Cascade response type immunomodulators and their applications

Abstract

The present invention relates to the field of biomedical technologies, and particularly relates to a preparation method and application of a cascade-responsive immunomodulator. After being taken up by tumor cells, the cascade-responsive immunomodulator of the present invention can undergo a cyclization reaction with overexpressed NQO1 in tumors under light illumination, and then release an active agonist to trigger an antigen-specific T cell anti-tumor immune response. Meanwhile, the singlet oxygen and heat generated by the photosensitizer are respectively used for photodynamic therapy and photothermal therapy. The cascade-responsive immunomodulator can achieve synergistic treatment of phototherapy and immunotherapy. The immunomodulator has high specificity for tumor cells and has very broad application prospects in designing tumor-targeted therapeutic materials, activating anti-tumor immune responses, etc.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to the preparation of a cascade-responsive immunomodulator and its application in cancer photo-immunotherapy. After being taken up by tumor cells, the cascade-responsive immunomodulator of the present invention can undergo a cyclization reaction with overexpressed NQO1 in tumors under light irradiation, and then release active agonists to trigger antigen-specific T cell anti-tumor immune responses. At the same time, the singlet oxygen and heat generated by the photosensitizer are used for photodynamic therapy and photothermal therapy respectively. The cascade-responsive immunomodulator can achieve synergistic therapy of phototherapy and immunotherapy. The immunomodulator has high specificity for tumor cells and has very broad application prospects in designing tumor-targeted therapeutic materials, activating anti-tumor immune responses, etc. Background Art

[0002] In recent years, immunotherapy, which inhibits tumor growth and metastasis by stimulating the host innate immune system and adaptive immune system and generating long-term immune memory effects, has been widely studied and achieved certain results. However, limited by the low immunogenicity of tumors, low immune responses of patients, immune escape, immune-suppressive tumor microenvironment and other characteristics, the effect of single immunotherapy is not ideal. In addition, a large number of immune-related adverse events such as colitis, metabolic disorders, organ failure, nervous system damage, and hypothyroidism caused during the treatment process have increased the difficulty of immunotherapy. Therefore, in order to improve the treatment effect of cancer, a large number of combined immunotherapy strategies have been widely explored by researchers to stimulate stronger anti-tumor immune responses.

[0003] Currently, a variety of cancer treatment methods including chemotherapy, radiotherapy, and phototherapy have been used in combination with immunotherapy, and good curative effects have also been achieved. However, the combination of chemotherapy and immunotherapy may not only cause drug resistance of tumor cells, but also inhibit the function of hematopoietic stem cells, thus affecting normal human functions. Due to the uncontrolled radiation range of rays, the combination of radiotherapy and immunotherapy may cause radiation pneumonitis and radiation pulmonary fibrosis. In addition, due to the lack of specificity for tumors, both chemotherapy and radiotherapy will cause unavoidable damage to normal tissues and organs, resulting in systemic adverse reactions and a series of complications. Therefore, the clinical transformation of these combination methods still faces huge challenges.

[0004] Phototherapy is a cancer treatment method with low side effects, non-invasiveness, and high spatiotemporal selectivity, and has significant advantages when combined with immunotherapy. Phototherapy includes photodynamic therapy and photothermal therapy. Photodynamic therapy uses photosensitizers to absorb light of a certain wavelength, resulting in the production of reactive oxygen species through intersystem crossing relaxation to achieve tumor ablation. Photothermal therapy is to convert light energy into heat energy through photothermal agents, causing local heating in the tumor area to kill tumors. Previous studies have shown that phototherapy can induce immunogenic cell death in tumors. The characteristics of immunogenic cell death are the secretion of a large number of damage-associated molecular patterns, which can promote the maturation of dendritic cells and improve their antigen presentation ability, while promoting the infiltration of effector T cells and inhibiting the activity of regulatory T cells. The effective triggering of immunogenic cell death by phototherapy in anti-cancer immunotherapy makes it a very promising combination therapy strategy to effectively enhance the immune response. However, most of the currently developed photo-immunotherapy strategies still have problems such as uncontrolled release and non-specific distribution of immunomodulators, posing certain biosafety hazards. Therefore, there is an urgent need to develop a spatiotemporally controllable and tumor-specific agonist to achieve precise and efficient combined immunotherapy. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art. A cascade-responsive immunomodulator is designed and synthesized, which is specifically activated in the tumor microenvironment after illumination, triggering the anti-tumor immune response of T cells and simultaneously exerting a phototherapy effect. The preparation process of the material is simple, and it can achieve precise and efficient tumor treatment, which has important guiding significance for the design and synthesis of materials with good tumor targeting performance.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] A preparation method of a cascade-responsive immunomodulator, dissolving an immunomodulator prodrug, a photosensitizer, and a phase change material in a solvent to obtain Solution 1; dispersing linear PEG phospholipid in an aqueous solution to obtain Solution 2; ultrasonically mixing the two solutions and then cooling and solidifying; after the solution is cooled to room temperature, removing tetrahydrofuran and filtering to obtain the cascade-responsive immunomodulator.

[0008] Preferably: The immunomodulator prodrug is R848-QPA.

[0009] Preferably: The photosensitizer is Aza-BODIPY.

[0010] Preferably: The phase change material is PCM.

[0011] Preferably: The linear PEG phospholipid is DSPE-mPEG 2000 .

[0012] Preferably: The cascade-responsive immunomodulator is obtained by filtering through a surfactant-free cellulose acetate membrane.

[0013] Preferably: The solvent is tetrahydrofuran.

[0014] Preferably: The two solutions are mixed under ultrasound at 50 °C for 10 minutes and then cooled and solidified on ice.

[0015] Preferably: The structural formulas of Aza-BODIPY and R848-QPA are shown as follows:

[0016]

[0017] In the preparation methods of Aza-BODIPY and R848-QPA, the synthetic routes are shown as follows:

[0018]

[0019]

[0020] Preferably: The synthesis method of the photosensitizer Aza-BODIPY includes the following specific steps:

[0021] (1) Dissolve 4-methoxyacetophenone, 4-(diethylamino)benzaldehyde, and potassium hydroxide in a solution composed of ethanol and water, and stir overnight at room temperature. The crude product is purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents, and yellow 3-(4-(diethylamino)phenyl)-1-(4-methoxyphenyl)prop-2-en-1-one (Compound 1) is obtained after filtration.

[0022] (2) Dissolve Compound 1, nitromethane, and diethylamine in methanol and reflux at 70 °C for 24 hours. Neutralize the cooled mixture to room temperature with dilute hydrochloric acid, then extract first with water and dichloromethane, and then dry with anhydrous sodium sulfate. After evaporation, an orange oily crude product is obtained. The crude product is further purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents to obtain white-green 3-(4-(diethylamino)phenyl)-1-(4-methoxyphenyl)-4-nitrobut-1-one (Compound 2).

[0023] (3) Add Compound 2 and ammonium acetate to a round-bottom flask containing n-butanol and reflux and heat the reaction for 24 hours. After the reaction solution is cooled to room temperature, the formed precipitate is purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents to obtain red-violet 4-(2-((3-(4-(diethylamino)phenyl)-5-(4-methoxyphenyl)-1H-pyrrol-2-yl)imino)-5-(-4-methoxybenzene)-2H-pyrrol-3-yl)-N,N-diethylaniline (Compound 3).

[0024] (4) Dissolve compound 3 and diisopropylethylamine in ultradry dichloromethane. Then, dropwise add boron trifluoride diethyl etherate under nitrogen and stir at room temperature for 24 hours. Finally, extract the reaction mixture with dichloromethane and water, collect the organic layer, dry it over anhydrous sodium sulfate first, and then perform rotary evaporation to remove the solvent. The crude product is purified by silica gel adsorption column chromatography to obtain red-violet Aza-BODIPY.

[0025] Preferably: The synthesis method of the immunomodulatory prodrug R848-QPA includes the following specific steps:

[0026] (1) Add 3-methylbut-2-enoic acid and 2,3,5-trimethylbenzene-1,4-diol to methanesulfonic acid under stirring. Then, heat the mixture to 85 °C and react under nitrogen for 3 hours. Add ice to the reaction solution to quench the reaction, and extract the precipitate with ethyl acetate. After combining the organic phases, wash them with saturated sodium bicarbonate solution and brine, and dry over anhydrous sodium sulfate. After filtration and evaporation, recrystallize the obtained residue with hexane and ethyl acetate to obtain white 6-hydroxy-4,4,5,7,8-pentamethylchroman-2-one (compound 4).

[0027] (2) Suspend compound 4 in a mixture composed of water and acetonitrile. Then, slowly add N-bromosuccinimide, and stir the mixture at room temperature for 1 hour. Extract the reaction mixture with water and dichloromethane, dry the combined organic phases over anhydrous sodium sulfate. Remove the solvent by evaporation under reduced pressure, and purify the crude product by silica gel adsorption column chromatography to obtain yellow 3-methyl-3-(2,4,5-trimethyl-3,6-dioxocyclohex-1,4-dien-1-yl)butyric acid (compound 5).

[0028] (3) Dissolve compound 5 in dichloromethane, and add dicyclohexylcarbodiimide, p-dimethylaminopyridine, and N-hydroxysuccinimide under stirring. After the reaction is complete, filter the white precipitate, dissolve the crude product in ethyl acetate and filter out the insoluble impurities, and separate it by silica gel chromatography column to obtain QPA-NHS.

[0029] (4) Dissolve QPA-NHS and R848 in anhydrous dimethylformamide. Then, add N,N-diisopropylethylamine to the mixture and stir at room temperature for 24 hours. Purify the crude product by silica gel adsorption column chromatography using dichloromethane and methanol as the eluent to obtain yellow powder-like R848-QPA.

[0030] Preferably: The synthesis method of the phase change material PCM includes the following specific steps:

[0031] Dissolve oleic acid and 1-hexadecanol in methanol at a mass ratio of 1:3.5. Then, mix the mixture with vigorous vortex and store it in a 4 °C refrigerator for further use.

[0032] Preferably, the mass ratio of the immunomodulator prodrug, photosensitizer, phase change material, and linear PEG phospholipid is 1:1:50:50; the volume ratio of the two solutions is 1:10.

[0033] The present invention also provides an application of the cascade-responsive immunomodulator prepared by the above preparation method in cancer photo-immunotherapy.

[0034] Beneficial effects:

[0035] (1) The cascade-responsive immunomodulator NPs provided by the present invention have the characteristics of strong specificity, good photostability, and high safety;

[0036] (2) After being taken up by tumor cells, the cascade-responsive immunomodulator NPs provided by the present invention can be controlled to release by laser;

[0037] (3) The cascade-responsive immunomodulator NPs provided by the present invention can be specifically activated by the tumor microenvironment, triggering immunogenic cell death;

[0038] (4) The cascade-responsive immunomodulator NPs provided by the present invention can reverse the immunosuppressive tumor microenvironment and activate the T cell-mediated immune cascade reaction;

[0039] (5) The cascade-responsive immunomodulator NPs provided by the present invention can achieve synergistic treatment of phototherapy and immunotherapy, improving the cancer treatment effect;

[0040] (6) The cascade-responsive immunomodulator NPs provided by the present invention have high specificity for tumor cells, can achieve precise treatment of tumors and simultaneously reduce damage to normal cells and tissues. Brief description of the drawings

[0041] Figure 1 is a schematic diagram of the composition of the cascade-responsive NPs of the present invention and its photo-immunotherapy combined treatment mechanism.

[0042] Figure 2 is the 1 1H NMR of Aza-BODIPY of the present invention.

[0043] Figure 3 is the 1 1H NMR of R848-QPA of the present invention.

[0044] Figure 4 is the morphology and photodynamic effect curve of the cascade-responsive NPs of the present invention.

[0045] Figure 5 is the photothermal performance and photostability performance curve of the cascade-responsive NPs of the present invention.

[0046] Figure 6 is the characteristic response of the R848-QPA of the present invention to NQO1.

[0047] Figure 7 is the evaluation of cell viability of the cascade response type NPs of the present invention before and after light irradiation.

[0048] Figure 8 are the immunofluorescence staining images and column statistical charts of Hsp70 after treating 4T1 cells with different schemes in the examples of the present invention.

[0049] Figure 9 are the immunofluorescence staining images and column statistical charts of CRT after treating 4T1 cells with different schemes in the examples of the present invention.

[0050] Figure 10 are the immunofluorescence staining images and column statistical charts of HMGB1 after treating 4T1 cells with different schemes in the examples of the present invention.

[0051] Figure 11 is the expression level of ATP after treating 4T1 cells with different schemes in the examples of the present invention.

[0052] Figure 12 are the proportion and column statistical chart of mature dendritic cells analyzed by flow cytometry after co-culturing 4T1 cells and BMDCs treated with different schemes in the examples of the present invention.

[0053] Figure 13 are the volumes of proximal tumors and distal tumors of mice after the anti-tumor treatment schemes and different schemes in the examples of the present invention.

[0054] Figure 14 are the proportion and column statistical chart of dendritic cells analyzed by flow cytometry in the tumor-draining lymph nodes of mice after treating mice with different schemes in the examples of the present invention.

[0055] Figure 15 are the proportion and column statistical chart of cytotoxic T lymphocytes analyzed by flow cytometry in the spleen of mice after treating mice with different schemes in the examples of the present invention.

[0056] Figure 16 are the proportion and column statistical chart of regulatory T cells analyzed by flow cytometry in the spleen of mice after treating mice with different schemes in the examples of the present invention.

[0057] Figure 17 are the proportion and column statistical chart of memory T cells analyzed by flow cytometry in the spleen of mice after treating mice with different schemes in the examples of the present invention. Detailed implementation manners

[0058] The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the essence of the present invention, modifications and substitutions made to the methods, steps or conditions of the present invention all fall within the scope of the present invention.

[0059] Example 1: Synthesis of Aza - BODIPY

[0060] Preparation of Compound 1:

[0061] Dissolve 3.00 g of 4 - methoxyacetophenone, 3.54 g of 4 - (diethylamino)benzaldehyde and 0.11 g of potassium hydroxide in a solution composed of 34 mL of ethanol and 6 mL of water, and stir overnight at room temperature. The crude product is purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents, and a yellow Compound 1 is obtained after filtration. 1 1H NMR(CDCl3, 400 MHz): δ 8.02(d, 2H), 7.78(d, 1H), 7.53(d, 2H), 7.33(d, 1H), 6.97(d, 2H), 6.66(d, 2H), 3.89(s, 3H), 3.41(m, 4H), 1.21(t, 6H).

[0062] Preparation of Compound 2:

[0063] Dissolve 3.09 g of Compound 1, 3.03 g of nitromethane and 3.67 g of diethylamine in 50 mL of methanol, and reflux at 70 °C. After 24 hours, cool the mixture to room temperature, first neutralize it with 2 mol / L dilute hydrochloric acid, then extract with water and dichloromethane, and then dry with anhydrous sodium sulfate. After evaporation, an orange - oil - like crude product is obtained. The crude product is further purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents, and a white - green Compound 2 is obtained after filtration. 1 1H NMR(CDCl3, 400 MHz): δ 7.51(d, 2H), 7.08(d, 2H), 6.91(d, 2H), 6.59(d, 2H), 4.77(m, 1H), 4.62(m, 1H), 3.86(s, 3H), 3.38(m, 2H), 3.31(m, 4H), 1.13(t, 6H).

[0064] Preparation of Compound 3:

[0065] Add 0.74 g of Compound 2 and 7.71 g of ammonium acetate to a 100 mL round - bottom flask containing 50 mL of n - butanol, and reflux and heat the reaction for 24 hours. After the reaction solution is cooled to room temperature, the formed precipitate is purified by silica gel adsorption column chromatography using dichloromethane and petroleum ether as eluents, and a red - purple Compound 3 is obtained. 11H NMR (CDCl3, 400 MHz): δ 8.05 (d, 2H), 7.88 (d, 2H), 7.03 (d, 2H), 6.96 (s, 1H), 6.73 (d, 2H), 3.91 (s, 3H), 3.43 (m, 4H), 1.23 (t, 6H).

[0066] Preparation of Aza - BODIPY:

[0067] Dissolve 0.33 g of Compound 3 and 0.58 g of diisopropylethylamine in 50 mL of ultradry dichloromethane. Then, add 0.85 g of boron trifluoride diethyl ether dropwise under nitrogen, and stir at room temperature for 24 hours. Finally, extract the reaction mixture with dichloromethane and water, collect the organic layer, dry it over anhydrous sodium sulfate first, and then perform rotary evaporation to remove the solvent. The crude product is purified by silica gel adsorption column chromatography to obtain red - purple Aza - BODIPY. 1 1H NMR (CDCl3, 400 MHz): δ 8.05 (t, 8H), 6.98 (d, 4H), 6.79 (s, 2H), 6.72 (d, 4H), 3.87 (s, 6H), 3.46 (m, 8H), 1.25 (t, 12H).

[0068] Example 2: Synthesis of R848 - QPA

[0069] Preparation of Compound 4:

[0070] Add 1.5 g of 3 - methylbut - 2 - enoic acid and 2.0 g of 2,3,5 - trimethylbenzene - 1,4 - diol to 30 mL of methanesulfonic acid with stirring. Then, heat the mixture to 85 °C and react under nitrogen for 3 hours. Add 100 g of ice to the reaction solution to quench the reaction, and extract the precipitate with ethyl acetate. After combining the organic phases, wash them with saturated sodium bicarbonate solution and brine, and dry over anhydrous sodium sulfate. After filtration and evaporation, recrystallize the resulting residue from hexane and ethyl acetate to obtain white intermediate Compound 4. 1 1H NMR (CDCl3, 400 MHz): δ 4.60 (s, 1H), 2.55 (s, 2H), 2.36 (s, 3H), 2.22 (s, 3H), 2.18 (s, 3H), 1.46 (s, 6H).

[0071] Preparation of Compound 5:

[0072] Suspend 2.5 g of Compound 4 in a mixture composed of 5 mL of water and 25 mL of acetonitrile. Then, slowly add 2.0 g of N - bromosuccinimide, and stir the mixture at room temperature for 1 hour. Extract the reaction mixture with water and dichloromethane. After combining the organic phases, dry them over anhydrous sodium sulfate. Evaporate the solvent under reduced pressure, and purify the crude product by silica gel adsorption column chromatography to obtain yellow Compound 5.1 1H NMR (CDCl3, 400 MHz): δ 3.02 (s, 2H), 2.15 (s, 3H), 1.93 (d, 6H), 1.43 (s, 6H).

[0073] Preparation of QPA-NHS:

[0074] Dissolve 3.25 g of compound 5 in dichloromethane. With stirring, add 2.7 g of dicyclohexylcarbodiimide, p-dimethylaminopyridine, and 1.52 g of N-hydroxysuccinimide. After the reaction is complete, filter the white precipitate. Dissolve the crude product in ethyl acetate and filter the insoluble impurities. QPA-NHS is obtained by separation on a silica gel chromatographic column. 1 1H NMR (CDCl3, 400 MHz): δ 3.28 (s, 2H), 2.79 (s, 4H), 2.17 (s, 3H), 1.96 (s, 6H), 1.53 (s, 6H).

[0075] Preparation of R848-QPA:

[0076] Dissolve 104 mg of QPA-NHS and 94 mg of R848 in 5 mL of anhydrous dimethylformamide. Then add 0.25 mL of N,N-diisopropylethylamine to the mixture and stir at room temperature for 24 hours. The crude product is purified by silica gel adsorption column chromatography using dichloromethane and methanol as eluents to obtain R848-QPA as a yellow powder. 1 1H NMR (CDCl3, 400 MHz): δ 8.06 (d, 1H), 7.83 (d, 1H), 7.52 (t, 1H), 7.33 (t, 1H), 4.84 (d, 4H), 3.66 (q, 2H), 2.16 (m, 8H), 1.28 (m, 18H).

[0077] Example 3: Preparation of PCM

[0078] Dissolve oleic acid and 1-hexadecanol in methanol at a mass ratio of 1:3.5. Then, mix the mixture with vigorous vortexing and store it in a refrigerator at 4 °C for further use.

[0079] Example 4: Preparation of NPs

[0080] Dissolve 0.4 mg of R848-QPA, 0.4 mg of Aza-BODIPY, and 20 mg of PCM in 2 mL of tetrahydrofuran solution as Solution 1. Disperse 20 mg of DSPE-mPEG in an aqueous solution 2000As Solution 2. Then, the two solutions were mixed under ultrasound at 50 °C for 10 minutes and then rapidly cooled and solidified on ice. After the solution was cooled to room temperature, tetrahydrofuran was removed, and NPs were obtained by filtering through a surfactant-free cellulose acetate membrane. The unencapsulated contents were removed using a centrifugal filter.

[0081] Example 5: Study on Photodynamic Performance

[0082] DPBF was used as a probe to detect the 1 O2 generated after irradiation of NPs. The probe (25 μM) was mixed with NPs (12.5 μg / mL), and the mixture was irradiated with an 808 nm laser at a light power density of 0.5 W cm -2 for 14 minutes. Then, the absorbance of DPBF at a wavelength of 420 nm was measured using a UV-visible spectrophotometer. After incubation with NPs, 1 the generation of O2 caused the absorption of DPBF at 420 nm to continuously decrease with the prolongation of the irradiation time.

[0083] Example 6: Study on Photothermal Performance and Photostability

[0084] NPs at different concentrations (0, 6.25, 12.5, and 25 μg / mL) in PBS buffers with pH = 5.0 and pH = 7.0 were irradiated with an 808 nm laser or NPs were irradiated at different light power densities (0.1, 0.3, and 0.5 W cm -2 ). After 10 minutes, the laser was turned off, and the solution was naturally cooled. The temperature of the NPs solution was monitored throughout the process using an infrared thermal imager. To evaluate the photostability, the NPs solution underwent 5 heating-cooling cycles. The temperature of the NPs solution increased significantly with the increase in concentration and light power density. In addition, the temperature of NPs increased faster at pH = 5.0 than at pH = 7.4. After undergoing 5 heating and natural cooling cycles, the thermal stability of NPs remained good.

[0085] Example 7: Characteristic Response of R848-QPA to NQO1

[0086] 20 μM R848-QPA, 2.5 μg / mL NQO1, and 100 μM NADH were incubated at 37 °C in PBS containing 0.1% fetal bovine serum for 1 hour. The changes in the emission spectrum and absorption spectrum were recorded using a fluorescence spectrophotometer and a UV-visible spectrophotometer, respectively. The release of R848-QPA in the reaction mixture was analyzed by high performance liquid chromatography. After incubation of R848-QPA with NQO1 and the cofactor NADH, the UV absorption peak increased significantly, the fluorescence intensity decreased significantly, and it could be converted into free R848 and cyclic lactone.

[0087] Example 8: ELISA assay for adenosine triphosphate (ATP)

[0088] 4T1 cells were seeded in cell culture dishes and incubated overnight. After the cells adhered to the wall, they were treated in different ways (i.e., PBS, NPs, R848-QPA, NQO1 inhibitor and NPs plus light irradiation, BDP plus light irradiation and NPs plus light irradiation) for 12 hours, and then irradiated or not irradiated with an 808 nm laser at a light power density of 0.5 W cm -2 for 10 minutes. After further incubation in an incubator for 12 hours, the cell supernatant was extracted, and the ATP content secreted by the cells was detected using an ATP detection kit. After laser irradiation, the ATP content secreted by the cells treated with NPs was the highest, about 2.28 times that of the PBS-treated group (blank control group).

[0089] Example 9: Immunofluorescence staining for heat shock protein 70 (Hsp70), calreticulin (CRT) and high mobility group box 1 protein (HMGB1)

[0090] 4T1 cells were seeded in cell culture dishes and incubated overnight. After the cells adhered to the wall, they were treated in different ways (i.e., PBS, NPs, R848-QPA, NQO1 inhibitor and NPs plus light irradiation, BDP plus light irradiation and NPs plus light irradiation) for 12 hours. Then, they were irradiated or not irradiated with an 808 nm laser at a light power density of 0.5 W cm -2 for 10 minutes, and then the cells were returned to the incubator for continued incubation. After 12 hours, the cells were washed 3 times with PBS, fixed in 4% paraformaldehyde for 20 minutes, permeabilized in 0.1% Triton X-100 for 20 minutes, and blocked in 3% fetal bovine serum for 1 hour. Subsequently, the cells were incubated with Hsp70 antibody, CRT antibody and HMGB1 antibody in a 4 °C refrigerator. On the second day, after washing the cells 3 times with PBS, the secondary antibody corresponding to each antibody was co-incubated with the cells in the dark for 1 hour. Subsequently, the cells were washed 3 times with PBS, co-incubated with DAPI for 10 minutes, washed 3 times again, and finally the fluorescence images of the cells were observed under a Nikon inverted microscope. After light irradiation, a large amount of Hsp70 secretion, obvious exposure of CRT on the cell surface, and a large amount of HMGB1 migrated from the nucleus to the extracellular environment were observed in the 4T1 cells treated with NPs.

[0091] Example 10: Flow cytometry for detecting mature dendritic cells

[0092] Bone marrow-derived dendritic cells (BMDCs) were obtained from the bone marrow of 8-week-old BALB / c mice, and the extracted cells were cultured in high-glucose DMEM medium containing 10 ng / mL IL-4 and 20 ng / mL GM-CSF. 4T1 cells were inoculated overnight. After the cells adhered to the wall, they were treated in different ways (i.e., PBS, NPs, R848-QPA, NQO1 inhibitor, NPs combined with light irradiation, BDP combined with light irradiation, and NPs combined with light irradiation) for 12 hours. Then, the cells were irradiated or not irradiated with a 808 nm laser at a light power density of 0.5 W cm -2 for 10 minutes and then returned to the incubator for continued incubation. On the 4th day of BMDCs culture, 4T1 cells and BMDCs were co-cultured at a cell number ratio of 1:2 for 24 hours. All suspended and semi-suspended cells in the culture dish were collected. First, the cells were stained with CD16 / 32 antibody at 4 °C for 15 minutes, then stained with APC-CD11c antibody, FITC-CD80 antibody, and PE-CD86 antibody for 30 minutes, and finally the maturation level of DCs was detected by flow cytometry. The expression levels of CD80 and CD86 in the NPs group after light irradiation were the highest (58.8%), about 1.2 - 8.6 times that of other treatment groups.

[0093] Example 11: Antitumor treatment effect of mouse treatment regimen

[0094] On the 10th day after tumor implantation, 4T1 tumor-bearing mice were randomly divided into 6 groups, and each group of mice was treated correspondingly every other day (i.e., injected with normal saline, NPs, R848-QPA, NQO1 inhibitor, NPs combined with light irradiation, BDP combined with light irradiation, and NPs combined with light irradiation), and the treatment was carried out three times in total. The volume of the mice's tumors was monitored every 2 days for 14 days. Compared with other groups, the growth of primary tumors and distal tumors in mice after NPs treatment and laser irradiation was significantly inhibited.

[0095] Example 12: Flow cytometry analysis of dendritic cells in tumor-draining lymph nodes and cytotoxic T lymphocytes, regulatory T cells, and memory T cells in the spleen

[0096] After various treatments of mice were completed (i.e., injection of normal saline, NPs, R848-QPA, NQO1 inhibitor and NPs followed by light exposure, BDP followed by light exposure, and NPs followed by light exposure), the 4T1 tumor-bearing mice in each group were euthanized. The tumor-draining lymph nodes and spleens of the mice were removed to prepare single-cell suspensions for flow cytometry analysis. First, the obtained single-cell suspensions were filtered through a 70-μm nylon mesh filter, and then the cells were incubated with CD16 / 32 antibody at 4 °C for 15 minutes to block non-specific binding. To analyze cytotoxic T lymphocytes, the cells were stained with FITC-CD3 antibody, PE-CD4 antibody, and APC-CD8a antibody for 30 minutes. To analyze regulatory T cells, the cells were stained with FITC-CD3 antibody, PE-CD4 antibody, and APC-FOXP3 antibody for 30 minutes. To analyze memory T cells, the cells were stained with FITC-CD3 antibody, APC-CD8a antibody, PE-CD44 antibody, and Pacific Blue TM -CD62L antibody for 30 minutes. To analyze dendritic cells, the cells were stained with APC-CD11c antibody, FITC-CD80 antibody, and PE-CD86 antibody for 30 minutes. Finally, these samples were detected by flow cytometry. The proportions of mature dendritic cells (66.2%), cytotoxic T lymphocytes (30.0%), and memory T cells (31.1%) induced by the NPs treatment and laser irradiation group were the highest, which were 2.9 times, 2.5 times, and 3.0 times that of the normal saline treatment group, respectively. The proportion of regulatory T cells (16.5%) was the lowest in the NPs group after light exposure, which was reduced by 2.2 times compared with the normal saline treatment group.

[0097] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A preparation method of a cascade response type immunomodulator, characterized in that: Dissolve the immunomodulator prodrug, photosensitizer, and phase change material in tetrahydrofuran to obtain Solution 1; disperse the linear PEG phospholipid in an aqueous solution to obtain Solution 2; ultrasonically mix the two solutions and then cool and solidify; after the solution is cooled to room temperature, remove the tetrahydrofuran and filter to obtain the cascade-responsive immunomodulator. The immunomodulatory prodrug is R848-QPA, and its structure is ; the photosensitizer is Aza-BODIPY; The synthesis method of the phase change material is as follows: dissolve oleic acid and 1-hexadecanol in methanol at a mass ratio of 1:3.5; then, vortex-mix the mixture and store it at 4 °C.

2. The preparation method of a cascade response type immunomodulator according to claim 1, characterized in that: The linear PEG phospholipid is DSPE-mPEG 2000 .

3. The preparation method of a cascade response type immunomodulator according to claim 1, characterized in that: Obtain the cascade-responsive immunomodulator by filtering with a surfactant-free cellulose acetate membrane.

4. The preparation method of a cascade response type immunomodulator according to claim 1, characterized in that: Mix the two solutions under ultrasound at 50 °C for 10 minutes and then cool and solidify on ice.

5. The preparation method of a cascade response type immunomodulator according to claim 1, characterized in that: The mass ratio of the immunomodulator prodrug, photosensitizer, phase change material, and linear PEG phospholipid is 1:1:50:50; the volume ratio of the two solutions is 1:10.

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