Immunogenic cell death enhanced nano-vesicle hydrogel and application thereof

Abstract

The invention relates to the technical field of biological medicine, in particular to immunogen cell death enhanced nano-vesicle hydrogel and application thereof. The invention provides a 3D-printable multifunctional synergistic immunogen cell death enhanced nano-vesicle hydrogel, which is characterized in that SerMA hydrogel is used as a matrix, cell membrane nano-vesicles (N (at) ILO) loaded with a photothermal agent IR1061, a chemotherapeutic drug OXA and a tumor acidic microenvironment regulator Lon are encapsulated, initial tumor immunogen cell death is triggered through a photothermal effect, and the tumor immunogen cell death enhanced nano-vesicle hydrogel can be used for 3D printing. OXA further enhances the ICD effect, Lon regulates the acidic tumor microenvironment to improve the T cell activity, and postoperative multi-mode synergistic anti-tumor treatment is achieved; meanwhile, the hydrogel also has excellent photocuring 3D printing capability, can be used for individually constructing a filling structure matched with a postoperative defect area, has a wide application prospect, and provides a new strategy and theoretical basis for postoperative comprehensive treatment of choroidal melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to immunogenic cell death-enhanced nanovesicle hydrogels and their applications. Background Art

[0002] Choroidal melanoma (CM) is the most common primary intraocular tumor in adults. Currently, surgical resection remains the main treatment method for CM. Especially when local lesions cannot be controlled by chemotherapy or radiotherapy, it is still regarded as the "gold standard" for treatment. However, there is still a high recurrence rate and metastasis risk after surgery, especially in the context of microscopic residual lesions at the resection margin and tumor cell metastasis. In addition, the eye has a delicate and complex organizational structure, and surgical operations often cause inevitable tissue defects, resulting in damage to the integrity and function of the eyeball, further affecting the patient's quality of life after surgery. Currently, a variety of postoperative fillers or drugs for ocular tumors have been developed, which can be used to assist in preventing tumor recurrence and metastasis after surgery.

[0003] However, the anti-recurrence strategies of existing postoperative fillers for ocular tumors are single and fail to synergistically enhance immunogenicity; the functions of drug delivery systems are also very single and cannot achieve multi-mechanism synergy; at the same time, the glycolysis of residual tumor cells after surgery is hyperactive, resulting in the accumulation of lactic acid in the tumor microenvironment, inhibiting DC antigen presentation and CD8⁺ T cell activity, and lacking metabolic intervention means, resulting in the immunosuppressive microenvironment weakening the ICD effect; moreover, traditional fillers or drugs cannot match the complex ocular defect morphology and do not integrate immunomodulatory functions, leading to postoperative recurrence. Therefore, developing a multifunctional comprehensive treatment strategy with both the ability to inhibit tumor recurrence and personalized filling of tissue defects has become a key problem in current clinical treatment. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide immunogenic cell death-enhanced nanovesicle hydrogels and their applications.

[0005] The present invention provides drug-loaded vesicles, including a photothermal agent, an ICD-inducing chemotherapeutic drug, a glycolysis inhibitor, and cell vesicles.

[0006] The photothermal agent includes IR1061, IR780, ICG, IR210, gold nanoparticles, copper nanoparticles, Prussian blue nanoparticles, black phosphorus nanosheets, and / or MXene nanosheets.

[0007] The ICD-inducing chemotherapeutic drug includes: oxaliplatin, doxorubicin, epirubicin, daunorubicin, idarubicin, cisplatin, carboplatin, paclitaxel, docetaxel, bortezomib, cyclophosphamide, and / or 5-fluorouracil;

[0008] The glycolysis inhibitors include: lonidamine, 2-deoxy-D-glucose, 3-bromopyruvic acid, D-mannoheptulose, and / or Lon-TK.

[0009] In the present invention, the cell vesicles include extracellular vesicles and / or cell membrane vesicles, etc. The extracellular vesicles include exosomes, microvesicles, and / or apoptotic bodies, etc.; the cell membrane vesicles include human periodontal ligament stem cell (PDLSCs) membrane vesicles. In a specific embodiment of the present invention, it is the human periodontal ligament stem cell (PDLSCs) membrane vesicles.

[0010] Furthermore, in the drug-loaded vesicles of the present invention, the mass ratio of the photothermal agent, ICD-inducing chemotherapeutic drug, glycolysis inhibitor, and cell vesicles is 1:1:1:(1-4), specifically 1:1:1:3.

[0011] The concentration of the photothermal agent is 10-30 μg / mL.

[0012] The present invention provides a hydrogel, which includes silk protein, a photoinitiator, and the drug-loaded vesicles of the present invention;

[0013] The silk protein includes chemically modified silk protein, etc.; in the present invention, specifically, it is methacrylated sericin; the methacrylated sericin is obtained by subjecting the four-corner protein obtained from natural cocoons to a methacrylation reaction to obtain the methacrylated sericin;

[0014] The photoinitiator includes: lithium phenyl-2,4,6-trimethylbenzoylphosphinate, TPO-L, and / or a cyanine / iodonium salt system, specifically lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP).

[0015] The present invention has optimized the composition components and ratios of the hydrogel. Specifically, the silk protein, photoinitiator, photothermal agent, ICD-inducing chemotherapeutic drug, glycolysis inhibitor, and cell vesicles have been screened and recombined as shown above; the test results show that the hydrogel prepared with methacrylated sericin, the photoinitiator LAP, the photothermal agent IR1061, the ICD-inducing chemotherapeutic drug oxaliplatin (OXA), the glycolysis inhibitor lonidamine, and human periodontal ligament stem cell membrane vesicles has the best stability; and through comparison of the tumor immunity and tumor inhibition test results of the above-mentioned single components (such as only OXA or lonidamine alone), combinations of some components (such as the combinations of OXA or lonidamine with methacrylated sericin respectively), and the hydrogel prepared by the present invention, it shows that the hydrogel of the components of the present invention has the best effect, can form continuous anti-tumor immunity, achieve multi-mechanism synergy, enhance the ICD effect, and can achieve personalized adaptation.

[0016] Furthermore, the mass ratio of the photothermal agent, ICD-inducing chemotherapeutic drug, glycolysis inhibitor, and cell membrane vesicles in the drug-loaded vesicles of the hydrogel of the present invention has been optimized, and their ratio is 1:1:1:(1-4); in the specific embodiments of the present invention, 1:1:1:1, 1:1:1:2, 1:1:1:3, and 1:1:1:4 were tried; the test results showed that the performance was the best and the most stable at 1:1:1:3.

[0017] Meanwhile, in the multifunctional drug-loaded hydrogel of the present invention, the concentration of the photothermal agent has been optimized; in the embodiments of the present invention, the concentration of the photothermal agent is 5-30 μg / mL. The test results of the present invention showed that the photothermal agent IR1061 with a concentration of 10 μg / mL rapidly increased the temperature from the initial 25.6 °C to 49.8 °C within 5 minutes, which has reached the temperature threshold for inducing thermal damage or even apoptosis of tumor cells. Therefore, the concentration of the photothermal agent is preferably 10-30 μg / mL.

[0018] The present invention provides a method for preparing the hydrogel, which is to mix the sericin, photoinitiator, and drug-loaded vesicles to obtain the hydrogel.

[0019] The present invention provides the application of at least one of the following I) - III) in the preparation of products for tumor wound filling and / or prevention and treatment:

[0020] I), the drug-loaded vesicles of the present invention;

[0021] II), the hydrogel of the present invention;

[0022] III), the hydrogel prepared by the preparation method of the present invention.

[0023] The present invention provides a product for tumor wound filling and / or prevention and treatment, which is characterized in that the raw materials include at least one of i) - iii) shown below:

[0024] i), the drug-loaded vesicles of the present invention;

[0025] ii), the hydrogel of the present invention;

[0026] iii), the hydrogel prepared by the preparation method of the present invention.

[0027] Furthermore, the tumor includes ocular tumors, specifically choroidal melanoma.

[0028] Preventing recurrence and filling tissue defects after choroidal melanoma surgery are major challenges in current clinical treatment. In this study, a multifunctional composite hydrogel system, SerMA-N@ILO, with 3D printing ability and immunogenic cell death (ICD) induction function, was designed and constructed for local treatment after choroidal melanoma resection. This system uses silk sericin modified with methacrylic anhydride (SerMA) as the matrix, encapsulating nanocell membrane vesicles (N@ILO) loaded with the photothermal reagent IR1061, the chemotherapeutic drug oxaliplatin (OXA), and the lactate metabolism inhibitor lonidamine (Lon). In vitro and in vivo experimental results show that the SerMA-N@ILO hydrogel can significantly enhance the ICD effect of tumor cells, promote the externalization of calreticulin (CRT) on the cell membrane and the secretion of high-mobility group box 1 (HMGB1), thereby increasing the maturation rate of dendritic cells (DCs) by 2.4 times compared with the control group, and significantly enhancing the specific infiltration of CD4⁺ T cells (3.1-fold increase) and CD8⁺ T cells (6.8-fold increase) into tumor tissues. In addition, this hydrogel can alleviate the acidic microenvironment of tumor cells, reshape the immunosuppressive tumor microenvironment, and further enhance the local anti-tumor immune response. More importantly, this hydrogel has photocurable 3D printing properties, enabling personalized customization for precise filling of complex postoperative tissue defects. In summary, the SerMA-N@ILO hydrogel exhibits great therapeutic potential for inhibiting recurrence after choroidal melanoma surgery by synergistically inducing the ICD effect and microenvironment regulation.

[0029] The present invention provides a 3D printable multifunctional and synergistic immunogenic cell death-enhanced nanovesicle hydrogel. The hydrogel uses a SerMA hydrogel as the matrix, encapsulating nanocell membrane vesicles (N@ILO) loaded with the photothermal agent IR1061, the chemotherapeutic drug OXA, and the microenvironment regulator Lon. The initial ICD is triggered by the photothermal effect, OXA further enhances the ICD effect, and Lon regulates the acidic tumor microenvironment (TME) to promote T cell activity, achieving multimodal synergistic anti-tumor treatment after surgery. At the same time, this hydrogel also has excellent photocurable 3D printing ability and can be used for personalized construction of filling structures matching the postoperative defect area, with broad application prospects, providing a new strategy and theoretical basis for the comprehensive treatment of choroidal melanoma after surgery. Brief Description of the Drawings

[0030] Figure 1 Showing specific immunofluorescence staining of human periodontal ligament stem cells (hPDLSCs);

[0031] Figure 2Preparation and physicochemical property characterization of N@ILO. Among them, a is the transmission electron microscope (TEM) image of N@ILO nanoparticles, scale bar: 100 nm; b is the hydrodynamic diameter distribution of N@ILO analyzed by dynamic light scattering (DLS); c is the quantitative analysis of the concentration and size distribution of N@ILO in aqueous suspension by nanoparticle tracking analysis (NTA); d is the thermal imaging of H2O and N@ILO formulations with different IR1061 loadings under 1064 nm laser irradiation (1.0 W / m 2 ), e is the time-dependent temperature rise curve of N@ILO suspension under 1064 nm laser irradiation with different power densities (0.5 - 1.5 W / m 2 ); f is the photostability evaluation through four consecutive laser on / off cycles, showing the thermal cycling performance of N@ILO;

[0032] Figure 3 Longitudinal monitoring of the hydrodynamic diameter of N@ILO by dynamic light scattering (DLS) over 9 days;

[0033] Figure 4 Visible-near-infrared (Vis-NIR) absorption spectrum of free IR1061 in aqueous solution;

[0034] Figure 5 Quantitative calibration curve of IR1061 determined by high performance liquid chromatography (HPLC) (wavelength λ = 808 nm, correlation coefficient R² > 0.99);

[0035] Figure 6 Standard curve of lonidamine (Lon) quantitatively analyzed by high performance liquid chromatography (HPLC);

[0036] Figure 7 Photothermal heating curves of N@ILO suspensions with different IR1061 loadings under 1064 nm laser irradiation (1.0 W / m 2 );

[0037] Figure 8 Lactic acid (LA) content in B16F10 cells determined by enzymatic colorimetry;

[0038] Figure 9In vitro cell evaluation of N@ILO; among them, a is to evaluate the cytotoxicity of N@ILO containing different concentrations of IR1061; b is the live / dead cell staining of B16F10 cells under different treatment conditions, scale bar: 100 μm; c is to analyze the apoptosis of B16F10 cells in different treatment groups by Annexin V-FITC / propidium iodide (PI) flow cytometry; in the figure, I: blank; II: IR1061; III: Lon; IV: OXA; V: N@ILO; VI: IR1061(+); VII: N@ILO(+);

[0039] Figure 10 Study on the in vitro immunogenic cell death (ICD) effect; among them, a is to perform immunofluorescence analysis on B16F10 cells treated with Blank (control group), IR1061, Lon, OXA, N@ILO, IR1061(+) and N@ILO(+) to detect the exposure of calreticulin (CRT) on the cell surface, scale bar: 50 μm; b is to perform immunofluorescence staining experiments on B16F10 cells in different treatment groups (Blank, IR1061, Lon, OXA, N@ILO, IR1061(+) and N@ILO(+)) to detect the release of high-mobility group protein B1 (HMGB1), scale bar: 50 μm;

[0040] Figure 11 Synthesis of SerMA-N@ILO hydrogel and characterization of its physicochemical properties; among them, a is a digital image recording the sol-gel transition process of SerMA-N@ILO hydrogel; b is the photopolymerization rheological curve of SerMA-N@ILO hydrogel under 405 nm light irradiation; c is a representative scanning electron microscope (SEM) image of the microstructure of SerMA-N@ILO hydrogel; d is a high-fidelity three-dimensional printed construct manufactured by processing the precursor solution of SerMA-N@ILO hydrogel through a digital light processing micro 3D printing system (Magic Square, Shenzhen, nanoArchS140);

[0041] Figure 12 Computer-aided design (CAD) model designed using SolidWorks software;

[0042] Figure 13Demonstration of the in vivo anti-tumor treatment efficacy evaluation of SerMA-N@ILO; where, a is the tumor volume progression curve of different treatment groups; b is the quantitative analysis of the resected tumor weight after treatment; c is the body weight change trajectory of tumor-bearing mice in the treatment group; d is the level of the cytokine tumor necrosis factor-α (TNF-α) in the serum of mice under different treatment regimens by enzyme-linked immunosorbent assay (ELISA); e is the level of the cytokine interferon-γ (IFN-γ) in the serum of mice under different treatment regimens by ELISA; f is the level of the cytokine interleukin-6 (IL-6) in the serum of mice under different treatment regimens by ELISA; g is the representative hematoxylin-eosin (H&E) staining and TUNEL immunofluorescence staining of tumor tissues, scale bar: 100 μm; in the figure, I: control group; II: SerMA-IR1061; III: SerMA-Lon; IV: SerMA-OXA; V: SerMA-N@ILO; VI: SerMA-IR1061(+); VII: SerMA-N@ILO(+);

[0043] Figure 14 Representative photographic records of tumors resected from different treatment groups at the treatment endpoint (day 16 after treatment).

[0044] Figure 15 Demonstration of the histopathological evaluation (hematoxylin-eosin staining, H&E staining) of major organs (heart, liver, spleen, lung, kidney) in the control group and the SerMA-N@ILO treatment group.

[0045] Figure 16 Demonstration of the complete blood count (CBC) analysis of tumor-bearing mice in the control group and the SerMA-N@ILO treatment group.

[0046] Figure 17 Demonstration of the quantitative analysis of interleukin-10 (IL-10) in the serum of mice receiving different treatment regimens by enzyme-linked immunosorbent assay (ELISA).

[0047] Figure 18Analysis of anti-tumor immune mechanisms; wherein, a is the quantitative analysis of the expression of dendritic cell (DC) maturation markers (CD86 / CD80) in tumor-infiltrating immune cells of different treatment groups by flow cytometry; b is the quantitative analysis of tumor-infiltrating lymphocytes by flow cytometry: CD4+ T cells and CD8+ T cells; c is the statistical analysis of tumor-specific T cell infiltration: the proportion of CD4+ T cells; d is the statistical analysis of tumor-specific T cell infiltration: the proportion of CD8+ T cells; e is the determination of lactic acid (LA) content in tumor lysates by enzymatic method; f is the multi-parameter tumor tissue analysis: immunofluorescence staining of CD8+ T cells, and detection of calreticulin (CRT) and high-mobility group protein B1 (HMGB1) by immunohistochemistry. Statistical significance was calculated by ordinary one-way analysis of variance (ANOVA) and Tukey's test, **P < 0.01; ***P < 0.001; In the figure: I: control group; II: SerMA-IR1061; III: SerMA-Lon; IV: SerMA-OXA; V: SerMA-N@ILO; VI: SerMA-IR1061(+); VII: SerMA-N@ILO(+);

[0048] Figure 19 Quantitative analysis of dendritic cell (DC) maturation markers (CD80+ / CD86+) in different treatment groups by flow cytometry;

[0049] Figure 20 Multi-dimensional transcriptomic analysis of anti-tumor immune mechanisms; wherein, a is the principal component analysis (PCA) comparing the transcriptional profiles of the control group and the test group (SerMA-N@ILO(+) group or Test group); b is the Pearson correlation coefficient heatmap evaluating the global gene expression similarity between experimental groups; c is the Venn diagram between the control group and the test group; d is the volcano plot showing the differential gene expression comparing the control group and the test group; e is the heatmap showing the differential expression of immune-related genes involved in the "immune response" pathway; Kyoto Encyclopedia of Genes and Genomes (KEGG) (f) and Gene Ontology (GO) pathway enrichment of differentially expressed genes, |Fold change| > 1.2, p < 0.05;

[0050] Figure 21 Venn diagram analysis of the common and unique differentially expressed genes (DEGs) between the control group and the SerMA-N@ILO treatment group;

[0051] Figure 22 Bar chart showing the statistical count of differential gene expression between the control group and the SerMA-N@ILO group;

[0052] Figure 23 Show Figure 20 Magnified view of e in

[0053] Figure 24 Shown Figure 20 Magnified view of f in;

[0054] Figure 25 Shown Figure 20 Magnified view of g in. Detailed implementation manner

[0055] The present invention provides an immunogenic cell death-enhanced nanocapsule hydrogel and its application. Those skilled in the art can draw on the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and those related can obviously make changes or appropriate changes and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0056] The hydrogel of the present invention (SerMA hydrogel composite system (SerMA-N@ILO)) solves the problem of insufficient induction of single-mechanism ICD: IR1061 (photothermal) triggers the initial ICD effect, OXA (chemotherapy) further amplifies the release of DAMPs (CRT / HMGB1), and Lon inhibits lactate efflux to reverse the acidic microenvironment. The three work together to increase the DC maturation rate by 2.4 times (32.7% vs 13.6%) and increase the infiltration of CD8⁺ T cells by 6.8 times (22.6% vs 3.3%).

[0057] The hydrogel (SerMA hydrogel composite system (SerMA-N@ILO)) can achieve personalized 3D printing; the hydrogel of the present invention uses methacrylic anhydride-modified sericin (SerMA) as the matrix, blends the photoinitiator LAP (0.5 mg / mL) with N@ILO nanovesicles, and forms a hydrogel by curing with 405 nm visible blue light, supporting photocuring 3D printing (exposure intensity 20~18 mW / cm²).

[0058] The hydrogel of the present invention has the functions of synchronous treatment and personalized filling of tissue defects: SerMA hydrogel provides support for postoperative defects, and at the same time, the photocuring 3D printing accuracy reaches ±50 μm, matching personalized repair needs.

[0059] The hydrogel of the present invention has photothermal responsiveness: SerMA-N@ILO heats up to 49.8 °C under a 1064 nm laser (0.5 W / cm²), triggering local photothermal killing, and avoiding secondary surgery for in-situ filling after surgery.

[0060] The hydrogel of the present invention has a synergistic mechanism of immunogenic cell death (ICD) and microenvironment regulation.

[0061] In the present invention, the cell membrane vesicles and IR1061, Lon and OXA are mixed at a mass ratio of 3:1:1:1, and the IR1061 concentration is maintained at 10 μg / mL, which means that after the mass ratio is 3:1:1:1, the IR1061 concentration is 10 μg / mL. According to the encapsulation efficiency of IR1061 of 56.8%, the encapsulation efficiency of Lon of 40.6%, and the encapsulation efficiency of OXA of 63.0%, it can be calculated that Lon (7 μg / mL) and OXA (11 μg / mL) can maintain the IR1061 concentration at 10 μg / mL.

[0062] The test materials used in the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0063] Example 1 Preparation of immunogenic cell death-enhancing nanovesicle hydrogel

[0064] 1. Materials

[0065] Silkworm cocoons were purchased from Suzhou Siruibao Biotechnology Co., Ltd. (Suzhou, China); lithium phenyl-2,4,6-trimethylbenzoylphosphine (LAP) was provided by Aladdin (Shanghai, China); ELISA kits were purchased from Shenggong Biotechnology Co., Ltd. (China); calcein-AM / PI detection kits were from Shanghai Yisheng Biotechnology Co., Ltd. (Shanghai, China); lonidamine (Lon) was purchased from MedChemExpress (MedChemExpress); methacrylic anhydride (MA), IR1061, and oxaliplatin (OXA) were purchased from Sigma-Aldrich, USA; DAPI was supplied by Dalian Meilun Biotechnology Co., Ltd. (Dalian, China); human PDLSCs were purchased from iCell Bioscience Inc (iCell Bioscience Co., Ltd., China). In addition, other conventional chemical reagents (such as buffers, culture media, etc.) were obtained from Sinopharm Chemical Reagent Co., Ltd. (China), and their specifications met the experimental requirements. The purity, storage conditions, and instructions for use of all reagents and materials strictly followed the technical standards provided by the suppliers.

[0066] 2. Experimental Methods

[0067] 1. Synthesis and characterization of N@ILO

[0068] A large number of human PDLSCs cells were collected, washed three times with PBS, resuspended in PBS, and placed on an ice-water mixture. An ultrasonic cell disruptor was used to intermittently disrupt the cells, and the cells were treated with ultrasonic waves at 40% power for 1.5 minutes (the pulse cycle was 1 second on and 2 seconds off), and then centrifuged at 4,000 g for 30 minutes to remove cell debris; then the obtained supernatant was ultracentrifuged at 150,000 g for 70 minutes at 4 °C to obtain cell membrane vesicles. The cell membrane vesicles were mixed with IR1061, Lon, and OXA at a mass ratio of 3:1:1:1 in PBS, ultrasonically dispersed in an ice-water bath, and then repeatedly extruded through porous membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm using a liposome extruder, and finally the drug-loaded vesicles (N@ILO) were collected by ultracentrifugation at 150,000 g for 70 minutes at 4 °C.

[0069] The morphology of the drug-loaded vesicles (N@ILO) was observed using a transmission electron microscope (TEM); the hydrated particle size of the drug-loaded vesicles (N@ILO) was measured using a dynamic light scattering (DLS) particle size analyzer; the particle size of the drug-loaded vesicles (N@ILO) was further characterized by nanoparticle tracking analysis (NTA). The encapsulation efficiency of IR1064 was analyzed using a UV-visible-near-infrared spectrophotometer, the encapsulation efficiency of Lon was analyzed using a liquid chromatograph, and the encapsulation efficiency of OXA was analyzed using ICP.

[0070] 2. Synthesis and characterization of SerMA-N@ILO hydrogel

[0071] The brief steps are as follows: 20 g of natural silkworm cocoons were washed with double-distilled water and then immersed in 800 mL of boiling 0.02 M Na2CO3 solution for 1 hour, and the insoluble residues were removed by centrifugation (3000 rpm, 10 minutes) and filtration. The obtained crude sericin solution was dialyzed against double-distilled water for 3 days using a dialysis bag with a molecular weight cut-off of 3.5 kDa and then lyophilized to obtain purified sericin protein. 10 g of the lyophilized sericin was dissolved in 40 mL of PBS buffer with a pH of 8.5 and stirred well for 3 hours. 2.93 g of methacrylic anhydride (MA) was dissolved in 100 mL of PBS with a pH of 8.5 to prepare an MA stock solution. The MA solution was reacted with the sericin solution overnight at room temperature, and then dialyzed against double-distilled water for 3 days using a dialysis bag with a molecular weight cut-off of 3.0 kDa and lyophilized to obtain SerMA for standby.

[0072] SerMA, LAP, and N@ILO were dissolved in 1 mL of PBS, where the concentration of SerMA was maintained at 150 mg / mL, the concentration of LAP was maintained at 0.5 mg / mL, and the concentration of IR1061 in N@ILO was maintained at 10 μg / mL. After thorough mixing, a SerMA-N@ILO precursor solution was formed and irradiated with 405 nm laser for 1 minute to form the SerMA-N@ILO hydrogel.

[0073] The microscopic morphology of SerMA-N@ILO freeze-dried hydrogel was observed by a Quanta 200 scanning electron microscope (SEM) from FEI Company in the Czech Republic. Rheological analysis was completed using a TA-DHR-2 rheometer from TA Instruments in the United States, equipped with a 405 nm ultraviolet lamp.

[0074] 3. Study on the 3D printing performance of SerMA-N@ILO

[0075] The 3D bioprinting test of the SerMA-N@ILO precursor solution was carried out using a digital light processing printer (nanoArchS140) equipped with a 405 nm laser. A three-dimensional CAD model was established using Solidworks software, and then the CAD model was sliced using BMF3D slicing software, followed by layer-by-layer printing. The morphology of the 3D printed samples was first observed using a digital microscope. Among them, the printing thickness was 1600 μm, the number of printing layers was 4, the exposure time was 30 s, and the exposure intensity of the first layer was 20 mW / cm 2 , and that of the other layers was 18 mW / cm 2 .

[0076] 4. Study on the photothermal properties of N@ILO

[0077] To systematically evaluate the photothermal conversion characteristics of N@ILO nanovesicles, a multi-dimensional experimental scheme was designed in this study. The N@ILO dispersion containing IR1061 gradient concentrations (0, 5, 10, 15, 20, 30 μg / mL) was aliquoted into 1.5 mL EP tubes and irradiated with a near-infrared laser with a wavelength of 1064 nm at a power density of 0.5 W / cm² for a duration of 5 minutes. The temperature change kinetics was monitored and recorded in real time through a FLIR E60 infrared thermal imaging system, and equal-volume deionized water was set as a negative control group. On this basis, the influence law of laser power density (0.25, 0.50, 0.75, 1.00 W / cm²) on the heating behavior of the 10 μg / mL N@ILO dispersion was systematically investigated. To verify the photothermal stability of the material, a periodic laser on-off experiment was designed: under the condition of a fixed power density (0.5 W / cm²), the sample was subjected to 4 consecutive laser on-off cycles, and the temperature fluctuation curve was recorded synchronously.

[0078] 5. Analysis of cytotoxicity, apoptosis and LA content

[0079] The cell viability was evaluated by the CCK-8 method. B16F10 cells were seeded in 96-well plates at a density of 5×10³ cells / well and pre-cultured in a 37°C, 5% CO₂ incubator for 24 hours. Seven groups of treatments were set up in the experiment: Blank (control group), IR1061 (10 μg / mL), Lon (7 μg / mL), OXA (11 μg / mL), N@ILO, IR1061 (+), and N@ILO (+), with the concentration of IR1061 kept consistent (10 μg / mL). After 24 hours of drug treatment, the IR1061 (+) and N@ILO (+) laser treatment groups were irradiated with a 1064 nm near-infrared laser (power density 0.5 W / cm²) for 10 minutes. After each group of treatments, 100 μL of CCK-8 working solution was added to each well, and the incubation was continued for 2 hours. The absorbance value at 450 nm was measured using a Multiskan SkyHigh microplate reader.

[0080] To further verify the cell viability status, fluorescence analysis was performed using a Calcein-AM / PI double staining kit. 1×10 5 B16F10 cells were seeded in 6-well plates. After 12 hours of adherent culture, the above seven groups of treatments were carried out respectively, with the concentration of IR1061 kept consistent (10 μg / mL) in all groups. Co-culture was continued for 24 h. After removing the medium, 2 mL of PBS staining solution containing 2 μM Calcein-AM and 1.5 μM PI was added to each well, and the incubation was carried out in the dark for 15 minutes. Fluorescence images were collected by a confocal microscope, where green fluorescence (Calcein-AM) labeled live cells and red fluorescence (PI) indicated dead cells.

[0081] For the study of the cell apoptosis mechanism, B16F10 cells from the above treatment groups (1×10 6 cells / tube) were collected and operated according to the instructions of the Annexin V-FITC / PI apoptosis detection kit. After the cells were washed twice with pre-cooled PBS, they were resuspended in 195 μL of binding buffer, and 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added successively, and the reaction was carried out in the dark for 15 minutes. Flow cytometry was used for detection, and the proportions of early apoptotic (Annexin V⁺ / PI⁻) and late apoptotic (Annexin V⁺ / PI⁺) cells were analyzed by FlowJo V software.

[0082] In addition, the method for detecting LA content was carried out according to the instructions of the lactate detection kit of Nanjing Jiancheng Bioengineering Institute. 1×10 51×10

[0083] 6. Immunofluorescence staining of calreticulin (CRT) and high-mobility group protein 1 (HMGB1)

[0084] To systematically evaluate the regulatory effect of N@ILO on ICD markers, in this study, CRT and HMGB1 in B16F10 cells were analyzed by immunofluorescence staining. Seven groups of treatments were set up: Blank (control group), IR1061 (10 μg / mL), Lon (7 μg / mL), OXA (11 μg / mL), N@ILO, IR1061 (+), and N@ILO (+), etc. The concentration of IR1061 was kept consistent (10 μg / mL) in all groups. Among them, IR1061 (+) and N@ILO (+) were continuously irradiated with a 1064 nm near-infrared laser (power density 0.5 W / cm²) for 10 minutes.

[0085] 1×10 5 1×10

[0086] 7. Evaluation of in vivo antitumor effect

[0087] C57BL / 6 mice (female, 6 - 8 weeks old) were purchased from Hunan Slack Jingda Experimental Animal Co., Ltd. (Changsha, China). 1×10 6B16F10 cells were used to establish a tumor-bearing model. When the tumor volume reached 50 mm³, the mice were randomly divided into 7 groups (n = 5): Blank (control group), SerMA-IR1061 (SerMA-IR1061 was a direct mixture of SerMA and IR1061, and similar expressions hereinafter were the same as here, with the concentration of IR1061 being 10 μg / mL), SerMA-Lon, SerMA-OXA, SerMA-N@ILO, SerMA-IR1061(+), and SerMA-N@ILO(+), etc. Among them, the Blank group received no treatment. SerMA-IR1061(+) and SerMA-N@ILO(+) were irradiated with 1064 nm laser (0.5 W / cm²) for 10 minutes on days 1, 3, and 5. To simulate the clinical surgical resection treatment of choroidal melanoma, a circular wound with a diameter of 8 mm was created at the tumor site of the mice, and then a hydrogel was formed by in-situ photocuring of the precursor solution using 405 nm laser. The tumor volume and the body weight of the mice were measured every other day. On the 16th day, the mice were sacrificed and the tumor tissues were collected for weighing analysis. Further, histological staining analysis of the tumor tissues was performed. The tumor tissues were stained with H&E to evaluate the tissue morphology, immunofluorescence TUNEL staining was used to detect apoptotic cells, immunohistochemical staining was used to detect the expression levels of calreticulin (CRT), high-mobility group protein 1 (HMGB1), and PD-L1, and CD8 + T cells were stained and analyzed by immunofluorescence staining. In addition, enzyme-linked immunosorbent assay (ELISA) was used to detect the concentration levels of tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, and IL-10 in the serum samples. Meanwhile, the LA content in the tumor tissues was analyzed, and the detection method of LA content was carried out according to the instruction manual of the lactate detection kit of Nanjing Jiancheng Bioengineering Institute. In addition, the safety of SerMA-N@ILO was evaluated. HE staining analysis was performed on the main organs of the mice in the Blank group and the SerMA-N@ILO(+) group, namely the heart, liver, spleen, stomach, and kidney. At the same time, blood routine analysis was performed on the blood of the mice.

[0088] Furthermore, flow cytometry analysis was performed on tumor tissues. The immunocyte analysis process was as follows: Tissue sections were digested with a mixture of collagenase (0.5 g / L), hyaluronidase (0.3 g / L), and DNase (0.15 g / L) for 1 hour (37 °C), and the cells were collected by centrifugation at 1200 rpm for 5 minutes after passing through a 200-mesh sieve. After washing with PBS, the cells were labeled with specific antibodies and analyzed by flow cytometry. A BDFACS instrument was used to detect tumor-infiltrating T lymphocytes, and the proportion of CD8+ T cells was quantified by labeling with anti-CD45-APC-Cy7, anti-CD11b-FITC, and anti-CD8-Cy5.5 antibodies; at the same time, the proportion of mature dendritic cells (DCs) in tumor tissues was evaluated by staining with anti-CD11c-Qdot 605, anti-CD80-Pacific Blue, and anti-CD86-PE antibodies.

[0089] 8. Sequencing analysis of tumor tissues

[0090] To systematically analyze the molecular mechanism of SerMA-N@ILO(+)-mediated therapy, tumor tissues from the control group (Blank group) and the SerMA-N@ILO(+)-treated group (Test group) were collected on the 7th day after treatment of tumor-bearing mice, quickly transferred to pre-cooled cryotubes, and snap-frozen in liquid nitrogen for 5 minutes, and then stored in a -80 °C ultra-low temperature freezer to maintain RNA integrity. Sample gene expression profiling sequencing analysis was completed by Guangzhou Kediao Biotechnology Co., Ltd. The differential gene analysis between the two groups was performed using the "edgeR" package in R language, and the screening criteria were p value < 0.05 and absolute value of fold change > 1.2. Gene ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the "clusterProfiler" package in R language to analyze the biological processes, molecular functions, cellular components, and potential signaling pathways involved in the differential genes, respectively.

[0091] III. Experimental data

[0092] 1. Synthesis and characterization of N@ILO

[0093] First, the stemness of successfully isolated and cultured human periodontal ligament stem cells (PDLSCs) was identified. Immunofluorescence staining was used to detect that PDLSCs highly expressed the stem cell-specific surface markers CD146 and STRO-1 ( Figure 1), confirming its good stem cell characteristics and laying a reliable biological foundation for the subsequent construction of cell membrane-derived nanovesicles. Subsequently, ultrasonic fragmentation was used to extract cell membrane vesicles from PDLSCs, and they were mixed with IR1061, Lon, and OXA at a mass ratio of 3:1:1:1. After thorough blending in PBS, they were repeatedly extruded through a polycarbonate membrane with a pore size of 100 nm to successfully construct the multi-drug co-delivery nanovesicle N@ILO. Transmission electron microscopy (TEM) imaging results showed that the constructed N@ILO presented a typical cup-shaped vesicle structure with regular morphology and an average particle size of about 100 nm ( Figure 2 as shown in a of Figure 2 ). Further, dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA) were used to characterize its particle size and dispersibility. The results showed that the N@ILO particles were evenly distributed, with the particle size concentrated between 100 and 200 nm, and had good monodispersity ( Figure 3 as shown in b and c of Figure 4 ). To further evaluate its colloidal stability, its particle size change was continuously monitored in PBS buffer for 9 days. The results showed that the particle size fluctuation range was small, indicating that N@ILO had excellent physical stability ( Figure 5 ). In terms of drug loading capacity, standard curves were plotted and quantitative analysis was carried out using ultraviolet-visible-near-infrared spectrophotometer, high performance liquid chromatography (HPLC), and inductively coupled plasma mass spectrometry (ICP). The results showed that the encapsulation efficiency of IR1061 was 56.8% ( Figure 6 ), the encapsulation efficiency of Lon was 40.6% ( ), and the encapsulation efficiency of OXA was 63.0%.

[0094]

[0095] The results of this study verified that the N@ILO nanovesicles constructed based on PDLSC cell membranes showed excellent performance in terms of structural morphology, particle size distribution, colloidal stability, and multi-component loading capacity, providing a solid foundation for its application as a multifunctional delivery system in tumor treatment.

[0096] As a non-invasive anti-tumor strategy, hyperthermia has shown good prospects in combined treatment systems in recent years. In this study, N@ILO utilized the near-infrared II (NIR-II) photosensitizer IR1061 it loaded to achieve effective photothermal conversion under 1064 nm wavelength laser irradiation. To verify its photothermal performance, first, it was irradiated for 5 minutes under the condition of a laser power density of 0.5 W / cm². The results showed that the temperature increase amplitude of the control group (H2O2 solution) was less than 2 °C, while the temperature of the N@ILO group increased significantly with the increase in the concentration of IR1061. When the concentration of IR1061 was 10 μg / mL, the temperature rapidly increased from the initial 25.6 °C to 49.8 °C (Figure 2 d in Figure 7 ), reaching the temperature threshold for inducing thermal damage and even apoptosis of tumor cells. In addition, under the conditions of laser power densities of 0.25, 0.50, 0.75, and 1.00 W / cm² respectively, the system temperature gradually increases with the increase of laser power ( Figure 2 e in Figure 2 ), showing good power responsiveness. At the same time, to verify its cycling stability, N@ILO was subjected to 4 rounds of laser irradiation-cooling cycle experiments, and the results showed that it could be repeatedly heated up in each round without significant attenuation of photothermal performance (

[0097] 3. Antitumor effect in vitro

[0098] To evaluate the in vitro antitumor activity of N@ILO, it was first found through a lactate detection kit that both Lon and N@ILO could effectively reduce the LA level produced by tumors ( Figure 8 ). Then, the CCK-8 method was used to detect the toxic effects of N@ILO loaded with different concentrations of IR1061 on melanoma cells B16F10. The results showed that with the increase of IR1061 concentration, the inhibitory effect of N@ILO on B16F10 cells gradually increased, showing typical dose-dependent cytotoxicity ( Figure 9 a in Figure 9 ). This indicates that it has a significant photothermal enhanced killing effect under near-infrared irradiation. Further, the Calcein-AM / PI live / dead cell double staining experiment was used to visually analyze the survival and death states of cells in different treatment groups. The results showed that under the non-irradiation condition, IR1061 and N@ILO had limited effects on cells, mainly green fluorescence. After laser irradiation, the red fluorescence in the N@ILO(+) group was significantly enhanced, indicating that a large number of cells died and the cell membrane integrity was significantly damaged ( Figure 9 b in ). This shows that N@ILO can effectively release heat energy under laser excitation, thereby inducing cell death. Subsequently, the Annexin V-FITC / PI double staining flow cytometry was used to further evaluate its apoptosis-promoting effect ( Figure 9 c in ). The analysis results showed that the late apoptosis rate induced by the N@ILO(+) group was as high as 65.0%, significantly higher than that of the IR1061(+) single treatment group (late apoptosis rate was 33.0%) and the Blank group (late apoptosis rate was only 4.2%). This indicates that N@ILO can significantly induce programmed cell death of melanoma cells under the synergy of photothermal action and chemotherapy, showing superior antitumor potential.

[0099] In addition, to investigate whether it can induce immunogenic cell death (ICD), the expressions of two key ICD markers were further detected: calreticulin (CRT) and high-mobility group protein B1 (HMGB1). CRT usually migrates from the endoplasmic reticulum to the cell membrane surface under stress, serving as an "eat me" signal to induce dendritic cells to recognize tumor antigens; while HMGB1 is released from the nucleus to the extracellular space, serving as a "find me" signal to promote the activation of macrophages and immature dendritic cells. Immunofluorescence results showed that under laser irradiation, the membrane expression of CRT in the N@ILO(+) group was significantly enhanced ( Figure 10 as shown in a) in

[0100] , presenting a strong fluorescence signal, indicating its effective induction of CRT externalization. Meanwhile, the fluorescence signal of HMGB1 in the N@ILO(+) group was significantly weaker than that in the control group, suggesting that it had been released to the extracellular space, further corroborating its ability to induce ICD.

[0100] This study systematically verified that N@ILO has significant in vitro anti-tumor activity under near-infrared laser irradiation, manifested as enhanced cytotoxicity, increased apoptosis rate, and altered expression of immunogenic cell death markers. In particular, its induction of CRT externalization and HMGB1 release suggest that N@ILO may not only directly kill tumor cells but also activate downstream immune responses, providing solid theoretical support for the realization of the "photothermal + chemotherapy + immunotherapy" triple synergistic anti-tumor strategy.

[0101] 4. Preparation of SerMA-N@ILO and Study on Its 3D Printing Performance

[0102] In this study, sericin was first extracted from silkworm cocoons by the hot alkali degumming method, and then SerMA was obtained by MA chemical graft modification. This SerMA hydrogel material has both good biocompatibility and degradability, and has broad biomedical application prospects. Subsequently, SerMA, photoinitiator LAP, and drug-loaded nanovesicles N@ILO were co-dissolved in PBS, and after uniform mixing, a SerMA-N@ILO precursor solution was prepared. This solution undergoes a photoinduced sol-gel transition under 405 nm blue light irradiation and rapidly forms a cross-linked hydrogel ( Figure 11 as shown in a) in Figure 11 . The results of rheological dynamic time sweep experiments showed that under the light intensity condition of 30 mW / cm², the SerMA-N@ILO system can complete the curing process within 1 minute, having good rapid gelation performance ( Figure 11In c), it was found that its interior presented a typical three-dimensional porous structure. This structure helps with the slow release of drugs, the penetration of nutrients, and the excretion of metabolites, thereby enhancing its functionality as a biological scaffold material in the in vivo environment.

[0103] The photocuring 3D printing technology is a highly precise and controllable manufacturing method, widely used in the construction of personalized tissue engineering scaffolds. It enables the rapid construction of complex geometric structures through computer-aided design (CAD), and is particularly suitable for the customized repair of tissue defect areas after tumor resection. Given that the SerMA-N@ILO precursor solution has good photocuring properties, the SLA (stereolithography) 3D bioprinting technology was used to evaluate its printing and forming ability. First, the required CAD model was designed using Solidworks software ( Figure 12 ), and then the SerMA-N@ILO biomaterial ink was loaded onto the printing platform and printed layer by layer according to the design drawing. As shown in d of Figure 11 , the obtained hydrogel sample had a complete structure and clear edges. The printed product was highly consistent with the original design model, showing excellent structural fidelity and forming accuracy.

[0104] The above results indicate that SerMA-N@ILO not only has good photocuring performance and a porous structure, but can also be highly integrated with the photocuring 3D printing technology to achieve the precise construction of personalized hydrogel scaffolds. This system is expected to be applied to various clinical scenarios such as local treatment after tumor surgery and tissue regeneration, showing good translational prospects.

[0105] 5. In vivo anti-tumor experimental study

[0106] To construct a multifunctional SerMA-N@ILO photosensitive hydrogel for the postoperative treatment of choroidal melanoma, the N@ILO nanovesicles were mixed with the SerMA hydrogel precursor solution (PBS containing SerMA) by physical mixing to obtain an injectable and photocurable SerMA-N@ILO composite system. In the experiment, a skin defect with a diameter of about 8 mm was prepared on the back melanoma model of C57BL / 6 mice using scissors to simulate the situation of tissue defect after clinical choroidal melanoma resection. Subsequently, the hydrogel precursor solutions of different groups were locally coated on the surgical wound, and irradiated with a 405 nm wavelength blue light flashlight to enable the in-situ photocuring and forming of the hydrogel at the wound site. During the treatment stage, the SerMA-IR1061(+) and SerMA-N@ILO(+) groups were further irradiated with a 1064 nm laser (power density 0.5 W / cm², irradiation time 10 minutes) to activate their photothermal therapy function. As shown in a of Figure 13 and Figure 14As shown, tumor growth was inhibited in both the SerMA-IR1061(+) group and the SerMA-N@ILO(+) group, and the SerMA-N@ILO(+) group showed the most significant tumor growth inhibitory effect, while the tumor volumes in the other groups continued to increase. The tumor weighing results further verified the anti-tumor advantage of the SerMA-N@ILO(+) group ( Figure 13 b in), indicating that the combination of photothermal and multi-drug synergistic therapy can significantly improve the anti-tumor efficiency.

[0107] To systematically evaluate the biosafety of SerMA-N@ILO, the body weight changes of mice in each group were monitored. As shown in Figure 13 c in, the body weights of all treatment groups remained stable, and no significant fluctuations were observed, suggesting that the treatment process did not cause obvious toxic and side effects. At the same time, H&E tissue section analysis of the main organs (heart, liver, spleen, lung, kidney) ( Figure 15 ), and the results of blood routine tests ( Figure 16 ) did not find organ damage or systemic toxicity, further confirming the good biocompatibility of the hydrogel.

[0108] To explore the role of SerMA-N@ILO hydrogel in inducing anti-tumor immune responses, ELISA technology was used to detect the expression levels of related cytokines in the sera of mice after treatment. The results showed that the SerMA-N@ILO(+) group significantly up-regulated the secretion of TNF-α, IFN-γ, and IL-6 after laser irradiation ( Figure 13 d - f in), while the level of the immunosuppressive factor IL-10 was significantly inhibited ( Figure 17 ). Among them, the increase in TNF-α can directly induce tumor cell necrosis, IFN-γ helps to enhance the body's immune recognition and killing ability, and IL-6 can participate in initiating the initial immune response, suggesting that the hydrogel not only plays a local photothermal treatment role but also has certain immune regulation potential. In addition, the results of further histological analysis showed that obvious structural damage occurred in the tumor tissues of the SerMA-N@ILO(+) group after PTT. The H&E staining images showed disordered cell arrangement and karyolysis ( Figure 13 g in); the results of TUNEL immunofluorescence staining also confirmed that a large number of cells in the tumor tissues underwent apoptosis ( Figure 13 g in), indicating that the hydrogel treatment can effectively induce programmed cell death.

[0109] In summary, the SerMA-N@ILO hydrogel not only shows significant photothermal synergistic anti-tumor ability in the postoperative melanoma model, but also has good biocompatibility and immune activation potential, and has high clinical translation prospects.

[0110] 6. Study on the anti-tumor immune mechanism in vivo

[0111] To deeply analyze the anti-tumor immune mechanism induced by SerMA-N@ILO hydrogel in the treatment of choroidal melanoma, the changes in the immune cascade reaction were systematically studied in a B16F10 mouse tumor model, and the activation levels of dendritic cell (DC) maturation and tumor-infiltrating T cells were mainly evaluated. As Figure 18 shown in a of Figure 19 below, the flow cytometry results showed that the DC maturation ratio in the Blank group was only 13.6%; it increased to 22.7% in the SerMA-IR1061(+) group; while the DC maturation ratio in the SerMA-N@ILO(+) group was the highest, reaching 32.7%. Compared with the Blank group, the DC maturation ratio in the SerMA-N@ILO(+) group increased by 2.4 times and was 1.4 times that of the SerMA-IR1061(+) group, suggesting that this hydrogel can significantly promote DC maturation, thereby effectively initiating the initial immune response. Further analysis was performed on the infiltration of T lymphocytes in the tumor microenvironment. Figure 18 As shown in b-c of Figure 18 below, the proportion of CD4⁺ T cells in the Blank group was 8.1%, 13.5% in the SerMA-IR1061(+) group, while it significantly increased to 24.8% in the SerMA-N@ILO(+) group, approximately 3.1 times that of the Blank group. Similarly, the infiltration levels of CD8⁺ T cells in the Blank, SerMA-IR1061(+), and SerMA-N@ILO(+) groups were 3.3%, 15.4%, and 22.6% respectively. The number of CD8⁺ T cells in the SerMA-N@ILO(+) group was approximately 6.8 times that of the Blank group, suggesting that this treatment strategy can effectively activate and recruit cytotoxic T lymphocytes (CTLs) and enhance the tumor clearance effect. In addition, the change in the content of lactic acid (LA) in tumor tissues (

[0112] To further verify the occurrence of T cell infiltration and immunogenic cell death (ICD) effects, CD8⁺ T cell immunofluorescence staining and immunohistochemical (IHC) staining analysis of CRT and HMGB1 were performed on tumor tissues ( Figure 18In f). The results showed that in the tumor tissues of the SerMA-N@ILO(+) group, the infiltration density of red fluorescence-labeled CD8⁺ T cells was significantly increased, indicating its significant advantage in inducing systemic immune responses. Meanwhile, the expression of CRT externalization and the extracellular release level of HMGB1 were also significantly enhanced in the SerMA-N@ILO(+) group, which was consistent with the in vitro experimental results. This phenomenon confirmed that the SerMA-N@ILO hydrogel could induce the ICD effect in tumor cells, thereby effectively activating the adaptive immune system and synergistically enhancing the remote immune clearance effect after photothermal therapy.

[0113] In summary, the SerMA-N@ILO hydrogel can not only directly kill tumor cells through the photothermal effect, but also construct an effective anti-tumor immune microenvironment by inducing ICD, promoting DC maturation and T cell activation, showing good potential for tumor treatment and immune regulation.

[0114] 7. RNA sequencing analysis of tumor tissues

[0115] To further reveal the potential molecular mechanism of the SerMA-N@ILO hydrogel in tumor immunotherapy, RNA sequencing (RNA-seq) analysis was performed on the tumor tissues of mice in the Blank (control group) and SerMA-N@ILO(+) group (hereinafter referred to as the Test group). The principal component analysis (PCA) plot showed that although there was a certain heterogeneity among the tumor tissue samples, the Blank group and the Test group were significantly separated at the transcriptome level, indicating that there were obvious differences in the gene expression profiles between the two groups ( Figure 20 In a). The sample quality control analysis showed that the sequencing depth was consistent, ensuring the reliability of subsequent data analysis ( Figure 20 In b and c), and at the same time showing a high degree of overlap in gene expression among different samples. A total of 11,725 co-expressed genes were detected in the tumor tissues of the two groups, and the Blank group and the Test group had 49 and 856 specifically expressed genes respectively ( Figure 21 ). Further differential expression analysis showed that compared with the Blank group, 1309 genes were significantly up-regulated and 217 genes were significantly down-regulated in the Test group ( Figure 20 In d, Figure 22 ). The expression changes of immune-related genes were mainly analyzed, especially the genes related to "immune response". The results showed that the expression levels of multiple key genes closely related to immune regulation were significantly increased in the Test group, indicating the immune activation effect induced by the hydrogel. Figure 20in e). To analyze the signaling pathways involved in these differentially expressed genes by the system, KEGG pathway analysis and GO enrichment analysis were further performed. KEGG analysis showed that the significantly enriched pathways in the Test group included multiple signaling pathways closely related to immune response and apoptosis, such as "IL-17 signaling pathway", "TNF signaling pathway", "T cell receptor signaling pathway", and "Apoptosis" ( Figure 20 in f). Meanwhile, GO functional enrichment analysis indicated that the differentially expressed genes in the Test group were involved in key biological processes such as "Immune response", "Positive regulation of immune response", "T cell activation", and "Cytokine-mediated signaling pathway" ( Figure 20 in g).

[0116] In summary, the RNA sequencing results further confirmed the ability of SerMA-N@ILO hydrogel to induce a significant anti-tumor immune response in vivo. Especially through mechanisms such as enhancing cytokine signaling and promoting T cell activation, it synergistically increased the activation level of cytotoxic T lymphocytes (CTLs), which was consistent with the aforementioned immunomics and histological results.

[0117] Example 2 Optimization in the Preparation of Immunogenic Cell Death-Enhanced Nanovesicle Hydrogel

[0118] Cell membrane vesicles were mixed with IR1061, Lon, and OXA at mass ratios of 1:1:1:1, 2:1:1:1, 3:1:1:1, and 4:1:1:1 in PBS. After ultrasonic dispersion in an ice-water bath, they were repeatedly extruded through porous membranes with pore sizes of 800 nm, 400 nm, 200 nm, and 100 nm using a liposome extruder. Finally, the drug-loaded nanovesicles were collected by ultracentrifugation at 150,000 g for 70 minutes at 4 °C, which were N@ILO. The stability (hydrated particle size) of the nanovesicles with different mass ratios was evaluated by dynamic light scattering (DLS), and the particle size changes were continuously monitored in PBS buffer for 9 days. The results are shown in Table 1. The results of the mass ratio of 3:1:1:1 showed a smaller particle size fluctuation range, indicating that N@ILO with a mass ratio of 3:1:1:1 had excellent physical stability. The mass ratio of 3:1:1:1 was preferred.

[0119] Table 1. Optimization in the preparation of immunogenic cell death-enhanced nanocapsule hydrogel (nm)

[0120] Mass ratio 1 (day) 3 (days) 5 (days) 7 (days) 9 (days) 1:1:1:1 220.3±10.0 248.6±7.6 281.5±7.1 296.9±5.7 303.7±5.9 2:1:1:1 244.7±3.6 255±4.4 295.9±4.8 303.3±6.2 314.5±4.0 3:1:1:1 197.4±2.9 199.3±3.2 198.8±1.2 194.0±1.4 192.5±0.5 4:1:1:1 215.0±5.0 263.6±3.5 285.7±3.7 292.6±3.3 312.7±9.3

[0121] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Drug-loaded vesicles, characterized in that, It includes a photothermal agent, an ICD-induced chemotherapeutic drug, a glycolysis inhibitor, and a cell vesicle.

2. The drug-loaded vesicle according to claim 1, wherein the photothermal agent includes IR1061, IR780, ICG, IR210, gold nanoparticles, copper nanoparticles, Prussian blue nanoparticles, black phosphorus nanosheets, and / or MXene nanosheets; the ICD-induced chemotherapeutic drug includes: oxaliplatin, doxorubicin, epirubicin, daunorubicin, idarubicin, cisplatin, carboplatin, paclitaxel, docetaxel, bortezomib, cyclophosphamide, and / or 5-fluorouracil; the glycolysis inhibitor includes: lonidamine, 2-deoxy-D-glucose, 3-bromopyruvate, D-mannoheptulose, and / or Lon-TK; the cell vesicle includes human periodontal ligament stem cell membrane vesicles.

3. The drug-loaded vesicle according to claim 2, wherein The mass ratio of the photothermal agent, the ICD-induced chemotherapeutic drug, the glycolysis inhibitor, and the cell vesicle is 1:1:1:(1-4).

4. The drug-loaded vesicle according to claim 3, wherein The concentration of the photothermal agent is 10-30 μg / mL.

5. Hydrogel, characterized in that, It includes silk protein, a photoinitiator, and the drug-loaded vesicle according to any one of claims 1-4.

6. The hydrogel according to claim 5, wherein the silk protein includes methacrylated sericin; the photoinitiator includes: lithium phenyl-2,4,6-trimethylbenzoylphosphinate, TPO-L, and / or a cyanine / iodonium salt system.

7. The method for preparing the hydrogel according to claim 5 or 6, characterized in that, Mix the sericin, the photoinitiator, and the drug-loaded vesicle to obtain the hydrogel.

8. Use of at least one of the following I) - III) in the preparation of a product for filling and / or preventing and treating a tumor wound: I), the drug-loaded vesicle according to any one of claims 1-4; II), the hydrogel according to claim 5 or 6; III), the hydrogel prepared by the preparation method according to claim 7.

9. A product for filling and / or preventing and treating tumor wounds, characterized in that, The raw material includes at least one of i) - iii) shown: i), the drug-loaded vesicle according to any one of claims 1-4; ii), the hydrogel according to claim 5 or 6; iii), the hydrogel prepared by the preparation method according to claim 7.

10. The product according to claim 9, wherein, The tumor includes an eye tumor.

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