Immunogenic cell death-enhancing nanovesicle hydrogel and its application

Abstract

The present invention relates to the field of biomedicine, and in particular to immunogenic cell death-enhancing nanovesicle hydrogels and their applications. The present invention provides a 3D-printable, multifunctional, synergistic immunogenic cell death-enhancing nanovesicle hydrogel. The hydrogel uses SerMA hydrogel as a matrix and encapsulates cell membrane nanovesicles (N@ILO) loaded with the photothermal agent IR1061, the chemotherapy drug OXA, and the tumor acidic microenvironment regulator Lon. The hydrogel triggers initial tumor immunogenic cell death through the photothermal effect, further enhances the ICD effect through OXA, and modulates the acidic tumor microenvironment through Lon to increase T cell activity, achieving multimodal synergistic anti-tumor therapy after surgery. The hydrogel also possesses excellent photocurable 3D printing capabilities and can be used to construct personalized filling structures that match postoperative defect areas. The hydrogel has broad application prospects and provides a new strategy and theoretical basis for the comprehensive postoperative treatment of choroidal melanoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to immunogenic cell death-enhancing nanovesicle hydrogel and applications thereof. Background Art

[0002] Choroidal melanoma (CM) is the most common primary intraocular tumor in adults. Currently, surgical resection remains the mainstay of treatment for CM, particularly when localized lesions cannot be controlled by chemotherapy or radiotherapy, and is still considered the "gold standard" of treatment. However, there is still a high risk of recurrence and metastasis after surgery, especially in the setting of tiny residual lesions at the resection margin and tumor cell metastasis. In addition, the ocular tissue structure is delicate and complex, and surgical procedures often cause unavoidable tissue defects, resulting in impaired eye integrity and function, further affecting the patient's quality of life after surgery. A variety of postoperative fillers or drugs for ocular tumors have been developed to help prevent postoperative tumor recurrence and metastasis.

[0003] However, the existing postoperative recurrence prevention strategies for ocular tumor fillers are single and fail to synergistically enhance immunogenicity; the drug delivery system is also single in function and cannot achieve multi-mechanism synergy; at the same time, the glycolysis of residual tumor cells after surgery is hyperactive, leading to lactic acid accumulation in the tumor microenvironment, inhibiting DC antigen presentation and CD8⁺ T cell activity, and lacking metabolic intervention methods, resulting in an immunosuppressive microenvironment that weakens the ICD effect; and traditional fillers or drugs cannot match the complex morphology of ocular defects and do not integrate immune regulation functions, leading to postoperative recurrence; therefore, developing a multifunctional comprehensive treatment strategy that has both the ability to inhibit tumor recurrence and the ability to fill tissue defects in a personalized way 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 an immunogenic cell death-enhancing nanovesicle hydrogel and its application.

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

[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 induction chemotherapy drugs include: oxaliplatin, doxorubicin, epirubicin, daunorubicin, idarubicin, cisplatin, carboplatin, paclitaxel, docetaxel, bortezomib, cyclophosphamide and / or 5-fluorouracil;

[0008] The glycolysis inhibitor includes lonidamine, 2-deoxy-D-glucose, 3-bromopyruvate, D-mannoheptulose and / or Lon-TK.

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

[0010] Furthermore, in the drug-loaded vesicles of the present invention, the mass ratio of the photothermal agent, ICD-inducing chemotherapy 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 comprising 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, the specific one is methacrylated sericin. The methacrylated sericin is obtained by methacrylating tetragonal protein obtained from natural silkworm cocoons.

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

[0015] The present invention optimizes the components and ratios of the hydrogel, specifically screening and recombining the above-mentioned substances, including silk protein, a photoinitiator, a photothermal agent, an ICD-inducing chemotherapy drug, a glycolysis inhibitor, and cell vesicles. Test results indicate that a hydrogel prepared with methacryloylated sericin, LAP as a photoinitiator, IR1061 as a photothermal agent, oxaliplatin (OXA) as an ICD-inducing chemotherapy drug, lonidamine as a glycolysis inhibitor, and human periodontal ligament stem cell membrane vesicles has the best stability. Furthermore, tumor immunity and tumor inhibition tests comparing single components (such as OXA or lonidamine alone), combinations of some components (such as OXA or lonidamine combined with methacryloylated sericin), and hydrogels prepared according to the present invention indicate that the hydrogel containing the components of the present invention is the most effective, capable of generating sustained anti-tumor immunity, achieving multi-mechanism synergy, enhancing the ICD effect, and enabling personalized adaptation.

[0016] Furthermore, the mass ratio of the photothermal agent, ICD-inducing chemotherapy drug, glycolysis inhibitor and cell membrane vesicles in the drug-loaded vesicles in the hydrogel of the present invention was optimized, and their ratio was 1:1:1:(1~4); in the specific embodiment 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 most stable when 1:1:1:3 was used.

[0017] The multifunctional drug-loaded hydrogel of the present invention also optimizes the concentration of the photothermal agent; in embodiments of the present invention, the concentration is 5 to 30 μg / mL. Experimental results show that a 10 μg / mL concentration of the photothermal agent IR1061 rapidly increases the temperature from an initial 25.6°C to 49.8°C within 5 minutes, reaching the temperature threshold for inducing thermal damage and even apoptosis in tumor cells. Therefore, the preferred concentration of the photothermal agent is 10 to 30 μg / mL.

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

[0019] The present invention provides the use of at least one of the following I) to III) in the preparation of a product for filling and / or preventing tumor wounds:

[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 filling and / or preventing tumor wounds, characterized in that the raw materials include at least one of i) to iii):

[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 an eye tumor, specifically choroidal melanoma.

[0028] Preventing recurrence and filling tissue defects after choroidal melanoma surgery are currently major challenges in clinical treatment. This study designed and constructed a 3D-printable, multifunctional composite hydrogel system, SerMA-N@ILO, with immunogenic cell death (ICD)-inducing properties, for localized treatment of choroidal melanoma after resection. This system utilizes methacrylic anhydride-modified sericin (SerMA) as a matrix, encapsulating nanostructured cell membrane vesicles (N@ILO) loaded with the photothermal agent IR1061, the chemotherapy drug oxaliplatin (OXA), and the lactate metabolism inhibitor lonidamine (Lon). In vitro and in vivo experimental results demonstrated that the SerMA-N@ILO hydrogel significantly enhanced the ICD effect of tumor cells, promoting calreticulin (CRT) membrane externalization and high-mobility group protein B1 (HMGB1) secretion, thereby increasing the maturation rate of dendritic cells (DCs) by 2.4 times compared to the control group. It also significantly enhanced the specific infiltration of CD4⁺ T cells (by 3.1 times) and CD8⁺ T cells (by 6.8 times) into tumor tissue. Furthermore, the hydrogel alleviated the acidic microenvironment of tumor cells, remodeling the immunosuppressive tumor microenvironment and further enhancing the local anti-tumor immune response. Importantly, the hydrogel possesses photocurable 3D printing capabilities, enabling personalized customization for the precise filling of complex postoperative tissue defects. In summary, by synergistically inducing the ICD effect and regulating the microenvironment, the SerMA-N@ILO hydrogel demonstrates significant therapeutic potential for inhibiting postoperative recurrence of choroidal melanoma.

[0029] The present invention provides a 3D-printable multifunctional synergistic immunogenic cell death-enhancing nanovesicle hydrogel. The hydrogel uses SerMA hydrogel as a matrix and encapsulates nanocell membrane vesicles (N@ILO) loaded with the photothermal agent IR1061, the chemotherapy drug OXA, and the microenvironment regulator Lon. The hydrogel triggers initial ICD through the photothermal effect, OXA further enhances the ICD effect, and Lon regulates the acidic TME to promote T cell activity, thereby achieving postoperative multimodal synergistic anti-tumor therapy. At the same time, the hydrogel also has excellent light-curing 3D printing capabilities and can be used to personalize the construction of filling structures that match postoperative defect areas. It has broad application prospects and provides a new strategy and theoretical basis for the comprehensive postoperative treatment of choroidal melanoma. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0031] Figure 2Figure 4 shows the preparation of N@ILO and the characterization of its physicochemical properties, where a is a transmission electron microscopy (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 fluorescence intensity of N@ILO nanoparticles under 1064 nm laser irradiation (1.0 W / m 2 ) under different IR1061 loading amounts of H2O and N@ILO formulations; e is the thermal imaging of different power densities (0.5~1.5W / m 2 ) is the time-dependent temperature rise curve of the N@ILO suspension under 1064 nm laser irradiation; f is the photostability evaluation through four consecutive laser on / off cycles, demonstrating 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 was shown;

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

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

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

[0036] Figure 7 The results show that N@ILO suspensions with different IR1061 loadings were irradiated with 1064 nm laser (1.0 W / m 2 ) under the photothermal heating curve;

[0037] Figure 8 shows the lactate (LA) content in B16F10 cells determined by enzymatic colorimetric method;

[0038] Figure 9Figure 3 shows the in vitro cell evaluation of N@ILO; a is the evaluation of 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 the analysis of 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 Figure 3 shows the study of in vitro immunogenic cell death (ICD) effects; a is an immunofluorescence analysis of B16F10 cells treated with Blank (control group), IR1061, Lon, OXA, N@ILO, IR1061(+), and N@ILO(+) to detect the exposure of cell surface calreticulin (CRT), scale bar: 50 μm; b is an immunofluorescence staining experiment of B16F10 cells in different treatment groups (Blank, IR1061, Lon, OXA, N@ILO, IR1061(+), and N@ILO(+)) to detect the release of high-mobility group box 1 (HMGB1), scale bar: 50 μm;

[0040] Figure 11 Figure 3 shows the synthesis and physicochemical characterization of SerMA-N@ILO hydrogels; (a) a digital image recording the sol-gel transition process of SerMA-N@ILO hydrogels; (b) a photopolymerization rheological curve of SerMA-N@ILO hydrogels under 405 nm light irradiation; (c) a representative scanning electron microscopy (SEM) image of the microstructure of SerMA-N@ILO hydrogels; and (d) a high-fidelity 3D-printed construct fabricated by processing the SerMA-N@ILO hydrogel precursor solution using a digital light processing micro 3D printing system (nanoArchS140, Shenzhen Magic Cube).

[0041] Figure 12 Display computer-aided design (CAD) models designed using SolidWorks software;

[0042] Figure 13Figure 3 shows the in vivo anti-tumor therapeutic effect evaluation of SerMA-N@ILO; a is the tumor volume progression curve of different treatment groups; b is the quantitative analysis of the weight of the resected tumor after treatment; c is the weight change trajectory of tumor-bearing mice in the treatment group; d is the level of 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 cytokine interferon-γ (IFN-γ) in the serum of mice under different treatment regimens by ELISA; f is the level of cytokine interferon-γ (IFN-γ) in the serum of mice under different treatment regimens by ELISA serum levels of cytokine interleukin-6 (IL-6) in mice under different treatment regimens; g is a 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 end point of treatment (day 16 after treatment) are shown;

[0044] Figure 15 Shown are histopathological evaluations (hematoxylin-eosin staining, H&E staining) of major organs (heart, liver, spleen, lung, and kidney) in the control group and the SerMA-N@ILO-treated group;

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

[0046] Figure 17 The results show that the quantification of interleukin-10 (IL-10) in the serum of mice receiving different treatment regimens was performed by enzyme-linked immunosorbent assay (ELISA);

[0047] Figure 18Figure 3 shows the analysis of the anti-tumor immune mechanism; a is the quantitative analysis of the expression of dendritic cell (DC) maturation markers (CD86 / CD80) in tumor-infiltrating immune cells in different treatment groups by flow cytometry; b is the quantitative analysis of tumor-infiltrating lymphocytes: CD4+ T cells and CD8+ T cells by flow cytometry; c is the statistical analysis of the proportion of tumor-specific T cell infiltration: CD4+ T cells; d is the statistical analysis of the proportion of tumor-specific T cell infiltration: CD8+ T cells; e is the enzymatic determination of lactic acid (LA) content in tumor lysates; f is the multiparameter tumor tissue analysis: immunofluorescence staining of CD8+ T cells, and immunohistochemistry detection of calreticulin (CRT) and high-mobility group box 1 (HMGB1). 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 Figure 4 shows the quantitative analysis of dendritic cell (DC) maturation markers (CD80+ / CD86+) in different treatment groups by flow cytometry;

[0049] Figure 20 Figure 3 shows a multidimensional transcriptomic analysis of anti-tumor immune mechanisms; (a) principal component analysis (PCA) comparing the transcriptional profiles of the control and test groups (SerMA-N@ILO(+) or Test groups); (b) a Pearson correlation coefficient heat map assessing the global gene expression similarity between the experimental groups; (c) a Venn diagram between the control and test groups; (d) a volcano plot showing the differential expression of genes between the control and test groups; (e) a heat map showing the differential expression of immune-related genes involved in the "immune response" pathway; (f) Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways enriched for differentially expressed genes, |Fold change| > 1.2, p < 0.05;

[0050] Figure 21 Shown Venn diagram analysis of shared and unique differentially expressed gene DEGs between the control group and SerMA-N@ILO treatment group;

[0051] Figure 22 The bar graph shows the statistical analysis of the number of genes differentially expressed between the control group and the SerMA-N@ILO group;

[0052] Figure 23 Show Figure 20 Enlarged view of e in ;

[0053] Figure 24 Show Figure 20 The enlarged view of f in ;

[0054] Figure 25 Show Figure 20 Magnified view of g in . DETAILED DESCRIPTION

[0055] The present invention provides immunogenic cell death-enhancing nanovesicle hydrogels and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve the desired effect. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications herein 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 ICD induction by a single mechanism: 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 synergistically increase the DC maturation rate by 2.4 times (32.7% vs. 13.6%) and CD8⁺ T cell infiltration 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 a matrix, and a photoinitiator LAP (0.5 mg / mL) is blended with N@ILO nanovesicles. The hydrogel is cured by 405 nm visible blue light to form a hydrogel, supporting photocuring 3D printing (exposure intensity 20-18 mW / cm²).

[0058] The hydrogel of the present invention has the functions of simultaneous treatment and personalized filling of tissue defects: SerMA hydrogel provides support for postoperative defects, while the light-curing 3D printing accuracy reaches ±50μm, matching personalized repair needs.

[0059] The hydrogel of the present invention has photothermal responsiveness: SerMA-N@ILO heats up to 49.8°C under 1064nm laser (0.5W / cm²), triggering local photothermal killing, and in situ filling after surgery avoids secondary 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 are mixed with IR1061, Lon, and OXA in a mass ratio of 3:1:1:1, and the IR1061 concentration is maintained at 10 μg / mL. This means that after mixing at a mass ratio of 3:1:1:1, the IR1061 concentration is 10 μg / mL. Based on the encapsulation efficiency of 56.8% for IR1061, 40.6% for Lon, and 63.0% for OXA, it can be calculated that Lon (7 μg / mL) and OXA (11 μg / mL) are sufficient to 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 with reference to the following examples:

[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 obtained 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 provided by Dalian Meilun Biotechnology Co., Ltd. (Dalian, China); and human PDLSCs were purchased from iCell Bioscience Inc. (iCell Bioscience, China). Other common chemical reagents (such as buffers and culture media) 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 adhered to the technical specifications provided by the suppliers.

[0066] 2. Experimental Methods

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

[0068] Human PDLSCs were harvested and washed three times with PBS. The cells were resuspended in PBS and placed on ice-water. The cells were intermittently permeabilized using an ultrasonic cell disruptor, using 40% ultrasonic power for 1.5 minutes (1 second on, 2 seconds off) and centrifuged at 4,000 g for 30 minutes to remove cell debris. The supernatant was then 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. After sonication in an ice-water bath, the cells were repeatedly extruded through 800 nm, 400 nm, 200 nm, and 100 nm porous membranes using a liposome extruder. Finally, the drug-loaded vesicles (N@ILO) were collected by ultracentrifugation at 150,000 g for 70 minutes at 4°C.

[0069] Transmission electron microscopy (TEM) was used to observe the morphology of the drug-loaded vesicles (N@ILO). Dynamic light scattering (DLS) particle size analyzer was used to determine the hydrated particle size of the drug-loaded vesicles (N@ILO). Nanoparticle tracking analysis (NTA) was used to further characterize the particle size of the drug-loaded vesicles (N@ILO). The encapsulation efficiency of IR1064, Lon, and OXA was analyzed using UV-Vis-NIR spectrophotometry, liquid chromatography, and 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 immersed in 800 mL of boiling 0.02 M Na₂CO₃ solution for 1 hour. Insoluble residues were removed by centrifugation (3000 rpm, 10 minutes) and filtration. The resulting crude sericin extract was dialyzed against double-distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 3.5 kDa. Purified sericin protein was obtained after lyophilization. 10 g of lyophilized sericin was dissolved in 40 mL of PBS buffer (pH 8.5) and stirred for 3 hours. 2.93 g of methacrylic anhydride (MA) was dissolved in 100 mL of PBS (pH 8.5) to prepare a MA stock solution. The MA solution and sericin solution were reacted overnight at room temperature. The solution was then dialyzed against double-distilled water for 3 days using a dialysis bag with a molecular weight cutoff of 3.0 kDa. SerMA was then lyophilized and used for further processing.

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

[0073] The micromorphology of the SerMA-N@ILO freeze-dried hydrogel was observed using a Czech FEI Quanta 200 scanning electron microscope (SEM). Rheological analysis was performed using a TA-DHR-2 rheometer (TA Instruments, USA) equipped with a 405 nm UV lamp.

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

[0075] The SerMA-N@ILO precursor solution was 3D bioprinted using a digital light processing printer (nanoArchS140) equipped with a 405nm laser. A 3D CAD model was created using Solidworks software, then sliced ​​using BMF3D slicing software before being printed layer by layer. The morphology of the 3D-printed sample was first observed using a digital microscope. The printing thickness was 1600μm, the number of layers was 4, the exposure time was 30s, and the exposure intensity for the first layer was 20mW / cm. 2 , other layers are 18mW / cm 2 .

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

[0077] To systematically evaluate the photothermal conversion properties of N@ILO nanovesicles, a multi-dimensional experimental protocol was designed. N@ILO dispersions containing gradient concentrations of IR1061 (0, 5, 10, 15, 20, and 30 μg / mL) were dispensed into 1.5 mL EP tubes and irradiated with a 1064 nm near-infrared laser at a power density of 0.5 W / cm² for 5 minutes. The temperature dynamics were monitored and recorded in real time using a FLIRE60 infrared thermal imaging system. An equal volume of deionized water was used as a negative control. Furthermore, the effect of laser power densities (0.25, 0.50, 0.75, and 1.00 W / cm²) on the temperature rise 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: at a fixed power density (0.5 W / cm²), the sample was subjected to four consecutive laser on-off cycles, with the temperature fluctuations simultaneously recorded.

[0078] 5. Cytotoxicity, Apoptosis and LA Content Analysis

[0079] Cell viability was assessed using the CCK-8 assay. B16F10 cells were seeded at a density of 5×10 cells / well in a 96-well plate and pre-cultured for 24 hours at 37°C in a 5% CO2 incubator. Seven treatment groups were set up: blank (control), IR1061 (10 μg / mL), Lon (7 μg / mL), OXA (11 μg / mL), N@ILO, IR1061 (+), and N@ILO (+). The IR1061 concentration remained constant at 10 μg / mL. After 24 hours of drug exposure, the plates were irradiated with a 1064 nm near-infrared laser (power density 0.5 W / cm²) for 10 minutes in the IR1061 (+) and N@ILO (+) laser-treated groups. After each treatment, 100 μL of CCK-8 working solution was added to each well, and the cells were incubated for an additional 2 hours. The absorbance at 450 nm was measured using a Multiskan SkyHigh microplate reader.

[0080] To further verify the cell activity status, Calcein-AM / PI double staining kit was used for fluorescence analysis. 5 B16F10 cells were seeded in 6-well plates and cultured for 12 hours after adherence. The seven treatments described above were then applied, maintaining a consistent IR1061 concentration (10 μg / mL) across all groups. Co-culture was continued for 24 hours. After removing the culture medium, 2 mL of a staining solution containing 2 μM Calcein-AM and 1.5 μM PI in PBS was added to each well and incubated in the dark for 15 minutes. Fluorescence images were acquired using a confocal microscope, with green fluorescence (Calcein-AM) marking live cells and red fluorescence (PI) indicating dead cells.

[0081] For the study of cell apoptosis mechanism, B16F10 cells (1×10 6 Cells were collected (100 cells / tube) according to the instructions for the Annexin V-FITC / PI apoptosis detection kit. After washing twice with pre-chilled PBS, the cells were resuspended in 195 μL of binding buffer. 5 μL of Annexin V-FITC and 10 μL of PI staining solution were added sequentially and incubated in the dark for 15 minutes. Flow cytometry was used to analyze the proportions of early apoptotic (Annexin V⁺ / PI⁻) and late apoptotic (Annexin V⁺ / PI⁺) cells using FlowJo V software.

[0082] In addition, the LA content was detected according to the instructions of the lactate detection kit of Nanjing Jiancheng Bioengineering Institute. 5B16F10 cells were seeded in confocal microplates and incubated for 24 hours. B16F10 cells were then exposed to culture medium containing IR1061 (10 μg / mL), Lon (7 μg / mL), OXA (11 μg / mL), and N@ILO (IR1061 at 10 μg / mL). After 12 hours, the culture supernatant was separated from the cells and centrifuged at 3000 rpm for 10 minutes. The resulting supernatant (20 μL) was mixed with 1 mL of enzyme solution and 200 μL of color development reagent. After incubation for 10 minutes, 2 mL of stop solution was added. LA concentration was determined by measuring the absorbance of the reaction mixture at 530 nm using a microplate reader.

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

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

[0085] 1×10 5 B16F10 cells were seeded in 6-well plates pre-mounted on glass slides and cultured for 12 hours at 37°C in 5% CO₂. The cells were then treated with the respective intervention drugs and cultured for an additional 24 hours. Laser treatment was performed after the completion of drug treatment. Cells were washed three times with pre-chilled PBS, fixed with 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 for 10 minutes, and then blocked with 5% goat serum for 1 hour at room temperature to block nonspecific binding. Cells were then incubated with either CRT or anti-HMGB1 antibodies at 4°C overnight, followed by incubation with a secondary antibody (goat anti-rabbit IgG H&L) for 1 hour. Nuclear staining with DAPI was performed and fluorescence signals were observed using confocal microscopy.

[0086] 7. In vivo anti-tumor effect evaluation

[0087] C57BL / 6 mice (female, 6–8 weeks old) were purchased from Hunan Slake Jingda Experimental Animal Co., Ltd. (Changsha, China). 1×10 6A tumor-bearing model was established using B16F10 cells. When tumors reached 50 mm³, they were randomly divided into seven groups (n=5): Blank (control group), SerMA-IR1061 (SerMA-IR1061 is a direct mixture of SerMA and IR1061; similar descriptions apply here, with an IR1061 concentration of 10 μg / mL), SerMA-Lon, SerMA-OXA, SerMA-N@ILO, SerMA-IR1061(+), and SerMA-N@ILO(+). The Blank group received no treatment. SerMA-IR1061(+) and SerMA-N@ILO(+) groups were irradiated with a 1064 nm laser (0.5 W / cm²) for 10 minutes on days 1, 3, and 5. To simulate the clinical surgical resection of choroidal melanoma, a circular wound with a diameter of 8 mm was created at the tumor site of mice, and then a 405 nm laser was used to in situ photocuring the precursor solution to form a hydrogel. The tumor volume and mouse weight were measured every other day. On the 16th day, the mice were killed and the tumor tissue was collected for weighing and analysis. Further, the tumor tissue was subjected to histological staining and analysis. The tumor tissue was evaluated for tissue morphology by H&E staining, apoptotic cells were detected by immunofluorescence TUNEL staining, and the expression levels of calreticulin (CRT), high mobility group protein 1 (HMGB1) and PD-L1 were detected by immunohistochemistry. CD8 + T cells were stained and analyzed. In addition, serum samples were assayed for tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), IL-6, and IL-10 levels using enzyme-linked immunosorbent assay (ELISA). LA content in tumor tissue was also analyzed using the lactate detection kit provided by the Nanjing Jiancheng Bioengineering Institute. Furthermore, to assess the safety of SerMA-N@ILO, HE staining of the heart, liver, spleen, stomach, and kidney of mice in the blank and SerMA-N@ILO (+) groups was performed. Blood samples were also collected for routine blood analysis.

[0088] Tumor tissues were further analyzed by flow cytometry. The immune cell analysis process was as follows: tissue sections were enzymatically digested with a mixture of collagenase (0.5 g / L), hyaluronidase (0.3 g / L), and DNase (0.15 g / L) for 1 hour at 37°C. Cells were then passed through a 200-mesh sieve and centrifuged at 1200 rpm for 5 minutes to collect the cells. After washing with PBS, the cells were labeled with specific antibodies and analyzed by flow cytometry. Tumor-infiltrating T lymphocytes were detected using a BD FACS instrument, and the proportion of CD8+ T cells was quantified using anti-CD45-APC-Cy7, anti-CD11b-FITC, and anti-CD8-Cy5.5 antibodies. The proportion of mature dendritic cells (DCs) in tumor tissue was assessed using anti-CD11c-Qdot 605, anti-CD80-Pacific Blue, and anti-CD86-PE antibodies.

[0089] 8. Tumor tissue sequencing analysis

[0090] To systematically elucidate the molecular mechanisms of SerMA-N@ILO(+) therapy, tumor tissues were collected from tumor-bearing mice on day 7 after treatment in the control (Blank) group and the SerMA-N@ILO(+) (Test) group. These tissues were rapidly transferred to pre-frozen tubes, snap-frozen in liquid nitrogen for 5 minutes, and then stored in a -80°C ultra-low temperature freezer to maintain RNA integrity. Gene expression profiling and sequencing analysis of these samples were performed by Guangzhou Kedior Biotechnology Co., Ltd. Differentially expressed genes between the two groups were analyzed using the "edgeR" R package, with a p-value < 0.05 and a fold-difference absolute value > 1.2. Gene Ontology (GO) functional enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis were performed using the "clusterProfiler" R package to elucidate the biological processes, molecular functions, cellular components, and potential signaling pathways involved in these differentially expressed genes, respectively.

[0091] 3. Experimental Data

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

[0093] First, the stemness of the successfully isolated and cultured human periodontal ligament stem cells (PDLSCs) was identified. Immunofluorescence staining revealed that PDLSCs highly expressed stem cell-specific surface markers CD146 and STRO-1 ( Figure 1), which confirmed its good stem cell properties and laid a reliable biological foundation for the subsequent construction of cell membrane-derived nanovesicles. Subsequently, the cell membrane vesicles were extracted from PDLSCs by ultrasonic fragmentation and mixed with IR1061, Lon and OXA at ​​a mass ratio of 3:1:1:1. After being fully blended in PBS, they were repeatedly extruded through a polycarbonate membrane with a pore size of 100nm to successfully construct the multi-drug synergistic delivery nanovesicles N@ILO. Transmission electron microscopy (TEM) imaging results showed that the constructed N@ILO exhibited a typical cup-shaped vesicle structure with a regular morphology and an average particle size of approximately 100nm ( Figure 2 The particle size and dispersibility of the N@ILO were further characterized by dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA). The results showed that the N@ILO particles were uniformly distributed, with a particle size concentrated between 100 and 200 nm, and had good monodispersity ( Figure 2 To further evaluate its colloidal stability, the particle size changes were continuously monitored in PBS buffer for 9 days. The results showed that the particle size fluctuation range was small, indicating that N@ILO has excellent physical stability ( Figure 3 In terms of drug loading capacity, UV-visible-near infrared spectrophotometer, high performance liquid chromatography (HPLC) and inductively coupled plasma mass spectrometry (ICP) were used to draw standard curves and conduct quantitative analysis. The results showed that the encapsulation efficiency of IR1061 was 56.8% ( Figure 4 、 Figure 5 ), the encapsulation efficiency of Lon was 40.6% ( Figure 6 ), the encapsulation efficiency of OXA was 63.0%.

[0094] The results of this study verified that the N@ILO nanovesicles constructed based on PDLSC cell membranes exhibited excellent performance in 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.

[0095] 2. Photothermal performance evaluation of N@ILO

[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 used its loaded near-infrared II (NIR-II) photosensitizer IR1061 to achieve effective photothermal conversion under 1064nm wavelength laser irradiation. In order to verify its photothermal performance, it was first irradiated for 5 minutes under a laser power density of 0.5W / cm². The results showed that the temperature increase of the control group (H2O2 solution) was less than 2°C, while the temperature of the N@ILO group increased significantly with the increase of IR1061 concentration. When the concentration of IR1061 was 10μg / mL, the temperature rapidly increased from the initial 25.6°C to 49.8°C ( Figure 2 d in Figure 7 ), has reached the temperature threshold for inducing thermal damage or even apoptosis of tumor cells. In addition, under the conditions of laser power density of 0.25, 0.50, 0.75 and 1.00 W / cm², the system temperature gradually increases with the increase of laser power ( Figure 2 At the same time, to verify its cyclic stability, N@ILO was subjected to four rounds of laser irradiation-cooling cycle experiments. The results showed that it could be repeatedly heated in each round without significant photothermal performance degradation ( Figure 2 f), reflecting its excellent cyclic photothermal stability and thermal conversion capacity. In summary, N@ILO exhibits concentration- and power-dependent efficient photothermal conversion under near-infrared laser irradiation, along with good thermal stability, indicating its potential as a photothermal therapeutic agent for tumor treatment.

[0097] 3. In vitro anti-tumor effect

[0098] To evaluate the in vitro anti-tumor activity of N@ILO, we first used a lactate detection kit to study and found that both Lon and N@ILO could effectively reduce the LA level produced by tumors ( Figure 8 ). Then, CCK-8 assay was used to detect the cytotoxic effects of N@ILO on melanoma B16F10 cells under different concentrations of IR1061. The results showed that with the increase of IR1061 concentration, the inhibitory effect of N@ILO on B16F10 cells gradually increased, showing a typical dose-dependent cytotoxicity ( Figure 9 a in the figure). This indicates that it has a significant photothermal enhancement killing effect under near-infrared irradiation. Furthermore, a Calcein-AM / PI live / dead cell double staining experiment was used to visualize the life and death status of cells in different treatment groups. The results showed that under non-irradiated conditions, IR1061 and N@ILO had limited effects on cells, with green fluorescence being the main luminescence. However, after laser irradiation, red fluorescence was significantly enhanced in the N@ILO (+) group, indicating that a large number of cells died and cell membrane integrity was significantly damaged ( Figure 9 b in the figure). This indicates that N@ILO can effectively release heat energy under laser excitation, thereby inducing cell death. Subsequently, Annexin V-FITC / PI double staining flow cytometry was used to further evaluate its pro-apoptotic effect ( Figure 9 (c) Analysis results showed that the N@ILO (+) group induced a late apoptosis rate of 65.0%, significantly higher than the IR1061 (+) alone group (late apoptosis rate of 33.0%) and the Blank group (late apoptosis rate of only 4.2%). This suggests that N@ILO, through the synergistic effect of photothermal therapy and chemotherapy, can significantly induce programmed cell death in melanoma cells, demonstrating superior anti-tumor potential.

[0099] In addition, to explore whether it can induce immunogenic cell death (ICD), the expression of two key ICD markers was 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 conditions, acting as an "eat me" signal to induce dendritic cells to recognize tumor antigens; while HMGB1 is released from the cell nucleus to the extracellular space, acting 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 (a) shows a strong fluorescence signal, indicating that it effectively induces CRT externalization. At the same time, the HMGB1 fluorescence signal in the N@ILO (+) group is significantly weakened compared with the control group, indicating that it has been released into the extracellular space, further confirming its ability to induce ICD.

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

[0101] 4. Preparation of SerMA-N@ILO and its 3D printing performance

[0102] This study first extracted sericin from silkworm cocoons using a hot alkaline degumming method. SerMA was then chemically grafted onto MA to produce a hydrogel material known as SerMA. This SerMA hydrogel exhibits excellent biocompatibility and biodegradability, promising broad biomedical applications. Subsequently, SerMA was dissolved in PBS with a photoinitiator, LAP, and drug-loaded nanovesicles, N@ILO, and mixed uniformly to create a SerMA-N@ILO precursor solution. This solution underwent a photoinduced sol-gel transition under 405nm blue light irradiation, rapidly forming a cross-linked hydrogel ( Figure 11 The results of the rheological dynamic time scanning experiment show that under the condition of 30mW / cm² light intensity, the SerMA-N@ILO system can complete the curing process within 1 minute and has good rapid gelation performance ( Figure 11 b). Further, the microscopic morphology of the freeze-dried SerMA-N@ILO hydrogel was observed by scanning electron microscopy (SEM). Figure 11(c) The typical three-dimensional porous structure is found inside the scaffold. This structure facilitates sustained drug release, nutrient penetration, and metabolic product excretion, thereby enhancing its functionality as a bioscaffold material in the in vivo environment.

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

[0104] These results demonstrate that SerMA-N@ILO not only possesses excellent photocuring performance and a porous structure, but also can be highly integrated with photocuring 3D printing technology to achieve precise construction of personalized hydrogel scaffolds. This system has the potential to be applied in a variety of clinical scenarios, including localized tumor treatment and tissue regeneration after tumor surgery, showing promising translational potential.

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

[0106] In order to construct a multifunctional SerMA-N@ILO photosensitive hydrogel for postoperative treatment of choroidal melanoma, N@ILO nanovesicles were mixed with SerMA hydrogel precursor solution (PBS containing SerMA) by physical mixing method to prepare an injectable, photocurable SerMA-N@ILO composite system. In the experiment, scissors were used to prepare a skin defect with a diameter of approximately 8 mm in the dorsal melanoma model of C57BL / 6 mice to simulate the situation of tissue defect after clinical choroidal melanoma resection. Subsequently, the hydrogel precursor solutions of different groups were topically applied to the surgical wound and irradiated with a 405nm wavelength blue light flashlight to photocurate the hydrogel in situ at the wound site. During the treatment phase, the SerMA-IR1061 (+) and SerMA-N@ILO (+) groups were further irradiated with 1064nm laser (power density 0.5W / cm², irradiation time 10 minutes) to activate their photothermal therapeutic function. Figure 13 a and Figure 14As shown, tumor growth was inhibited in the SerMA-IR1061 (+) group and the SerMA-N@ILO (+) group, and the SerMA-N@ILO (+) group showed the most significant tumor growth inhibition effect, while the tumor volume of the other groups continued to expand. Tumor weighing results further verified the anti-tumor advantage of the SerMA-N@ILO (+) group ( Figure 13 b), indicating that combined photothermal and multi-drug synergistic therapy can significantly improve the anti-tumor efficiency.

[0107] To systematically evaluate the biosafety of SerMA-N@ILO, the weight changes of mice in each group were monitored, e.g. Figure 13 As shown in Figure c, the body weights of all treatment groups remained stable with no significant fluctuations, suggesting that the treatment process did not cause significant toxic side effects. Figure 15 ) and blood test results ( Figure 16 ) No organ damage or systemic toxicity was found, further confirming the good biocompatibility of the hydrogel.

[0108] To explore the role of SerMA-N@ILO hydrogel in inducing anti-tumor immune response, ELISA was used to detect the expression levels of related cytokines in the serum of mice after treatment. The results showed that the SerMA-N@ILO (+) group significantly upregulated the secretion of TNF-α, IFN-γ, and IL-6 after laser irradiation ( Figure 13 d~f in the figure), while the level of immunosuppressive factor IL-10 was significantly suppressed ( Figure 17 ). Among them, the increase of 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 the initiation of the initial immune response, suggesting that the hydrogel has a certain immunomodulatory potential while exerting a local photothermal therapeutic effect. In addition, further histological analysis results showed that the tumor tissue in the SerMA-N@ILO (+) group underwent obvious structural damage after receiving PTT. H&E staining images showed that the cells were disordered and the cell nuclei were dissolved ( Figure 13 TUNEL immunofluorescence staining also confirmed that a large number of cells in the tumor tissue underwent apoptosis ( Figure 13 (g), indicating that hydrogel treatment can effectively induce programmed cell death.

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

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

[0111] To further 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 the B16F10 mouse tumor model, focusing on the evaluation of dendritic cell (DC) maturation and the activation level of tumor-infiltrating T cells. Figure 18 a in Figure 19 As shown, flow cytometry results revealed that the DC maturation rate in the blank group was only 13.6%, while it increased to 22.7% in the SerMA-IR1061(+) group. The SerMA-N@ILO(+) group had the highest DC maturation rate, reaching 32.7%. Compared to the blank group, the DC maturation rate in the SerMA-N@ILO(+) group increased by 2.4 times, and was 1.4 times that of the SerMA-IR1061(+) group, suggesting that the hydrogel significantly promotes DC maturation, effectively initiating an initial immune response. Further analysis was conducted to determine T lymphocyte infiltration within the tumor microenvironment. Figure 18 Figures b~c show that the proportion of CD4⁺T cells in the Blank group was 8.1%, 13.5% in the SerMA-IR1061(+) group, and significantly increased to 24.8% in the SerMA-N@ILO(+) group, which was approximately 3.1 times that of the Blank group. Similarly, the infiltration levels of CD8⁺T cells were 3.3%, 15.4% and 22.6% in the Blank, SerMA-IR1061(+) and SerMA-N@ILO(+) groups, 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 tumor clearance effects. In addition, changes in lactic acid (LA) content in tumor tissue ( Figure 18 e in the figure), the results showed that the SerMA-N@ILO (+) group could significantly reduce the accumulation of lactic acid in the tumor site, help alleviate the immunosuppressive microenvironment, thereby enhancing the function of immune cells and improving the efficiency of anti-tumor immune response.

[0112] To further verify the occurrence of T cell infiltration and immunogenic cell death (ICD) effect, CD8⁺T cell immunofluorescence staining and CRT and HMGB1 immunohistochemistry (IHC) staining analysis were performed on tumor tissues ( Figure 18(f) The results showed that the density of red fluorescently labeled CD8⁺ T cells infiltrating tumor tissues in the SerMA-N@ILO (+) group was significantly increased, demonstrating its significant advantage in inducing a systemic immune response. Furthermore, CRT externalization expression and extracellular release of HMGB1 were significantly enhanced in the SerMA-N@ILO (+) group, consistent with in vitro experimental results. This phenomenon confirms that the SerMA-N@ILO hydrogel can induce an ICD effect in tumor cells, effectively activating the adaptive immune system and synergistically enhancing the remote immune clearance effect after photothermal therapy.

[0113] In summary, SerMA-N@ILO hydrogel can not only directly kill tumor cells through 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. Tumor tissue RNA sequencing analysis

[0115] To further reveal the potential molecular mechanisms of SerMA-N@ILO hydrogel in tumor immunotherapy, RNA sequencing (RNA-seq) analysis was performed on tumor tissues from mice in the Blank (control) group and the SerMA-N@ILO (+) group (hereinafter referred to as the Test group). Principal component analysis (PCA) plots showed that, despite some heterogeneity among tumor tissue samples, the Blank and Test groups exhibited significant separation at the transcriptome level, indicating significant differences in gene expression profiles between the two groups. Figure 20 Sample quality control analysis showed that the sequencing depth was consistent, ensuring the reliability of subsequent data analysis ( Figure 20 b, c), and also showed a high degree of overlap in gene expression between different samples. A total of 11,725 ​​commonly expressed genes were detected in the two groups of tumor tissues, of which 49 and 856 were specifically expressed genes in the Blank group and the Test group, respectively ( Figure 21 Further differential expression analysis showed that compared with the Blank group, 1,309 genes were significantly upregulated and 217 genes were significantly downregulated in the Test group ( Figure 20 d in Figure 22 The focus was on analyzing the expression changes of immune-related genes, especially genes related to "immune response". The results showed that the expression levels of several key genes closely related to immune regulation in the Test group increased significantly, indicating the immune activation effect induced by the hydrogel ( Figure 20e in the figure). To systematically analyze the signaling pathways involved in these differentially expressed genes, KEGG pathway analysis and GO enrichment analysis were further performed. KEGG analysis showed that the pathways significantly enriched in the Test group included "IL-17 signaling pathway", "TNF signaling pathway", "T cell receptor signaling pathway" and "Apoptosis", which are signaling pathways closely related to immune response and cell apoptosis ( Figure 20 At the same time, GO functional enrichment analysis showed 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 g in the .

[0116] In summary, the RNA sequencing results further confirmed the ability of SerMA-N@ILO hydrogel to induce significant anti-tumor immune responses in vivo, especially by enhancing cytokine signaling and promoting T cell activation, which synergistically increased the activation level of cytotoxic T lymphocytes (CTLs), which is consistent with the aforementioned immunogenomic and histological results.

[0117] Example 2 Optimization in the preparation of immunogenic cell death-enhancing nanovesicle hydrogels

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

[0119] Table 1. Optimization in the preparation of immunogenic cell death-enhancing nanovesicle hydrogels (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 preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drug-loaded vesicle, characterized in that: It is composed of photothermal agents, ICD-inducing chemotherapy drugs, glycolysis inhibitors and cell vesicles; The photothermal agent is IR1061; The ICD induction chemotherapy drug is oxaliplatin; The glycolysis inhibitor is lonidamine; The cell vesicles are human periodontal ligament stem cell membrane vesicles; The mass ratio of the photothermal agent, ICD-inducing chemotherapy drug, glycolysis inhibitor and cell vesicles is 1:1:1:3; The concentration of the photothermal agent is 10-30 μg / mL.

2. A hydrogel, characterized in that Composed of silk protein, a photoinitiator and the drug-loaded vesicle according to claim 1; The silk protein is methacrylated sericin; The photoinitiator is lithium phenyl-2,4,6-trimethylbenzoylphosphinate.

3. The method for preparing the hydrogel according to claim 2, wherein: The sericin, photoinitiator and drug-loaded vesicles are mixed to obtain the hydrogel.

4. Use of at least one of the following I) to III) in the preparation of a drug for wound filling and / or prevention of choroidal melanoma: 1), the drug-loaded vesicle according to claim 1; II), the hydrogel according to claim 2; III), the hydrogel prepared by the preparation method according to claim 3.

5. A drug for filling and / or preventing and treating choroidal melanoma wounds, characterized in that: The raw materials include at least one of i) to iii): i) The drug-loaded vesicle according to claim 1; ii), the hydrogel according to claim 2; iii) The hydrogel prepared by the preparation method according to claim 3.

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