescence intensity in the bladder region, indicating that it had the best tumor suppression effect. Figure 9 E). Notably, in the HSS-PCPDTBT@MM + Laser group, fluorescence intensity initially increased and then decreased, rebounding on day 14. This further illustrates that while photothermal therapy alone can inhibit tumor growth, it cannot produce a durable anti-tumor effect. Analysis of the survival curves for each group revealed ( Figure 9 In the control group, all mice died within 24 days, while all mice in the HSS-PCPDTBT / anti-PDL1@MM + Laser group survived, significantly prolonging the survival time of the mice.

[0107] Example 12: Evaluation of anti-tumor recurrence effect

[0108] On day 28 of the initial treatment, cured tumor-bearing mice and age-matched control mice were challenged again with MB49-Luc cells (second challenge). Tumor volume was measured in all mice every other day. On day 56, all mice were sacrificed, and tumor tissue was collected for immune-related assays. To assess biosafety in the mice, major organs (heart, liver, spleen, lungs, and kidneys) were collected from all groups of mice on day 56 for H&E staining for histological analysis.

[0109] A key feature of the immune system is immune memory, which enables the body to rapidly trigger an immune response to clear antigens upon secondary attacks. Therefore, this study further investigated whether HSS-PCPDTBT / anti-PDL1@MM photothermal combined with immunotherapy could inhibit bladder tumor recurrence. The experimental design is as follows: Figure 10 As shown in Figure A, MB49 cells were inoculated into the bladder to simulate tumor recurrence, and changes in secondary tumor volume were monitored.

[0110] like Figure 10As shown in B, compared with the control treatment group, both the HSS-PCPDTBT / anti-PDL1 + Laser treatment group and the HSS-PCPDTBT / anti-PDL1@MM + Laser combined treatment group inhibited tumor recurrence in tumor-bearing mice, but the HSS-PCPDTBT / anti-PDL1@MM + Laser treatment group was significantly more effective in inhibiting tumor growth.

[0111] Memory T cells can be divided into central memory T cells and effector memory T cells. TCM cells are mainly distributed in secondary lymphoid tissues and provide protection through clonal expansion, differentiation, and transport in response to antigen stimulation. TEM cells, on the other hand, exist in both lymphoid and non-lymphoid tissues and can immediately induce immune protection by producing cytokines such as TNF-α and IFN-γ. Flow cytometry was used to detect the expression of TEM and TCM cells in the spleen. Figure 10 C). For example Figure 10 As shown in Figure D, compared with the control group, the proportions of TEM and TCM cells were increased in both the HSS-PCPDTBT / anti-PDL1 + Laser treatment group and the HSS-PCPDTBT / anti-PDL1@MM + Laser combination treatment group, and the proportions in the HSS-PCPDTBT / anti-PDL1@MM + Laser combination treatment group were significantly higher than those in the HSS-PCPDTBT / anti-PDL1 + Laser treatment group. These results indicate that the HSS-PCPDTBT / anti-PDL1@MM + Laser combination therapy can significantly enhance the immune memory effect.

[0112] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A pH-responsive biomimetic nanoparticle coated with a macrophage membrane, characterized in that, The biomimetic nanoparticles have a core-shell structure, wherein the core is a polymer nanoparticle loaded with a photothermal agent and an immune checkpoint inhibitor, and the polymer is HA-SD-SA containing Se-Se bonds; the shell is a macrophage membrane; the photothermal agent is a semiconductor particle PCPDTBT; and the immune checkpoint inhibitor is an anti-PD-L1 antibody.

2. The biomimetic nanoparticles according to claim 1, characterized in that, The core is composed of nanoparticles formed by the self-assembly of a polymer long chain HA-SD-SA, loaded with a photothermal agent and an immune checkpoint inhibitor; the polymer long chain HA-SD-SA is synthesized from hyaluronic acid, selenocysteine ​​and stearic acid through an amidation reaction.

3. The biomimetic nanoparticles according to claim 1, characterized in that, The macrophage membrane described was derived from RAW264.7 cells.

4. A method for preparing pH-responsive biomimetic nanoparticles coated with macrophage membranes as described in claim 1, characterized in that, Includes the following steps: Step S1: Synthesize the polymer HA-SD-SA containing Se-Se bonds; Step S2, Preparation of nanoparticle core HSS-PCPDTBT / anti-PDL1: The polymer obtained in step S1 is used to load PCPDTBT and anti-PD-L1 antibody through self-assembly to form a nanoparticle core; Step S3: Extract macrophage cell membranes; Step S4: Preparation of biomimetic nanoparticles HSS-PCPDTBT / anti-PDL1@MM: The nanoparticle core and the macrophage membrane are fused together by co-extrusion and ultrasonic treatment to obtain biomimetic nanoparticles.

5. The preparation method according to claim 4, characterized in that, Step S1 includes the following steps: 1) Stearic acid (SA) was dissolved in anhydrous DMSO, and the carboxyl group was activated by adding condensing agent EDC. Then, selenocysteine ​​(SD) was added to react and the SD-SA conjugate was obtained. 2) Dissolve hyaluronic acid (HA) in MES buffer, activate the carboxyl group with EDC, and then slowly add DMSO solution of SD-SA to react. After the reaction is complete, precipitate with acetone, wash, and vacuum dry to obtain HA-SD-SA polymer.

6. The preparation method according to claim 4, characterized in that, Step S2 involves dispersing the polymer HA-SD-SA obtained in step S1 in water, adding PCPDTBT and anti-PD-L1 antibody, and forming a drug-loaded nanoparticle core through hydrophobic-hydrophobic interactions and self-assembly.

7. The preparation method according to claim 4, characterized in that, The method for extracting macrophage membranes in step S3 is as follows: RAW264.7 macrophages are cultured, and after the cells are collected, they are broken up using hypotonic lysis buffer and repeated freeze-thaw cycles. Cell membrane fragments are then obtained by differential centrifugation.

8. The preparation method according to claim 4, characterized in that, Step S4 involves mixing the nanoparticle core obtained in step S2 with the macrophage membrane obtained in step S3, repeatedly extruding the mixture using a micro extruder, and then subjecting it to ultrasonic treatment to ensure that the cell membrane completely coats the surface of the nanoparticles, thus obtaining the target product.

9. The use of the macrophage membrane-coated pH-responsive biomimetic nanoparticles as described in claim 1 in the preparation of bladder cancer therapeutic drugs.

10. The application of a pH-responsive biomimetic nanoparticle coated with a macrophage membrane as described in claim 1, combined with near-infrared laser, in the preparation of a bladder cancer therapeutic drug.

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