Optimized light-enhanced carrier-free tumor vaccine, preparation method and application thereof

Abstract

The present application relates to a kind of light-enhanced carrier-free tumor vaccine and its preparation method and application, belong to tumor immunotherapy technical field.The light-enhanced carrier-free tumor vaccine is prepared by the precipitation of photosensitizer and immunoadjuvant in antigen protein / polypeptide cavity.The vaccine is drained to lymph node after subcutaneous administration or incubated with antigen presenting cells such as dendritic cells, and can produce singlet oxygen under light to destroy lysosome, promote intracellular transport of tumor antigen and immunoadjuvant, and enhance proteasome activity, enhance tumor antigen presentation, induce CD8 + T cell-mediated anti-tumor immune response.Therefore, the vaccine can be prepared by extracting antigen from tumor tissue removed by surgery, achieve individual customization, and can be used with PD-L1 antibody, etc., has the effects of prevention, treatment and prevention of recurrence, has the advantages of high drug loading, high safety, time and space controllable light-enhanced immune activation characteristics and one-to-one personalized treatment.

Description

Technical Field

[0001] This invention relates to the field of tumor immunotherapy technology, and in particular to a photoenhanced carrier-free tumor vaccine, its preparation method, and its application. Background Technology

[0002] Tumor vaccines are a key technology in tumor immunotherapy. Compared to traditional surgical treatments, chemotherapy, and radiotherapy, tumor vaccines have significant advantages in fighting tumors, inhibiting metastasis, and preventing recurrence. Tumor vaccines mainly include whole-cell tumor vaccines, dendritic cell vaccines, protein peptide vaccines, and gene vaccines. By providing the body with tumor antigens or neoantigens in conjunction with immune adjuvants, they can activate specific cytotoxic T cells and establish immune memory, and are considered a core means of next-generation cancer immunotherapy. However, vaccines prepared using traditional methods with cell lysates or single antigens have limited immunogenicity due to insufficient targeting in lymph nodes, low antigen uptake, and excessive degradation of antigens in lysosomes.

[0003] To overcome the limitation of insufficient lymph node targeting in tumor vaccines, researchers have developed nanotumor vaccines based on polymer or lipid carriers, improving lymph node accumulation and antigen delivery efficiency through particle size regulation and surface functionalization. However, issues such as the potential toxicity or immunogenicity of the carrier itself, limited drug loading capacity, difficulty in precisely controlling drug release, molecular and storage instability, and systemic toxicity caused by accumulation in non-target organs still hinder their clinical translation.

[0004] Tumor vaccines are first captured in the lymph nodes and transported to the lysosomes of antigen-presenting cells. However, antigen proteins can only be presented via the MHC-I pathway in the cytoplasm, and immune adjuvants also need to be in the cytoplasm to function. Therefore, achieving lysosomal escape and cytoplasmic transport of tumor vaccines is a key problem that urgently needs to be solved to achieve efficient antigen presentation. Previous studies have found that photosensitizers, under light irradiation, induce photodynamic effects to generate reactive oxygen species (ROS, such as singlet oxygen), which, through photochemical internalization, cause lysosomal rupture and induce drug transport into the cytoplasm, achieving photoresponsive cytoplasmic drug delivery. Therefore, integrating photosensitizers into tumor vaccines to construct photoenhanced tumor vaccines, by inducing ROS production through light irradiation and utilizing photochemical internalization effects to promote cytoplasmic transport of tumor antigens and immune adjuvants, can improve antigen presentation efficiency and potentially achieve a high level of anti-tumor immune response.

[0005] In summary, developing a carrier-free tumor vaccine with high lymph node targeting and strong antigen presentation efficiency, which achieves precise, controllable, and efficient lysosomal escape of tumor antigens and immune adjuvants through photochemical internalization, promotes the maturation of antigen-presenting cells, enhances antigen cross-presentation levels, effectively activates antigen-specific T cells, and achieves specific immune killing of tumor tissues, is of great significance for tumor prevention, treatment, and postoperative recurrence prevention.

[0006] Carrier-free tumor vaccines are assembled from antigens (proteins / peptides) derived from the patient's tumor, overcoming the safety risks of exogenous carriers and achieving ultra-high drug loading. However, existing technologies still face the following problems during in vivo delivery: (1) insufficient lymph node targeting and uptake efficiency by antigen-presenting cells (APCs) such as dendritic cells (DCs); (2) lysosomal capture leads to the destruction of antigen epitopes and difficulty in adjuvants reaching cytoplasmic targets; (3) limited proteasome activity in the cytoplasm restricts the presentation of major histocompatibility complex class I molecules (MHC-I). However, existing methods using pH-sensitive carriers, cationic polymers, or bacterial cleavage are not suitable for completely carrier-free systems. Therefore, there is an urgent need for a carrier-free tumor vaccine that can maintain antigen integrity while achieving light-controlled lysosomal escape and enhancing proteasome activity. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a light-enhanced carrier-free tumor vaccine, its preparation method, and its application. Specifically, it is a tumor vaccine that does not require carrier loading, can promote antigen presentation and enhance anti-tumor immune response under light conditions, and its application in the prevention and treatment of solid tumors. The tumor vaccine is formed by the self-assembly of tumor-derived protein antigen molecules into nanoparticles. Photosensitizers and adjuvants are co-precipitated within the antigen nanoparticles using the positive and negative charges and hydrophobic regions on the protein surface, forming a near-spherical carrier-free tumor vaccine (containing three components: photosensitizer, adjuvant, and protein antigen) with a size of 30-80 nm. The photosensitizer may be selected from hematoporphyrin (Hp) and hematoporphyrin derivatives, 5-aminolevulinic acid, verteporfen, temoporfen, dihydroporphyrin E6, methylene blue (MB), indocyanine green (ICG), IR-780, IR-825, hypericin, bamboo red chlorophyll, pyrophyllite-A, loxenan sodium, tetrachlorotetraiodofluorescein disodium salt, and tetrasulfonated aluminum phthalocyanine; the immunoadjuvant may be selected from SR-717 and Mn. 2+ Ions, PolyI:C, R848, Imiquimod, CpG ODN, G100, ADU-S100, MSA-2.

[0008] The tumor vaccine of this invention remains stable at pH 5.0–7.5 and is also stable under specific wavelengths of light (e.g., methylene blue excitation wavelength of 660 nm) and appropriate excitation power densities (e.g., 0.05–5.0 W·cm⁻¹). - ²) When the vaccine is administered, it generates reactive oxygen species such as singlet oxygen¹O2, which triggers lysosomal membrane rupture and enhances the degradation activity of cytoplasmic proteasomes, achieving efficient cytoplasmic transport of antigens and adjuvants. After subcutaneous injection and drainage to lymph nodes or incubation with antigen-presenting cells (including but not limited to dendritic cells, macrophages, B cells, endothelial cells, and fibroblasts), the vaccine generates singlet oxygen under light, which destroys lysosomes, promotes intracellular transport of tumor antigens and immune adjuvants, enhances proteasome activity, strengthens tumor antigen presentation, and induces CD8+. + T-cell-mediated anti-tumor immune responses produce immunotherapeutic effects. Furthermore, the light-enhanced carrier-free tumor vaccine system of this invention can achieve: (1) efficient lymph node localization and APC uptake; (2) generating an appropriate amount of reactive oxygen species under low-intensity light to disrupt lysosomal membranes, achieving cytoplasmic delivery of antigens and immune adjuvants; (3) enhancing proteasome activity through ROS, promoting effective processing of antigen peptides and enhancing MHC-I cross-presentation, thereby inducing potent CD8+. + T-cell responses are used in the treatment and prevention of solid tumors.

[0009] This invention is achieved through the following technical solution:

[0010] The first objective of this invention is to provide a light-enhanced carrier-free tumor vaccine, comprising a tumor antigen protein or polypeptide, a photosensitizer, and an immune adjuvant.

[0011] In one embodiment of the present invention, the photosensitizer is selected from one or more of the following: hematoporphyrin derivatives, 5-aminolevulinic acid, verteporfen, temoporfen, dihydroporphyrin E6, methylene blue, indocyanine green, IR-780, IR-825, hypericin, bamboo red chlorophyll, pyrophyllite-A, loxenan sodium, tetrachlorotetraiodofluorescein disodium salt, and tetrasulfonate aluminum phthalocyanine.

[0012] And / or, the encapsulation rate of the photosensitizer is 30% to 99%; preferably 70% to 99%.

[0013] In one embodiment of the present invention, the immune adjuvant is selected from SR-717 and Mn. 2+ One or more of the following: ions, PolyI:C, R848, imiquimod, CpG ODN, G100, ADU-S100, and MSA-2;

[0014] And / or, the encapsulation rate of the immune adjuvant is 30% to 99%; preferably 80% to 99%.

[0015] In one embodiment of the present invention, the tumor antigen protein or polypeptide is prepared by chemical synthesis, biosynthesis, or direct lysis of tumor cells or tissues. The tumor may be melanoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer, or thyroid cancer; the tumor antigen protein or polypeptide may include PSMA with an amino acid sequence as shown in SEQ ID NO:1, breast cancer tissue lysate, etc.

[0016] In one embodiment of the present invention, the light-enhanced carrier-free tumor vaccine is nearly spherical;

[0017] And / or, the particle size of the light-enhanced carrier-free tumor vaccine is 30-80 nm, preferably 40-70 nm.

[0018] The second objective of this invention is to provide a method for preparing the aforementioned photo-enhanced carrier-free tumor vaccine, comprising the following steps: mixing a photosensitizer, an immune adjuvant, and a tumor antigen protein or polypeptide, and forming the photo-enhanced carrier-free tumor vaccine through nanoprecipitation self-assembly;

[0019] The mass ratio of the photosensitizer, the immune adjuvant, and the tumor antigen protein or polypeptide is 0.5~20:0.5~50:100.

[0020] A third objective of this invention is to provide a photo-enhanced carrier-free tumor vaccine lyophilized powder, prepared from the photo-enhanced carrier-free tumor vaccine.

[0021] The fourth objective of this invention is to provide the application of the aforementioned photo-enhanced carrier-free tumor vaccine or the aforementioned photo-enhanced carrier-free tumor vaccine lyophilized powder in the preparation of tumor prevention or treatment drugs.

[0022] In one embodiment of the present invention, the light-enhanced carrier-free tumor vaccine is combined with light irradiation to prevent or treat tumors;

[0023] The wavelength of the illumination is 200~2000 nm, and the power density is 0.05~5.0 W·cm⁻¹. - ², the irradiation time is 0.5~60 minutes.

[0024] In one embodiment of the present invention, the tumor is melanoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer, or thyroid cancer.

[0025] Compared with the prior art, the above-described technical solution of the present invention has the following advantages:

[0026] 1) This invention provides a photoenhanced carrier-free tumor vaccine, its preparation method, and its application. The schematic diagrams illustrating the preparation principle of the photoenhanced carrier-free tumor vaccine and the mechanism by which the photoenhanced carrier-free tumor vaccine enhances the anti-tumor immunotherapy effect by achieving lysosomal escape from antigen-presenting cells such as dendritic cells are shown below. Figure 1 As shown. This invention uses the tumor antigen itself as a nanocarrier material, and after introducing photosensitizers and immune adjuvants, achieves a drug loading rate of >80%, significantly improving delivery efficiency and avoiding the potential toxicity of traditional carriers.

[0027] 2) The light-induced moderate ROS of this invention can efficiently destroy lysosomes without causing cell death, and promote the entry of antigens and adjuvants into the cytoplasm; at the same time, ROS can upregulate proteasome activity, making it easier for antigens to be processed into MHC-I binding peptides of 8-13 amino acids.

[0028] 3) After incubation and light exposure with dendritic cells (DCs), the photoenhanced carrier-free tumor vaccine of the present invention significantly improved DC maturation, MHC-I / SIINFEKL complex expression, and CD8 expression compared with the control containing only photosensitizers or adjuvants. + T-cell specific killing ability; tumor suppression, prolonged survival and immune memory were achieved in various solid tumor models such as B16-OVA, 4T1 and KPC pancreatic cancer.

[0029] 4) By injecting the vaccine subcutaneously into the lymph nodes near the groin and then applying local light, the tumor vaccine can be precisely released into the lymph nodes, reducing systemic side effects; light intensity 0.15 W·cm. - ²It has been confirmed that it causes no significant damage to normal tissues.

[0030] 5) The tumor vaccine of the present invention can be used in combination with PD-L1 antibody to achieve synergistic effect of immune checkpoint blockade and vaccine activation, and significantly improve the survival of refractory pancreatic cancer models. Attached Figure Description

[0031] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0032] Figure 1 The diagram shows the preparation principle of the photo-enhanced carrier-free tumor vaccine of the present invention (a) and the mechanism by which the photo-enhanced carrier-free tumor vaccine enhances the anti-tumor immunotherapy effect by achieving lysosomal escape in antigen-presenting cells such as dendritic cells (b).

[0033] Figure 2 The tumor vaccine of this invention OVA NV M / S , 4T1 NVI / R , PSMA NV I / R , 4T1 NV H / Mn Transmission electron microscope images;

[0034] Figure 3 This is the co-localization result of the tumor vaccine and lysosomes after laser irradiation according to the present invention; where a is OVA NV M / S Subcellular localization map within dendritic cells DC2.4; b is... OVA NV M / S Colocalization rate with lysosomes within DC2.4 cells;

[0035] Figure 4 For the present invention OVA NV M / S Results of the immune activation effect of combined light irradiation: a) shows the expression of p-TBK1 and p-IRF3 in dendritic cells (DC2.4) after different treatments; b) shows... OVA NV M / S Lymph node targeting effect; c is OVA NV M / S The combined effects of light irradiation at different wattages on reactive oxygen species production and apoptosis in lymph node tissue; d is OVA NV M / S Combined light exposure promotes dendritic cell maturation; e is OVA NV M / S Combined light exposure promotes antigen cross-presentation in dendritic cells;

[0036] Figure 5 For the present invention OVA NV M / S The therapeutic and preventive effects of combined light irradiation on B16-OVA model mice were investigated; where a is... OVA NV M / S The experimental procedure for combined light irradiation in the treatment of a B16-OVA melanoma model; b shows the graph of changes in subcutaneous tumor volume of B16-OVA after different treatments; c shows photographs of changes in subcutaneous tumor volume of B16-OVA after different treatments; d shows... OVA NV M / S Experimental procedure for the prevention of B16-OVA subcutaneous tumors by combined light exposure; e represents the change in B16-OVA subcutaneous tumor volume; f represents the mouse survival curve;

[0037] Figure 6 This is the result of an investigation into the therapeutic and preventive effects of the tumor tissue-derived tumor vaccine combined with light irradiation in 4T1 model mice; a is... 4T1 NV M / SThe experimental procedure for combined light irradiation in the treatment of 4T1 breast cancer in situ tumors; b shows the volume change of 4T1 in situ tumors in the treatment experiment; c shows the survival curve of mice in the treatment experiment; d shows... 4T1 NV M / S The experimental procedure for the prevention of 4T1 orthotopic tumors by combined light irradiation; e represents the change in volume of 4T1 orthotopic tumors in the prevention experiment; f represents the survival curve of mice in the prevention experiment;

[0038] Figure 7 This presents the results of an investigation into the therapeutic effects of different combinations of photosensitizers and immune adjuvants in combination with phototherapy on a tumor recurrence model after surgery; where a is... 4T1 NV M / S Survival curves of mice with a 4T1 breast cancer orthotopic tumor recurrence model treated with combined light irradiation; b represents... 4T1 NV I / R Survival curves of mice with a 4T1 orthotopic tumor recurrence model treated with combined light irradiation; c represents... 4T1 NV H / Mn Survival curves of mice with a 4T1 in situ tumor recurrence model after combined light irradiation. Detailed Implementation

[0039] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0040] This invention relates to tumor immunotherapy technology, specifically to a tumor vaccine that does not require a traditional carrier, can generate reactive oxygen species under light conditions to achieve antigen cytoplasmic delivery and enhance proteasome activity, and its application in the prevention and treatment of solid tumors.

[0041] In response to the challenges posed by existing technologies in the clinical treatment of tumors, which urgently require highly targeted and personalized solutions, and the problems encountered during drug delivery in vivo, such as: (1) immunogenicity caused by the carrier and limited drug loading capacity; (2) insufficient lymph node targeting and APC uptake efficiency; (3) lysosomal capture leading to destruction of antigen epitopes and difficulty for adjuvants to reach cytoplasmic targets; and (4) limited proteasome activity in the cytoplasm, which restricts MHC-I presentation, this invention provides a carrier-free tumor vaccine that can target and maintain the integrity of antigens in specific individuals with tumors while achieving light-controlled lysosomal escape and enhancing proteasome activity.

[0042] The preparation method of the photo-enhanced carrier-free tumor vaccine of the present invention is as follows:

[0043] (1) At room temperature, the whole protein antigen extracted from the tumor tissue was dissolved in phosphate buffer, and the photosensitizer and immune adjuvant were dissolved in water.

[0044] (2) Place the whole protein antigen solution on a magnetic stirrer. During the stirring process, slowly add NaOH to adjust the pH to neutral. Continue to add photosensitizer solution. After stirring for 1-20 minutes, slowly add immune adjuvant solution. Then stir at room temperature so that the two react inside the antigen protein cavity to form a precipitate, thus obtaining a light-enhanced carrier-free tumor vaccine.

[0045] In step (1) above, the concentration of the three solutions is 1~50 mg / mL.

[0046] Specifically, the total protein antigens include, but are not limited to, chicken ovalbumin OVA, highly immunogenic peptides of prostate-specific membrane antigen PSMA, 4T1 breast cancer tissue lysate, and KPC pancreatic cancer tissue lysate.

[0047] Specifically, the photosensitizers include, but are not limited to, hematoporphyrin (Hp) and hematoporphyrin derivatives, 5-aminolevulinic acid, verteporfen, temoporfen, dihydroporphyrin E6, methylene blue (MB), indocyanine green (ICG), IR-780, IR-825, hypericin, bamboo red chlorophyll, pyrophyllite-A, loxenyl anthracene sodium, tetrachlorotetraiodofluorescein disodium salt, and tetrasulfonated aluminum phthalocyanine.

[0048] Specifically, the immune adjuvants include, but are not limited to, SR-717 and Mn. 2+ Ions, PolyI:C, R848, Imiquimod, CpG ODN, G100, ADU-S100, MSA-2.

[0049] Preferably, the immune adjuvant and photosensitizer form a precipitate inside the cavity of the whole protein antigen, and then the product is purified, for example by centrifugation in an ultrafiltration tube, wherein the centrifugation speed is 1500~3000 rpm.

[0050] In the carrier-free tumor vaccine prepared by the present invention, the drug loading of photosensitizer is 0.5%~20%; preferably 3%~20%; in the carrier-free tumor vaccine prepared by the present invention, the drug loading of adjuvant is 0.5%~50%; preferably 20%~50%.

[0051] In this invention, the photosensitizer encapsulation rate in the prepared carrier-free tumor vaccine is 30%~99%; preferably 70%~99%.

[0052] In this invention, the encapsulation rate of the adjuvant in the prepared carrier-free tumor vaccine is 30%~99%; preferably 80%~99%.

[0053] In this invention, the prepared carrier-free tumor vaccine has a size of 30-80 nm, preferably 40-70 nm.

[0054] The photo-enhanced carrier-free tumor vaccine of the present invention can be made into an injection, and can also be further freeze-dried into a lyophilized powder for injection, and made into a semi-solid preparation, a solid preparation, and other dosage forms.

[0055] In this invention, the protective agents used during freeze drying include one or more of mannitol, glucose, sucrose, lactose, etc.

[0056] The tumor vaccine, after being irradiated with a laser of a specific wavelength (e.g., the optimal excitation wavelength for methylene blue is 660 nm) and an appropriate power density (e.g., 0.05~5.0 W / cm²) for a certain period of time (e.g., 0.5~60 minutes), can achieve [the desired effect]. 1 O2 production ≥30µM leads to lysosomal membrane rupture and promotes rapid entry of antigens and adjuvants into the cytoplasm; ROS simultaneously upregulates proteasome activity by 20-30%.

[0057] Specifically, the specific wavelength refers to the optimal excitation wavelength of the photosensitizer contained in the tumor vaccine.

[0058] The specific embodiments of the present invention will be described in further detail below with reference to the accompanying drawings and examples.

[0059] The following detailed embodiments and examples are used to illustrate the present invention, but do not limit the scope of the invention. The raw materials used are existing products, and the specific preparation operations, performance tests, and data analysis are all conventional techniques. Animal experiments meet the relevant requirements of Soochow University.

[0060] Example 1: Preparation of 4T1 breast cancer tissue lysate

[0061] (1) Inoculation of 4T1 breast cancer cells: 4T1 (mouse breast cancer tumor cells) in logarithmic growth phase were collected at a density of 1.0 × 10⁻⁶. 7 Cells / mL, digested and centrifuged. Using 6-8 week old female Balb / c mice as animal models, the left armpit / right leg was shaved with electric clippers, and the remaining hair was removed with depilatory cream, revealing bare smooth skin. Cell suspension was injected into the breast / right leg muscle of the mice, 50 µL per mouse.

[0062] (2) Extraction of lysate from 4T1 breast cancer tissue: tumor-bearing mice were harvested, and the tumor volume was increased to approximately 500 mm. 3At approximately 10:00 AM, euthanasia was performed and tumor tissue was harvested. The tumor tissue was minced, and blood was washed away with saline solution. The saline solution was removed by centrifugation, and 3 mL of PBS was added. The tissue was homogenized on ice for 5 minutes, then centrifuged again to remove the PBS. 1 mL of lysis buffer (containing protease and phosphatase inhibitors, diluted according to the manufacturer's instructions) was added, and the tissue was lysed at low temperature on a shaker for 60 minutes, vortexing every 15 minutes. After lysis, the tissue was centrifuged at 4°C and 15,000 rpm for 30 minutes. 3 mL of the supernatant was collected and quantified using the Beyotime BCA protein quantification kit. Based on the quantification results, the remaining supernatant was diluted with distilled water to 5 mg / mL to obtain the 4T1 breast cancer tissue lysate for later use.

[0063] Example 2: Photoenhanced Carrier-Free Tumor Vaccine OVA NV M / S Preparation

[0064] OVA NV M / S The preparation method is as follows:

[0065] (1) Solution preparation: Prepare 5 mg / mL OVA (ovalbumin) aqueous solution, 10 mg / mL MB (methylene blue) aqueous solution and 5 mg / mL SR-717 aqueous solution at room temperature.

[0066] (2) Preparation of tumor vaccine: 5 mL of OVA solution was placed on a magnetic stirrer. During stirring, 0.2 μM NaOH was slowly added to adjust the pH to neutral. Then, 200 μL of MB solution was added. After stirring for 1 min, 2 mL of SR-717 solution was slowly added. The mixture was then stirred at room temperature for 12 h to allow MB and SR-717 to react inside the cavity of the antigen protein (ovalbumin) to form a precipitate, thus obtaining the vaccine. OVA NV M / S Nanoparticles.

[0067] (3) Determination of the encapsulation efficiency of tumor vaccine nanoparticles: Take freshly prepared... OVA NV M / S Nanoparticles were centrifuged at 3500 rpm for 5 min to remove the precipitate. The supernatant was then diluted with DMSO and methanol to prepare the test sample at a specific concentration. The concentrations were then determined by UV-Vis spectrophotometry and high-performance liquid chromatography, respectively. OVA NV M / S The contents of MB and SR-717 were determined. The encapsulation efficiencies of MB and SR-717 were calculated to be 75.87% and 86.82%, respectively.

[0068] (4) Determination of drug loading capacity of tumor vaccine nanoparticles: Take freshly prepared... OVA NV M / SThe nanoparticles were centrifuged at 3500 rpm for 5 min to remove the precipitate, and then lyophilized in 50 mL centrifuge tubes. The mass of the lyophilized powder (M0) was weighed, and the masses (M) of MB and SR-717 in the lyophilized powder were calculated by UV-Vis and HPLC, respectively. The drug loading was calculated using the formula (drug loading = M / M0 × 100%). The drug loading of MB was 4.69%; the drug loading of SR-717 was 25.34%.

[0069] (5) Morphological characterization of tumor vaccines: The newly prepared... OVA NV M / S Nanoparticles were diluted to a certain concentration, dropped onto a copper mesh carbon support film, and allowed to air dry. The morphology and structure of the nanoparticles were observed using a 120 kV transmission electron microscope. The prepared nanoparticles... OVA NV M / S Transmission electron microscopy images of nanoparticles as follows Figure 2 As shown in a, the particle size of the carrier-free tumor vaccine prepared in this embodiment is 65.7 nm.

[0070] Example 3: Photoenhanced Carrier-Free Tumor Vaccine 4T1 NV I / R Preparation

[0071] This embodiment describes the preparation of a light-enhanced carrier-free tumor vaccine. 4T1 NV I / R Similar to Example 2, the only difference is the selection of different components: the whole protein antigen is the lysate of 4T1 breast cancer tissue extracted according to Example 1, the photosensitizer is ICG, and the immune adjuvant is R848; the rest is the same as in Example 2.

[0072] Prepared 4T1 NV I / R Transmission electron microscopy images of nanoparticles as follows Figure 2 As shown in b in the figure, the particle size of the carrier-free tumor vaccine prepared in this embodiment is 48.4 nm.

[0073] Example 4: Photoenhanced Carrier-Free Tumor Vaccine PSMA NV I / R Preparation

[0074] This embodiment describes the preparation of a light-enhanced carrier-free tumor vaccine. PSMA NV I / R The specific preparation method is as follows:

[0075] (1) Solution preparation: Prepare 5 mg / mL PSMA aqueous solution, 2 mg / mL ICG aqueous solution and 1 mg / mL LR848 aqueous solution at room temperature. The amino acid sequence of PSMA (prostate-specific membrane antigen) is shown in SEQ ID NO:1, specifically: KYADKIYSI; the PSMA is the amino acid fragment of the full-length human prostate-specific membrane antigen (PSMA) shown in UniProt database accession number Q04609.1 (GI: 548615), which was chemically synthesized by Nanjing Jietai Biotechnology Co., Ltd.

[0076] (2) Preparation of tumor vaccine: 5 mL of PSMA solution was placed in a water bath sonicator, 10 μL of Tween 80 was added, and after sonication for 1 min, 200 μL of ICG solution was added, followed by the slow addition of 2 mL of R848 solution. The mixture was then sonicated in an ice bath for 10 min to obtain the vaccine. PSMA NV I / R Nanoparticles, also known as photo-enhanced carrier-free tumor vaccines.

[0077] Prepared PSMA NV I / R Transmission electron microscopy images of nanoparticles as follows Figure 1 As shown in c, the particle size of the light-enhanced carrier-free tumor vaccine prepared in this embodiment is 45.5 nm.

[0078] Example 5: Photoenhanced Carrier-Free Tumor Vaccine 4T1 NV H / Mn Preparation

[0079] This embodiment prepared a light-enhanced carrier-free tumor vaccine, similar to Example 2, except that the selected components were different: the source of the whole protein antigen was the lysate of 4T1 breast cancer tissue extracted according to Example 1, the photosensitizer was heme, and the immune adjuvant was MnCl2·4H2O (purchased from Saen Chemical Technology (Shanghai) Co., Ltd., catalog number DH270043).

[0080] The prepared light-enhanced carrier-free tumor vaccine 4T1 NV H / Mn Transmission electron microscope images such as Figure 2 As shown in d, the particle size of the light-enhanced carrier-free tumor vaccine prepared in this embodiment is 42.4 nm.

[0081] Example 6 OVA NV M / S Investigation of lysosomal escape under combined light irradiation

[0082] Investigating using fluorescence imaging OVA NV M / S The lysosomal escape ability is specifically implemented as follows:

[0083] (1) Coupling of fluorescent probe: Accurately weigh 5.0 mg of rhodamine and dissolve it in 0.5 mL of DMSO to prepare a 10 mg / mL rhodamine solution; take freshly prepared... OVA NV M / S The concentration of OVA protein was quantified using a BCA kit. Rhodamine solution was added to the nanoparticles at a ratio of 0.01 mg rhodamine-labeled 1 mg protein, with stirring applied. OVA NV M / S In nanoparticles, after stirring overnight, OVA NV M / S Ultrafiltration purification was performed to finally obtain OVA NV M / S -RhB nanoparticles.

[0084] (2) Cytoplasmic transport of antigens: DC2.4 (mouse dendritic cells derived from mouse transgenic tumors) were seeded into confocal glass dishes (1×10⁻⁶ cells / mL). 5 (1 cell / well), add 1 mL of DMEM medium (brand: Gibco; catalog number: C11995500BT) containing 10% FBS to each well, and incubate overnight. Then, set up a non-light group and a light group, both of which added MB at a concentration of 5 μg / mL. OVA NV M / S After incubation in RhB solution in the dark for 0, 0.5 h, 1 h, 3 h, and 6 h, the drug-containing culture medium was removed, and the cells were washed three times with PBS and replaced with fresh culture medium. The non-light-treated group continued culturing, while the light-treated group was exposed to light (660 nm, 0.15 W / cm²). 2 After 3 min of light exposure, the cells were incubated for another 2 h, and both groups of cells were stained simultaneously. First, the nuclear dye Hurst fluorescent dye 33342 (5 μg / mL, 1 mL) was added and incubated in the dark for 6 min. After washing three times with PBS, the lysosomal tracer red fluorescent probe (50 nM, 1 mL) was added and incubated in the dark for 10 min. After washing three times with PBS, the fluorescence distribution in the cells before and after light exposure was immediately observed using CLSM.

[0085] The colocation rates of the illuminated group at 0, 0.5 h, 1 h, 3 h, and 6 h were 89.77%, 76%, 70.93%, 59.7%, and 36.17%, respectively; while those of the unilluminated group at 0, 0.5 h, 1 h, 3 h, and 6 h were 89.27%, 88.53%, 85.3%, 79.83%, and 72.4%, respectively. (Note: The last sentence appears to be incomplete and possibly refers to a specific concentration of W / cm².) 2 Three minutes after laser irradiation, the colocalization rate of the tumor vaccine with lysosomes decreased significantly compared to the non-light-irradiated group, dropping to 36.17% within 6 hours (see details). Figure 3This indicates that the prepared tumor vaccine can effectively destroy the lysosomal membrane after being exposed to light, enabling lysosomal escape and cytoplasmic transport.

[0086] Example 7 OVA NV M / S Investigation of the immune activation effect of combined light irradiation

[0087] (1) Western blotting was used to investigate the effects of... OVA NV M / S The expression of key proteins in the STING pathway in cells after action on DC2.4 was investigated using the following method:

[0088] 1) Cell drug delivery: DC2.4 cells were seeded into 6-well plates (2.0 × 10⁻⁶ cells / well). 5 (cells / well), after the cells adhered, the following groups were set up: PBS group (control group), OVA group, OVA+SR-717 group, OVA+MB physical mixture + light group. OVA NV M / S Group, OVA NV M / S +Light group, administered SR-717 at 25 μg / mL, MB concentration corresponding to OVA NV M / S The dosage of MB was determined. Cells were co-incubated with each group of drugs for 12 h, then the medium was replaced with fresh medium. The non-light-treated groups received no treatment, while the light-treated groups were exposed to light (660 nm, 0.15 W / cm²). 2 Cells were collected 3 min later and 12 h later.

[0089] 2) Protein Sample Preparation: Add 300 μL of cell lysis buffer (containing protease inhibitors and phosphatase inhibitors, added to the lysis buffer according to the dilution ratio specified in the instructions) to each of the cell samples collected in the previous step. Incubate on a shaker for 45 min at low temperature for lysis, followed by centrifugation at 15,000 rpm for 15 min. Collect the supernatant. Quantify the protein concentration in the supernatant using a BCA kit and dilute the samples to the same concentration with cell lysis buffer. Take 200 μL of each sample, add 50 μL of 5× loading buffer, vortex to mix, heat in a 95°C water bath for 15 min to completely denature the protein, and store at -20°C.

[0090] 3) SDS-PAGE gel electrophoresis: First, prepare a 10% separating gel, add it to a glass plate, and seal it with 1 mL of anhydrous ethanol. After the separating gel solidifies, prepare a 10% stacking gel, add it to a glass plate, and insert a 15-well sampler. After the stacking gel solidifies, place it in an electrophoresis tank filled with electrophoresis buffer, remove the sampler, take 3 μL of pre-stained protein molecular weight standard, add it to the sample well, and add 20 μg of protein sample to the remaining wells. Electrophoresis at 80 V for 2.5 h until the bromophenol blue is close to the bottom of the separating gel.

[0091] 4) Transfer: Prepare a 60×80 mm polyvinylidene fluoride (PVDF) membrane and activate it by soaking it in methanol for 2 min. Remove the gel after electrophoresis, cut off the excess, place the black plate of the transfer clamp at the bottom, and then place the sponge, filter paper, gel, PVDF membrane, filter paper, and sponge in sequence. Remove any air bubbles between the gel and the PVDF membrane, fix the transfer clamp, insert it into the transfer tank, fill the tank with rapid transfer buffer, and transfer at a constant current of 400 mA for 45 min.

[0092] 5) Blocking: After the transfer is completed, take out the PVDF membrane, rinse with TBST for 5 min, immerse the membrane in blocking solution (5% skim milk powder), place it on a shaker, block at room temperature for 1.5 h, rinse with TBST 3 times, 5 min each time.

[0093] 6) Antibody incubation: Based on the bands displayed by the pre-stained protein molecular weight standard, identify the internal control protein and target protein bands. Cut the PVDF membrane and place it in the antibody incubation box, add the corresponding primary antibody, and incubate overnight at 4°C. Wash three times with TBST, then add secondary antibody diluted with 5% skim milk powder, incubate at room temperature for 1.5 h, and wash three times with TBST.

[0094] 7) Development: Apply freshly prepared ECL (enhanced chemiluminescence reagent) developer solution evenly to the surface of the PVDF membrane, develop it in a Bio-rad gel imaging system, set an appropriate exposure time, and continue until the bands are clear.

[0095] Thanks to the rapid cytoplasmic transport following light exposure as described in Example 4, SR-717 efficiently targets the STING protein in the endoplasmic reticulum, activating downstream TBK1. Activated TBK1 then autophosphorylates and recruits and phosphorylates interferon regulatory factor 3 (IRF3), which translocates to the nucleus to drive the transcription of the type I interferon gene, leading to the secretion of type I interferon. Type I interferon regulates CD8... + T cells play a crucial role in cross-presentation of T cells and in protective immunity against tumors and viral infections.

[0096] The expression of p-TBK1 and p-IRF3 in dendritic cells (DC2.4) after different treatments is as follows: Figure 4 As shown in Figure a, it can be seen that the IRF-3 bands in the illumination group exhibit a trend from weak to strong. Among them, OVA NV M / S The lighting treatment group showed the best results.

[0097] (2) The marker imaging method was used to investigate OVA NV M / S The lymph node targeting effect is achieved. The specific implementation method is as follows:

[0098] 1) Prepare Cy7 (anthocyanin 7) solution and use the BCA kit to... OVA NV M / S The protein in the sample was quantified at a ratio of 0.01 mg Cy7 labeled with 1 mg of protein. OVA NV M / S After labeling, the mixture was stirred at room temperature for 4 h, and then the free fluorescein was removed by ultrafiltration using a 10 kD ultrafiltration tube. Cy7-labeled free OVA was obtained using the same method.

[0099] 2) Cy7 markers were successfully injected bilaterally into the groin of normal C57BL / 6 female mice via subcutaneous injection. OVA NV M / S The dosage was 100 μg / animal (calculated based on the OVA dosage). After 12 h of injection, the animals were euthanized, and the heart, liver, spleen, lungs, kidneys, and inguinal lymph nodes were dissected. Small animal fluorescence imaging system was used to photograph them, with the excitation wavelength set at 438 nm and the emission wavelength at 512 nm. The fluorescence intensity of different organs was counted.

[0100] Imaging results showed that the fluorescence intensities of the free OVA group in the heart, liver, spleen, lungs, kidneys, and lymph nodes were (unit: ×10). 7 p / s / cm 2 / sr): 1.10, 4.83, 1.06, 1.55, 5.51, 25.27; OVA NV M / S The fluorescence intensities of the group in the heart, liver, spleen, lung, kidney, and lymph nodes were respectively (unit: ×10). 7 p / s / cm 2 / sr): 0.92, 3.57, 1.09, 1.32, 4.24, 42.49. See Table 1 for details.

[0101] Table 1 OVA NV M / S Lymph node targeting effect (unit: ×10) 7 p / s / cm 2 / sr)

[0102]

[0103] Fluorescence intensities in various organs of mice in the free OVA group and the tumor vaccine group are as follows: Figure 4 As shown in b in the diagram. Where, OVA NV M / S The fluorescence intensity in mouse lymph nodes was increased by 68.14% compared to free OVA, indicating that the tumor vaccine prepared in this invention successfully achieved lymph node accumulation.

[0104] (3) DHE staining and TUNEL staining were used to investigate OVA NV M / S The effects of reactive oxygen species (ROS) production and apoptosis on DC2.4 were investigated. The specific implementation method is as follows:

[0105] 1) DHE staining: DC2.4 was seeded in a confocal glass dish (1.0 × 10⁻⁶). 5 (1 cell / well), add 1 mL of DMEM medium containing 10% FBS to each well, incubate overnight, and then set up a control group, OVA NV M / S Non-illuminated group, OVA NV M / S 0.15 W / cm 2 Light group and OVA NV M / S 0.3 W / cm 2 Light-treated group. After incubation at a final MB concentration of 5 μg / mL for 6 h, the drug-containing culture medium was removed, and the cells were washed three times with PBS. Fresh culture medium was then added, and the cells were exposed to light (660 nm, 0.15 W / cm²). 2 Immediately after illumination, all three groups were stained simultaneously. Dihydroethidium (DHE) staining solution (10 μM, 1 mL) was added to a glass dish and incubated in the dark for 10 min. The staining solution was then removed, and the cells were washed three times with PBS. Subsequently, Hoechst 33342 nuclear staining solution (1 μg / mL, 1 mL) was added and incubated in the dark for 8 min. The staining solution was removed, and the cells were washed three times with PBS. The intensity of intracellular red fluorescence was immediately observed using a confocal laser microscope (CLSM). (Note: The excitation wavelength of DHE is 488 nm.)

[0106] 2) TUNEL staining: Follow the steps in (1) for cell seeding, drug administration, and illumination. Immediately after illumination, add 4% paraformaldehyde to each well and fix cells at room temperature for 20 min. Discard the fixative and wash three times with PBS for 5 min each time. Discard the PBS and add pre-cooled 0.1% Triton X-100 solution to each well, incubate at 4℃ for 10 min. Discard the permeabilization solution and wash three times with PBS for 5 min each time. Add the prepared TUNEL reaction solution, place in a light-protected humidified chamber, and incubate at 37℃ for 60 min, avoiding light throughout to prevent fluorescence quenching. After incubation, discard the reaction solution and wash cells three times with PBS for 5 min each time. Discard the PBS and add 500 μL of DAPI staining solution (5 μg / mL) to each well, incubate at room temperature in the dark for 10 min. Discard the DAPI staining solution and wash twice with PBS for 5 min each time to remove excess dye. Immediately observe the fluorescence intensity using a laser confocal microscope (CLSM).

[0107] Laser confocal imaging results as follows Figure 4 As shown in c in the figure. 0.15 W / cm 2 The laser only induces a small amount of superoxide anion production, with virtually no cell apoptosis. This indicates that the present invention has good biocompatibility under light-free conditions; at 0.15 W / cm²... 2 It is basically biosafety under laser operating conditions.

[0108] Example 8 OVA NV M / S Investigation on cell maturation and antigen presentation effects after incubation with dendritic cells combined with light irradiation

[0109] 1) Dendritic cell maturation: DC2.4 cells in logarithmic growth phase were dispersed into a single-cell suspension and seeded in six-well plates (2 mL of DMEM medium containing 10% FBS per well), at a cell density of 2.0 × 10⁵ cells / well. The cells were incubated overnight until fully adhered. Then, the cells were grouped into four groups: PBS group, OVA group, OVA + SR-717 group, and OVA + MB physical mixture + light group. OVA NV M / S Group, OVA NV M / S In the light-exposed group, SR-717 was administered at a concentration of 25 μg / mL, and the corresponding MB concentration was... OVA NV M / S After incubating with the appropriate dose of MB in the dark for 12 hours, the drug-containing culture medium was removed and fresh culture medium was added. The non-light-treated group received no treatment, while the light-treated group was exposed to light (660 nm, 0.15 W / cm²). 2After culturing for 12 h, cells were collected. Flow cytometry antibodies against CD80-PE and CD86-FITC (diluted 1:300 with PBS containing 2% FBS) were added. Cells were stained at 37°C in the dark for 30 min, then centrifuged to collect the cells. After washing twice with PBS, the cells were resuspended in PBS containing 2% FBS. The proportion of CD80 and CD86 double-positive cells in each group was detected by flow cytometry.

[0110] 2) Antigen cross-presentation in dendritic cells: Cell culture, grouping, drug administration, and illumination were performed according to method (1). After 12 h of illumination, cells were collected, and flow cytometry antibody for SIINFEKL-H2Kb-PE (diluted 1:300 with PBS containing 2% FBS) was added. After staining at 37°C in the dark for 30 min, cells were collected by centrifugation, washed twice with PBS, and resuspended in PBS containing 2% FBS. The expression of SIINFEKL-H2Kb in each group was detected by flow cytometry. The results of flow cytometry measurements are shown in Table 2.

[0111] Table 2. Experimental results of tumor vaccine promoting DC maturation and antigen presentation

[0112]

[0113] The experimental results show that, OVA NV M / S +The light-exposed group showed the best effect in promoting DC maturation and antigen presentation. For example... Figure 4 As shown in d and e.

[0114] Example 9 OVA NV M / S Efficacy of combined light irradiation in the treatment and prevention of B16-OVA model mice

[0115] (1) Investigation OVA NV M / S The specific implementation method for the treatment effect of B16-OVA is as follows:

[0116] 1) Establishment of a mouse skin cancer model: B16-OVA cells (mouse melanoma cells with high ovalbumin expression) in logarithmic growth phase were taken, resuspended in serum-free 1640 medium, and the cell density was adjusted to 1.2 × 10⁻⁶ cells / year. 6 Cells / mL. Using 3-5 week old C57BL / 6 female mice as an animal model, the backs of the mice were shaved, and 50 μL of cell suspension was injected subcutaneously into the shaved backs of each mouse. When the tumor size reached 80 mm... 3 When the time is left or right, the experiment can be conducted.

[0117] 2) Investigation of the tumor-suppressing effect of B16-OVA: 21 tumor-bearing mice were used, and the tumor volume was approximately 80 mm. 3Mice were randomly divided into 3 groups of 7 mice each: PBS group, MB+SR-717+OVA physical mixture group, and light group. OVA NV M / S Light irradiation group. The initial tumor volume was recorded for each group before drug administration. Using the PBS group as a control, mice in each group were subcutaneously administered SR-717 (30 mg / kg) bilaterally in the groin every 7 days. Twelve hours after administration, the lymph nodes in the light irradiation group were irradiated with a 660 nm laser at an intensity of 0.15 W / cm². 2 The light exposure time was 3 minutes, and the non-light-exposed group received no treatment. The length and width of the tumor in each mouse were recorded regularly, and the tumor volume was calculated. At 18 days, all mice were euthanized, and the tumors were dissected and photographed under white light.

[0118] Based on the experimental results, the tumor inhibition rate of the MB+SR-717+OVA physical mixture + light irradiation group was calculated to be 38.10%. OVA NV M / S The tumor inhibition rate in the light-irradiated group was 71.58%, such as Figure 5 As shown in a, b, and c. During the 18-day observation period, the... OVA NV M / S The invention achieved good anti-tumor effects under light irradiation, verifying that it has a highly efficient and specific immune stimulation ability.

[0119] (2) Investigation OVA NV M / S The specific implementation method for the preventive effect against B16-OVA is as follows:

[0120] 1) Normal C57BL / 6 female mice were randomly divided into 3 groups of 7 mice each. The specific groups were: PBS group, MB+SR-717+OVA physical mixture + light group, and... OVA NV M / S In the light-treated group, mice were immunized with SR-717 30 mg / kg via bilateral subcutaneous injection in the groin. Twelve hours after administration, the lymph nodes of the mice in the light-treated group were irradiated with a 660 nm laser (0.15 W / cm²). 2 Administer the medication once every 7 days (3 min), for a total of 3 doses.

[0121] 2) Seven days after the last inoculation, prepare a concentration of 1.2 × 10⁻⁶. 6 Melanoma B16-OVA cell suspension (containing cancer cells and PBS) was administered at a rate of 50 μL / mL. Mice were dehaired on their backs, and the cell suspension was injected subcutaneously into the dehaired backs. Tumor volume was measured every two days after inoculation, and mouse survival curves were monitored.

[0122] Experimental results are as follows Figure 5 As shown in d, e, and f, Table 3 is calculated based on the results.

[0123] Table 3 OVA NV M / S Preventive effect against B16-OVA

[0124]

[0125] It can be seen that, compared with other groups, OVA NV M / S The light-exposed group showed the greatest inhibition rate of tumor growth in mice and the longest survival time, verifying that the present invention has a good tumor prevention effect.

[0126] Example 10: Evaluation of the therapeutic and preventive effects of tumor tissue-derived tumor vaccines combined with light irradiation in 4T1 model mice.

[0127] A photoenhanced carrier-free tumor vaccine for the treatment of homologous tumors was developed by replacing the model antigen OVA with a specific tumor antigen. The photoenhanced carrier-free tumor vaccine prepared in this embodiment is similar to that in Example 2, except for the selected components: the whole-protein antigen is lysate of 4T1 breast cancer tissue extracted according to Example 1, the photosensitizer is methylene blue, and the immunoadjuvant is SR-717. 4T1 NV M / S Nanoparticles.

[0128] (1) Investigation 4T1 NV M / S The specific implementation method for the treatment effect of 4T1 is as follows: Take tumor-bearing mice and model them as described in step (1) of Example 1. When the tumor volume is about 80 mm 3 At that time, the tumor-bearing mice were divided into 3 groups, with 7 mice in each group. The specific groups were: PBS group, 4T1 NV M / S Group, 4T1 NV M / S Light-exposed group. The PBS group served as a negative control, and the group was not exposed to light. 4T1 NV M / S The first group served as a positive control. The initial tumor volume was recorded for each group before administration. Then, SR-717 30 mg / kg was administered subcutaneously to the groin. Twelve hours after administration, the lymph nodes in the light-irradiated group were irradiated with a 660 nm laser at an intensity of 0.15 W / cm². 2 The light exposure time was 3 minutes; the non-light exposure group received no light. The length and width of the tumor in each mouse were recorded periodically, and the tumor growth volume was calculated and a tumor volume growth curve was plotted. When the mouse tumor volume reached 1500 mm... 3 In accordance with the ethical requirements for laboratory animals, euthanasia was performed, and the number of days since the mice died was recorded and survival curves were plotted.

[0129] Experimental results are as follows Figure 6 As shown in a, b, and c, Table 4 is calculated based on the results.

[0130] Table 4 4T1 NV M / S Treatment efficacy for 4T1

[0131]

[0132] It is evident that, without illumination 4T1 NV M / S It could only moderately delay tumor progression; all mice died within 28 days due to excessive tumor size. 4T1 NV M / S Combined light irradiation showed excellent immunotherapy effects, extending the average survival time of tumor-bearing mice to 51.43 days.

[0133] (2) Investigation 4T1 NV M / S The specific implementation methods for preventing 4T1 are as follows:

[0134] 1) Take 21 healthy mice and randomly divide them into 3 groups of 7 mice each. The specific groups are: PBS group, non-light-exposed group, and non-light-exposed group. 4T1 NV M / S Group, 4T1 NV M / S Light irradiation group. Mice in each group were subcutaneously administered SR-717 (30 mg / kg) bilaterally in the groin every 7 days. Twelve hours after administration, the lymph nodes in the light irradiation group were irradiated with a 660 nm laser at an intensity of 0.15 W / cm². 2 The light exposure time was 3 minutes; the non-light-exposed group received no treatment. A total of three vaccinations were administered.

[0135] 2) Seven days after the last inoculation. Prepare a concentration of 1.2 × 10⁻⁶. 7 A 4T1 cell suspension (containing 4T1 cells and PBS) was administered at a rate of 50 µL / mL to mice after hair removal from the left axilla / right leg and injection into the mammary / right leg muscle. Tumor volume was measured every two days after inoculation, and mouse survival curves were monitored.

[0136] Experimental results are as follows Figure 6 As shown in d, e, and f, Table 5 is calculated based on the results.

[0137] Table 5 4T1 NV M / S Prevention of 4T1

[0138]

[0139] Compared with other groups, 4T1 NV M / S The light-exposed group showed the greatest inhibition rate of tumor growth in mice, and the mice also had the longest survival time. Therefore, this demonstrates that the present invention has a good tumor prevention effect.

[0140] Example 11: Investigation of the therapeutic effect of different combinations of photosensitizers and immune adjuvants combined with tumor vaccines and light irradiation on a tumor recurrence model after surgery.

[0141] To demonstrate the universality and recurrence-inhibiting effect of the photo-enhancing properties of this invention in the application of various photosensitizers and immune agonists in tumor treatment, a study was conducted... 4T1 NV M / S , 4T1 NV I / R , 4T1 NV H / Mn A tumor suppression experiment was conducted using a 4T1 tumor recurrence model. The specific implementation method is as follows:

[0142] (1) Establishment of mouse breast cancer orthotopic tumor model: 4T1 tumor cells in logarithmic growth phase were collected at a density of 1.0 × 10⁻⁶. 7 Cells / mL, digested and centrifuged. Using 35 healthy female Balb / c mice around 6-8 weeks old as an animal model, hair was removed from the left armpit / right leg of the mice, and the cell suspension was injected into the breast / right leg muscle of each mouse, 50 µL.

[0143] (2) Investigation of the tumor-suppressing effect of orthotopic / subcutaneous breast cancer tumors: Tumor-bearing mice were used until the tumor volume reached approximately 100 mm. 3 In this procedure, most of the tumor tissue was surgically removed, leaving a small amount of tissue to simulate postoperative recurrence. The removed tumor tissue was prepared according to the methods described in Examples 2, 3, and 7. 4T1 NV I / R , 4T1 NV H / Mn , 4T1 NV M / S Tumor-bearing mice were divided into 7 groups, with 5 mice in each group. The specific groups were: PBS group, ... 4T1 NV M / S Group, non-light 4T1 NV I / R Group, non-light 4T1 NV H / Mn Group, 4T1 NV M / S +Lighting group 4T1 NV I / R +Lighting group 4T1 NV H / Mn+ Irradiation group. The initial tumor volume was recorded for each group before drug administration. Subcutaneous drug administration was then performed in the groin area. Twelve hours after drug administration, the lymph nodes in the irradiation group were irradiated with a 660 nm laser at an intensity of 0.15 W / cm². 2 The light exposure time was 3 minutes; the non-light exposure group received no light. When the mouse tumor volume reached 1500 mm... 3 In accordance with the ethical requirements for laboratory animals, euthanasia was performed, and the number of days since the mice died was recorded and survival curves were plotted.

[0144] Experimental results are as follows Figure 7 As shown in a, b, and c, Table 6 is calculated based on the results.

[0145] Table 6 The therapeutic effects of different photosensitizer and immune adjuvant combination vaccines on tumor recurrence models

[0146]

[0147] As can be seen, the survival time of the light-exposed group was significantly longer than that of the non-light-exposed group, which verifies the universality of the present invention in the application of different photosensitizers and immune adjuvants in tumor treatment and its significant effect in inhibiting treatment recurrence.

[0148] Due to the highly complex biological characteristics of tumors involving multiple genes and multiple steps, it is crucial and urgent to establish one-to-one clinical treatment plans to block the development and progression of tumors in each individual. Furthermore, it is necessary to address three major problems encountered by existing tumor treatment technologies in vivo delivery: (1) insufficient lymph node targeting and antigen-presenting cell (APC) uptake efficiency; (2) lysosomal capture leading to the destruction of antigen epitopes and difficulty in adjuvants reaching cytoplasmic targets; and (3) limited proteasome activity in the cytoplasm, restricting the presentation of major histocompatibility complex class I molecules (MHC-I). Moreover, a comprehensive upgrade of existing carrier systems utilizing pH-sensitive carriers, cationic polymers, or bacterial cleavage membranes is required. Therefore, this invention provides a photo-enhanced carrier-free tumor vaccine. As it is a carrier-free system based on one-to-one individualized treatment, widely adaptable to tumor antigens from different sources, and constructed together with photosensitizers and immune adjuvants, it possesses high load capacity, high safety, and spatiotemporally controllable photo-enhanced immune activation characteristics, providing a novel treatment option with clinical translational potential for the prevention, treatment, and recurrence prevention of solid tumors.

[0149] Obviously, the above embodiments are merely examples for clearly illustrating the present invention and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A photo-enhanced carrier-free tumor vaccine, characterized in that, These include tumor antigen proteins or peptides, photosensitizers, and immune adjuvants.

2. The photo-enhanced carrier-free tumor vaccine according to claim 1, characterized in that, The photosensitizer is selected from one or more of the following: hematoporphyrin derivatives, 5-aminolevulinic acid, verteporfen, temoporfen, dihydroporphyrin E6, methylene blue, indocyanine green, IR-780, IR-825, hypericin, bamboo red chlorophyll, pyrophyllite-A, loxenan sodium, tetrachlorotetraiodofluorescein disodium salt, and tetrasulfonate aluminum phthalocyanine. And / or, the encapsulation rate of the photosensitizer is 30% to 99%; preferably 70% to 99%.

3. The photo-enhanced carrier-free tumor vaccine according to claim 1, characterized in that, The immune adjuvant is selected from SR-717 and Mn. 2+ One or more of the following: ions, PolyI:C, R848, imiquimod, CpG ODN, G100, ADU-S100, and MSA-2; And / or, the encapsulation rate of the immune adjuvant is 30% to 99%; preferably 80% to 99%.

4. The photo-enhanced carrier-free tumor vaccine according to claim 1, characterized in that, The tumor antigen protein or polypeptide is obtained through chemical synthesis, biosynthesis, or direct lysis of tumor cells or tissues.

5. The photo-enhanced carrier-free tumor vaccine according to claim 1, characterized in that, The light-enhanced carrier-free tumor vaccine is nearly spherical; And / or, the particle size of the light-enhanced carrier-free tumor vaccine is 30-80 nm, preferably 40-70 nm.

6. The method for preparing the photo-enhanced carrier-free tumor vaccine according to any one of claims 1-5, characterized in that, Includes the following steps: The photosensitizer, immune adjuvant, and tumor antigen protein or peptide are mixed and self-assembled by nanoprecipitation to form the light-enhanced carrier-free tumor vaccine. The mass ratio of the photosensitizer, the immune adjuvant, and the tumor antigen protein or polypeptide is 0.5~20:0.5~50:

100.

7. A photo-enhanced carrier-free tumor vaccine lyophilized powder, prepared from the photo-enhanced carrier-free tumor vaccine according to any one of claims 1-5.

8. The use of the photo-enhanced carrier-free tumor vaccine according to any one of claims 1-5 or the lyophilized powder of the photo-enhanced carrier-free tumor vaccine according to claim 7 in the preparation of tumor prevention or treatment drugs.

9. The application according to claim 8, characterized in that, The light-enhanced carrier-free tumor vaccine, combined with light irradiation, can prevent or treat tumors. The wavelength of the illumination is 200~2000 nm, and the power density is 0.05~5.0 W·cm⁻¹. - ², the irradiation time is 0.5~60 minutes.

10. The application according to claim 8, characterized in that, The tumors mentioned are melanoma, breast cancer, pancreatic cancer, colon cancer, lung cancer, liver cancer, stomach cancer, esophageal cancer, bladder cancer, cervical cancer, ovarian cancer, prostate cancer, kidney cancer, or thyroid cancer.

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