Photosensitizer drug for treating tumor, preparation method and application thereof

Abstract

The application provides a photosensitizer drug for treating tumors, a preparation method and application, and belongs to the technical field of biological medicines. The photosensitizer drug for treating tumors is prepared into TSA@, which can effectively induce necrotic apoptosis of tumors under light activation. After being treated by light, TSA@ can induce more efficient maturation of DC cells in the necrotic apoptosis mode than in the apoptosis mode, thereby promoting stronger antigen presentation and stimulating T cell activation, exerting more favorable anti-tumor immune response, and having strong treatment effect on tumors. TSA@HH prepared from the photosensitizer drug for treating tumors has good in-vivo biocompatibility and biosafety, and can more effectively enhance the anti-tumor treatment effect compared with TSA@.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a photosensitizer drug for treating tumors, its preparation method and application. Background Technology

[0002] Bladder cancer is a common malignant tumor of the urinary system, with urothelial carcinoma being the main histological type, characterized by a high recurrence rate. Due to the low immunogenicity of bladder cancer cells, insufficient infiltration of cytotoxic T cells in the tumor microenvironment, and a high proportion of immunosuppressive cells, patients exhibit low response rates and poor immune memory effects to immune checkpoint blockade (ICB) therapy. Therefore, there is an urgent need for treatment strategies that can eradicate the primary lesion while simultaneously generating potent immune memory and preventing recurrence and metastasis.

[0003] Photodynamic therapy (PDT) has advantages such as high selectivity, minimal invasiveness, low toxicity, reusability, and low drug resistance. Studies have shown that tumor cells can undergo immunogenic cell death (ICD) after receiving PDT; however, PDT mainly induces apoptosis, which is a non-inflammatory cell death, resulting in a significant reduction in the ICD effect during PDT.

[0004] Necrophage, or apoptosis, is a programmed inflammatory cell death process that indirectly promotes the activation of T-cell anti-tumor immune responses by releasing damage-associated molecular patterns (DAMPs) as adjuvants. The classic death receptor-mediated necroptosis pathway consists of RIPK1-RIPK3-MLKL, with RIPK3 and MLKL being core components regardless of upstream triggers. However, multiple studies have found that RIPK3 expression is significantly decreased in various cancer tissues, and this low expression is closely related to hypermethylation of CpG islands in the RIPK3 promoter region. Therefore, upregulating RIPK3 expression and remodeling the sensitivity of cancer cells to necroptosis remains a pressing technical challenge.

[0005] Azacitidine (AZA, 5-Azacytidine) is a water-soluble demethylating antitumor drug that restores the expression of genes silenced by methylation in tumor cells by inhibiting DNA methyltransferases. However, when exerting its demethylating antitumor effect, the effective drug concentration of AZA is often accompanied by significant systemic adverse reactions such as bone marrow suppression and gastrointestinal discomfort. Therefore, effective tumor-targeted delivery and precise release are prerequisites for AZA to fully exert its antitumor effect.

[0006] In summary, how to synergize the epigenetic sensitization effect of demethylating drugs on RIPK3 with the reactive oxygen species killing effect of PDT in a spatiotemporal manner, so as to switch the death mode of tumor cells from the less immunogenic apoptosis to the more immunogenic necrotizing apoptosis, while avoiding the systemic toxicity of demethylating drugs, is a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0007] In view of the above problems, the purpose of this invention is to provide a photosensitizer drug for treating tumors, its preparation method, and its application. To achieve the above-mentioned objective, this invention provides the following technical solution: A photosensitizer for treating tumors, wherein the chemical structural formula of the photosensitizer is as follows: It was named TSA.

[0008] The present invention also provides a method for preparing the above-mentioned photosensitizer drug TSA, which is formed by coupling the AIE photosensitizer TPAPy with the demethylating drug azacitidine via a disulfide bond, specifically including the following steps: using TPAPy and compound SS-AZA as reactants, and using sodium ascorbate and CuSO4 as catalysts, TSA is obtained through a "click" reaction; The chemical structural formula of TPAPy is: ; The chemical structural formula of the compound SS-AZA is as follows: The molar ratio of TPAPy, SS-AZA, sodium ascorbate and CuSO4 is (15~25):(15~25):(4~8):(2~4); the "click" reaction conditions are nitrogen protection, reaction at 20~25℃ for 20~26 hours.

[0009] More preferably, the molar ratio of TPAPy, SS-AZA, sodium ascorbate and CuSO4 is (17~22):(17~22):(5~7):(2.5~3.5), even more preferably 20:20:6:3; further preferably, the "click" reaction is carried out at 20~25℃ for 22~25 hours, even more preferably at 20~25℃ for 24 hours.

[0010] Preferably, the preparation method of TPAPy includes the following steps: condensing 4'-[bis(4-methoxyphenyl)amino]-[1,1'-biphenyl]-4-carboxaldehyde and 1-(4-azidobutyl)-4-methylpyridine-1-onium iodide, then performing ion exchange with saturated KPF6 aqueous solution, and purifying to obtain TPAPy.

[0011] Preferably, the molar ratio of 4'-[bis(4-methoxyphenyl)amino]-[1,1'-biphenyl]-4-carboxaldehyde and 1-(4-azidobutyl)-4-methylpyridine-1-onium iodide is (0.5~1.5):(0.5~1.5); more preferably 0.7~1.3:0.7~1.3, and even more preferably 1:1, wherein the condensation reaction is a reflux reaction for 40~50 h, and more preferably a reflux reaction for 47~49 h.

[0012] Preferably, the preparation method of the compound SS-AZA includes the following steps: dissolving propyne-monoethylene glycol, N,N-diisopropylethylamine and 4-dimethylaminopyridine in anhydrous dichloromethane, then adding p-nitrophenyl chloroformate to carry out a first reaction, obtaining a reaction stock solution, concentrating it and dissolving it in N,N-dimethylformamide to obtain a reaction solution, then mixing azacitidine, the reaction solution and triethylamine to carry out a second reaction, obtaining a mixed solution; the mixed solution is then concentrated, extracted and purified to obtain the SS-AZA. The mass-to-volume ratio of 4-dimethylaminopyridine to anhydrous dichloromethane is (1~4) mg:(8~12) mL, more preferably (1.5~3.5) mg:(9~11) mL, and more preferably 3 mg:10 mL.

[0013] Preferably, the molar ratio of propyne-monoethylene glycol, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and p-nitrophenyl chloroformate is (0.1~0.5):(1~1.4):(0.01~0.05):(0.4~0.8); the addition ratio of azacitidine, reaction solution, and triethylamine is (1~1.5) mmol:(5~10) mL:(1~1.5) mmol, more preferably (1.1~1.4) mmol:(6~8) mL:(1.1~1.4) mmol, and even more preferably 1.2 mmol:7 mL:1.2 mmol.

[0014] The present invention also provides a pharmaceutical composition for treating tumors, comprising an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is the photosensitizer drug TSA or nanoparticles formed by self-assembly of TSA prepared by the above method and the amphiphilic block copolymer DSPE-PEG2000 and its derivatives.

[0015] Preferably, as a specific embodiment, the preparation method of the above-mentioned TSA@nanoparticles includes the following steps: mixing the above-mentioned TSA or the TSA prepared by the above-mentioned method with DSPE-PEG. 2000 The TSA nanoparticles were obtained by dissolving the TSA nanoparticles in tetrahydrofuran. The TSA nanoparticles were then added to ultrapure water, and the mixture was dialyzed, washed, concentrated, and purified. 2000The preferred mass-to-volume ratio of the mixed solution to tetrahydrofuran is (0.5~1.5) mg:(2~4) mg:(0.5~1.5) mL, more preferably (0.7~1.3) mg:(2.5~3.5) mg:(0.7~1.3) mL, and even more preferably 1 mg:3 mg:1 mL. The preferred volume ratio of the mixed solution to ultrapure water is 1:(8~10), more preferably 1:(8.5~9.5), and even more preferably 1:9. The solution is added dropwise using a 1 mL syringe at a rate of 1 drop per second, which improves particle size uniformity.

[0016] Preferably, the amphiphilic block copolymer DSPE-PEG2000 derivative is DSPE-PEG2000-Mal, and the surface of the nanoparticles is further coupled with a PD-L1 blocking peptide, the amino acid sequence of which is HWFHRHH as shown in SEQ ID NO.1.

[0017] Preferably, as a specific embodiment, a PD-L1 blocking peptide (HH peptide) is coupled with a TSA@-Mal nanoparticle solution to obtain a conjugate, which is then washed to obtain TSA@HH nanoparticles. The mass-to-volume ratio of the PD-L1 blocking peptide to the TSA@-Mal nanoparticle solution is 2-3 mg:1 mL, more preferably 2.2-2.8 mg:1 mL, and even more preferably 2.5 mg:1 mL; the concentration of the TSA@-Mal nanoparticle solution, calculated in TPAPy, is 0.5-2 mg / mL, more preferably 0.8-1.5 mg / mL, and even more preferably 0.9, 1, or 1.1 mg / mL. The coupling reaction is carried out at room temperature for 10-14 h, more preferably 11-13 h, and even more preferably 12 h; room temperature refers to 20-25°C. After obtaining the conjugate, the conjugate was centrifuged and ultrafiltered. The molecular weight cutoff of the ultrafiltration tube was 100 kDa. After washing 5 times, the TSA@HH nanoparticles in the ultrafiltration tube were collected and named TSA@HH.

[0018] The present invention also provides the application of the photosensitizer drug TSA or the TSA prepared by the above preparation method or the drug composition thereof in the preparation of a drug for photodynamic therapy of tumors, wherein the drug or drug composition, after being activated by light, induces the death mode of tumor cells to change from apoptosis to necrotizing apoptosis.

[0019] Preferably, the tumor cells are bladder cancer cells, and the photoactivation is photodynamic drug activation by applying light within a 12-hour time window after drug administration.

[0020] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention combines photodynamic therapy (PDT) with the regulation of the RIPK3 / MLKL necrosis-apoptosis pathway. Through the synergistic effect of PDT mediated by TPAPy and the upregulation of RIPK3 expression induced by AZA, it can directionally trigger the immunogenic necrosis-apoptosis of tumor cells. Compared with the traditional apoptosis pathway, this method can more efficiently promote the maturation of dendritic cells (DCs), greatly improve the antigen presentation efficiency and T cell activation level, and strengthen the initiation ability of anti-tumor immune response from the source, thus solving the core problem of insufficient efficiency of conventional photosensitizer-induced immunogenic death.

[0021] (2) Based on the above mechanism, this invention uses the DSPE-PEG2000-Mal active site on the surface of the nanocarrier to directionally couple PD-L1 targeting HH peptides to obtain TSA@HH functionalized nanoparticles. This design deeply combines PDT-induced immunogenic necrotizing apoptosis with the in situ immune checkpoint blockade (ICB) effect. Compared with unmodified TSA@ nanoparticles, it can significantly increase the infiltration level of cytotoxic T cells in the tumor, effectively break the immunosuppressive microenvironment of bladder cancer, and inhibit the growth of primary tumor and the risk of distant metastasis.

[0022] (3) The present invention further proposes a dual-target delivery strategy of tumor microenvironment response prodrug encapsulation + active targeting modification of carrier surface. Relying on the EPR effect of nanocarrier and the active targeting recognition of HH peptide, the enrichment of photosensitizer and pathway regulation drug in tumor site is greatly improved, the drug exposure and toxic side effects in healthy tissue are significantly reduced, and it has compatibility with other anti-tumor drugs, which can effectively overcome the common clinical problem of tumor drug resistance.

[0023] (4) The photodynamic-necrotic apoptosis activation-immune checkpoint blockade synergistic treatment scheme finally formed in this invention has verified the ability to establish a lasting anti-tumor immune memory in a bladder cancer model. It has excellent in vivo biocompatibility and therapeutic effect. It not only provides an innovative photoimmunotherapy pathway with clear clinical translation potential for bladder cancer, but also provides a brand-new technical idea for the immunogenicity activation research of immunosuppressive "cold tumors". Attached Figure Description

[0024] Figure 1 For TPAPy 1 H NMR spectrum.

[0025] Figure 2 Alkyne-SS-Azacitidine (SS-AZA for short) 1 H NMR spectrum.

[0026] Figure 3 For TSA 1 H NMR spectrum.

[0027] Figure 4 The image shows the HR-MS spectrum of the TSA.

[0028] Figure 5 This is a synthetic route diagram for compounds TPAPy and TSA.

[0029] Figure 6 Characterization results of TSA@ nanophotosensitizer: a) UV-Vis absorption spectrum of TPAPy in DMSO; b) fluorescence spectrum in DMSO / toluene mixture; c) fluorescence intensity of DCF at 520 nm as a function of light irradiation time of TSA@ in aqueous solution; d) relative quantification of ROS capabilities of the three photosensitizing drugs; e) particle size distribution of TSA@ under dynamic light scattering; f) representative image of TSA@ under transmission electron microscopy.

[0030] Figure 7 The cumulative release curves of AZA in TSA@ are shown under different simulated physiological conditions.

[0031] Figure 8 Representative CLSM images of ROS detection at the cellular level for different groups. Scale bar: 50 μm.

[0032] Figure 9 Results of DCF fluorescence intensity detection in different groups of cells using flow cytometry.

[0033] Figure 10 Cell viability of MB49 cells under different treatment conditions is expressed as mean ± standard deviation (n=3), *p<0.05 and ****p<0.0001.

[0034] Figure 11 The images show representative AO staining and light micrographs of MB49 cells under different treatment conditions under a laser confocal microscope. The cells indicated by the cyan arrows are AO-stained necrotic and apoptotic cells, while the cells indicated by the white arrows are apoptotic cells.

[0035] Figure 12 Representative Western blot (WB) results for key signaling molecules in the necroptosis pathway after different treatment groups.

[0036] Figure 13 The study analyzed the intracellular caspase 8 activity level and LDH release level of MB49 cells after different treatment groups. a) shows the intracellular caspase 8 activity level of MB49 cells after different treatment groups, and b) shows the LDH release level of MB49 cells after different treatment groups.

[0037] Figure 14Representative confocal images of MB49 cells at the cellular level using ecto-CRT after treatment with different conditions. Red fluorescence: ecto-CRT signal; blue fluorescence: DAPI signal. Scale bar: 25 μm.

[0038] Figure 15 The results of HMGB1, HSP70 and ATP release levels after different treatment groups are shown. a) The release levels of HMGB1 and HSP70 in MB49 cells after different treatment conditions were detected by Western blot. b) The release levels of ATP in MB49 cells after different treatment conditions were detected.

[0039] Figure 16 The changes in PD-L1 receptor in MB49 cells after PDT treatment are shown in Figure a. Flow cytometry analysis of PD-L1 changes in different treatment groups; Figure b. Flow cytometry analysis of MFI changes in different treatment groups; Figure c. Western blot representations of PD-L1 in different treatment groups.

[0040] Figure 17 The left figure shows the results of DC maturation level detection under different treatments in the in vitro BMDC stimulation maturation experiment. The left figure shows the CD80 maturation level of DCs under different treatments. + Representative flow cytometry images; the right image shows the maturation level of DCs under different treatments and CD80. + The quantitative results are expressed as mean ± standard deviation (n=3), with ***p<0.001 and ****p<0.0001.

[0041] Figure 18 Quantitative data of IL-12p70 in the supernatant under different treatments in the in vitro BMDCs stimulation maturation experiment are presented as mean ± standard deviation (n=4), with ***p<0.001 and ****p<0.0001.

[0042] Figure 19 The structural formula of the C-HH (C-HWFHRHH) polypeptide is given.

[0043] Figure 20 The results are from mass spectrometry analysis of the C-HH (C-HWFHRHH) polypeptide.

[0044] Figure 21 The liquid chromatogram is for C-HH (C-HWFHRHH).

[0045] Figure 22 This is a transmission electron microscope image of TSA@HH.

[0046] Figure 23 The results of blood routine and blood biochemical index detection in mice after intravenous administration in different treatment groups are shown. Error bar: mean ± standard deviation (n=3).

[0047] Figure 24 To evaluate the in vivo distribution and tumor enrichment of TSA@HH nanophotosensitizer, a shows the enrichment results of TSA@HH in tumor tissues at different time points after intravenous injection in subcutaneous tumor-bearing mice, and b shows the quantitative data of TSA@HH in different tissues at different time points after intravenous injection in subcutaneous tumor-bearing mice. Error bar: mean ± standard deviation (n=3).

[0048] Figure 25 Tumor growth curves of MB49 subcutaneous tumor-bearing mice with bladder cancer after receiving different treatment groups are shown. Data are expressed as mean ± standard deviation (n=5), with **p<0.01 and ****p<0.0001.

[0049] Figure 26 The survival and weight changes of MB49 subcutaneous tumor-bearing mice with bladder cancer after receiving different treatment groups are shown in Figure a. The survival curves of MB49 subcutaneous tumor-bearing mice with bladder cancer after receiving different treatment groups are shown in Figure b.

[0050] Figure 27 Representative confocal images of ecto-CRT in tumor tissues of MB49 mice with subcutaneous bladder cancer after different treatment groups, scale bar 20 μm.

[0051] Figure 28 To detect the maturation level of DCs in TDLNs by flow cytometry, a) is a representative flow cytometry image of DC maturation level (CD80+ and CD86+ cells) in TDLNs of MB49 subcutaneous tumor-bearing mice with bladder cancer after different treatment groups; b) is the quantitative result of DC maturation level (CD80+ and CD86+ cells) in TDLNs of MB49 subcutaneous tumor-bearing mice with bladder cancer after different treatment groups. The results are expressed as mean ± standard deviation (n=3), with ***p<0.001 and ****p<0.0001.

[0052] Figure 29 To investigate the effects of different treatment groups on the growth of MB49 orthotopic bladder cancer tumors, a) shows bioluminescence images of orthotopic bladder tumors in mice bearing MB49 orthotopic bladder cancer at different time points after receiving different treatment groups (n=5) using a small animal in vivo imaging system. b) shows the tumor growth curves after quantification of bioluminescence signals following different treatment groups. Error bars are expressed as mean ± standard deviation (n=5), with *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001. Detailed Implementation

[0053] In this invention, unless otherwise specified, all raw material components are commercially available products well-known to those skilled in the art. Specifically, the anhydrous dichloromethane was purchased from Bailingwei Technology, product number: 913078; the propyne-monoethylene glycol, officially named 2-((2-(prop-2-yn-1-yloxy)ethyl)dithioalkyl)ethanol, was purchased from Bide Pharmaceuticals, catalog number BD00838989; DSPE-PEG2000 and DSPE-PEG2000-Mal were both purchased from Bide Pharmaceuticals, catalog numbers BD01106217 and BD01289340, respectively. Other components are not listed individually. The technical solutions provided by this invention are described in detail below with reference to embodiments, but these should not be construed as limiting the scope of protection of this invention.

[0054] Statistical analysis: GraphPad Prism 10 software was used for plotting and analyzing statistical significance. FlowJov 10.8.1 software was used for flow cytometry analysis. Kaplan-Meier analysis was used to plot survival curves. t-tests were used for statistical comparisons. Data are expressed as mean ± standard deviation (SD), with *p<0.05, **p<0.01, ***p<0.001, and ****p<0.0001 as criteria.

[0055] Example 1 1.1 Synthesis of TPAPy and TSA molecules 1. Synthesis of compound TPAPy: Under nitrogen protection, 225 mg and 0.55 mmol of 4'-[bis(4-methoxyphenyl)amino]-[1,1'-biphenyl]-4-carboxaldehyde and 175 mg and 0.55 mmol of 1-(4-azidobutyl)-4-methylpyridine-1-onium iodide were dissolved in 10 mL of anhydrous ethanol (EtOH). After reflux for 48 hours, the mixture was cooled to room temperature, and the solvent ethanol was removed by vacuum distillation. The remaining solid was redissolved in 10 mL of acetone, and 1 mL of saturated KPF6 aqueous solution was added for ion exchange. After stirring at room temperature for 30 minutes, the acetone and water were removed by vacuum evaporation to obtain crude product 1. Finally, the purified crude product 1, namely the target compound TPAPy, was obtained by silica gel column chromatography (using a 200-300 mesh silica gel column, and isocratic elution by mixing dichloromethane and anhydrous methanol at a volume ratio of 20:1). 228 mg of TPAPy was obtained by weighing, with a yield of 57%.

[0056] The 1H NMR spectrum of TPAPy is shown below. Figure 1 , Figure 1The results show that 1H NMR (400 MHz, Chloroform-d) δ8.40 (d, J = 6.2 Hz, 2H), 7.84 (d, J = 6.2 Hz, 2H), 7.63 - 7.48 (m, 5H), 7.37(d, J = 8.4 Hz, 2H), 7.10 - 1.99 (t, J = 7.6 Hz, 2H), 1.59 (t, J = 7.6 Hz, 2H).

[0057] The chemical structural formula of TPAPy is as follows: .

[0058] 2. Synthesis of compound Alkyne-SS-Azacitidine (SS-AZA): First, 58 mg of propyne-monoethylene glycol (0.30 mmol), 155 mg of N,N-diisopropylethylamine (DIEA) (1.20 mmol), and 3 mg of 4-dimethylaminopyridine (DMAP) (0.03 mmol) were dissolved in 10 mL of anhydrous dichloromethane (DCM). Then, under nitrogen protection and stirring at 0 °C, 120 mg of p-nitrophenyl chloroformate (0.60 mmol) was added dropwise to the above anhydrous DCM. After the addition was complete, the mixture was brought back to room temperature and stirred for 4 hours to obtain the stock solution. The stock solution was concentrated under reduced pressure until the solvent was completely evaporated. The residue was redissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF) to obtain the reaction solution. Subsequently, 293 mg and 1.20 mmol of azacitidine were dissolved in 5 mL of the above reaction solution and 121 mg and 1.20 mmol of triethylamine (TEA) were added. The reaction system was stirred at room temperature for 24 hours to ensure that the reaction proceeded fully and a mixture was obtained.

[0059] First, the mixture was concentrated under reduced pressure until the solvent was completely evaporated. Then, liquid-liquid extraction was performed using an extract obtained by mixing equal volumes of dichloromethane and saturated brine. The organic layer (the lower layer of the extract) was collected and dried with anhydrous sodium sulfate to remove moisture. The dried organic phase was then concentrated under reduced pressure again until the solvent was completely evaporated to obtain crude product 2. Finally, it was purified by silica gel column chromatography (using a 200-300 mesh silica gel column, isocratic elution was performed by mixing dichloromethane and anhydrous methanol at a volume ratio of 20:1). Crude product 2 yielded the target compound Alkyne-SS-Azacitidine, and 23 mg of Alkyne-SS-Azacitidine (SS-AZA) was obtained, with a yield of 17%.

[0060] The 1H NMR spectrum of Alkyne-SS-Azacitidine (SS-AZA) is shown below. Figure 2 , Figure 2 The results showed that the ¹H NMR (400 MHz, Methanol-d⁴) δ values ​​were 8.26 (s, ¹H), 5.63 (s, ¹H), 4.40 (d, J = 12.2, 2.1 Hz, ¹H), 4.35–4.27 (m, 3H), 4.17 (t, J = 3.7 Hz, 1H), 4.12–4.04 (m, 4H), 3.68 (t, J = 5.5 Hz, 2H), 2.92 (t, J = 6.4 Hz, 2H), 2.84 (t, J = 7.1 Hz, 2H), 2.77 (d, J = 2.1 Hz, 1H), and 1.19 (d, J = 3.0 Hz, 1H).

[0061] The chemical structural formula of Alkyne-SS-Azacitidine (SS-AZA for short) is as follows: .

[0062] 3. Synthesis of compound TSA: 14.5 mg and 20 µmol of TPAPy synthesized in step 1 above and 6.9 mg and 20 µmol of Alkyne-SS-Azacitidine synthesized in step 2 above were dissolved in 50 µL of DMSO. Subsequently, 1.0 mL of a mixed solvent of DMSO and H2O prepared at a volume ratio of 1:1 was added, and the solution was shaken until clear. Then, 1.2 mg and 6.0 µmol of sodium ascorbate and 0.48 mg and 3.0 µmol of anhydrous CuSO4 were added sequentially to initiate the "click" reaction. After reacting at room temperature under nitrogen protection for 24 hours, the mixture was extracted with an extract obtained by mixing equal volumes of dichloromethane and saturated brine. The organic layer (i.e., the lower layer of the extract) was separated and collected, and then dried with anhydrous sodium sulfate to remove water. The dried organic phase was concentrated under reduced pressure until the solvent was completely evaporated to obtain crude product 3. The crude product 3 was purified by preparative thin-layer chromatography using a developing solvent of dichloromethane and anhydrous methanol at a volume ratio of 20:1 to obtain the target compound TSA. The yield of TSA was 19.1 mg, with a yield of 89.3%.

[0063] The TSA 1H NMR spectrum is shown below. Figure 3 , Figure 3The results showed that: ¹H NMR (400 MHz, DMSO-d⁶) δ 8.94 (d, J = 5.3 Hz, 2H), 8.53 (s, 1H), 8.29 (s, 1H), 8.24 (d, J = 6.1 Hz, 2H), 8.11 (s, 1H), 8.05 (d, J = 16.3 Hz, 1H), 7.80 (d, J = 6.3 Hz, 2H), 7.75 (d, J = 6.2 Hz, 2H), 7.61 (d, J = 5.9 Hz, 4H), 7.53 (d, J = 14.6 Hz, 1H), 7.08 (d, J = 8.9 Hz, 4H), 6.95 (d, J = 6.5 Hz, 2H). 4H), 6.84 (d, J = 6.4 Hz, 2H), 5.63 (t,J = 3.2 Hz, 1H), 4.62 - 4.47 (m, 4H), 4.42 (t, J = 7.1 Hz, 2H), 4.36 - 4.24(m, 4H), 4.17 (t, 1H), 4.04 - 3.96 (m, 2H), 3.76 (s, 6H), 3.67 (t, J = 5.2Hz, 2H), 3.51 (s, 1H), 2.97 (t, J = 7.2, 3.6 Hz, 2H), 2.92 (t, J = 6.4, 2.4Hz, 2H), 1.93 - 1.82 (m, 4H).

[0064] Figure 4 The results show that HRMS (ESI) m / z calculates C 53 H 58 N9O 10 S 2+ The actual detected mass-to-charge ratio (m / z) value is 1044.3743.

[0065] The chemical structural formula of the target compound TSA is as follows: .

[0066] The synthetic route diagram for compounds TPAPy and TSA is shown below. Figure 5 .

[0067] 1.2 Preparation and characterization of T@ and TSA@ nanoparticles 1.2.1 In this embodiment, a nano-precipitation method is used to prepare nano-photosensitizers. Taking TSA@ as an example, the specific steps are as follows: 1. Raw material dissolution: Dissolve 1 mg TSA and 3 mg of the amphiphilic copolymer DSPE-PEG. 2000 Add 1 mL of tetrahydrofuran (THF) solvent and sonicate in an ultrasonic cleaner for 10 min to completely dissolve it, ensuring that the solution is homogeneous and free of precipitate, thus obtaining a mixed solution.

[0068] 2. Preparation of mixed solution: Mix the above mixed solution into a 1.5 mL EP tube, and use a pipette to repeatedly blow and swirl 30 times to mix it thoroughly, so as to obtain a uniform 1 mL raw material mixed solution.

[0069] 3. Nanoparticle formation by nanoprecipitation: Under ultrasonic conditions, the above raw material mixture solution is added dropwise to 9 mL of ultrapure water using a 1 mL syringe. The dropping rate is controlled (1 drop per second) to improve particle size uniformity. After ultrasonication for 3 min, TSA@ nanoparticle solution is formed by self-assembly through nanoprecipitation. The nanoparticle solution is clear and transparent.

[0070] 4. Dialysis to remove organic solvent: Transfer the TSA@nanoparticle solution to a dialysis bag (molecular weight cutoff 5000 Da) and dialyze in ddH2O at pH 7.4 for 24 h to remove THF solvent. During dialysis, replace the ddH2O every 6 h to maintain good solvent exchange efficiency. Collect the dialyzed TSA@nanoparticle solution in the dialysis bag.

[0071] 5. Purification and Concentration: After dialysis, an air pump was used to accelerate THF evaporation to reduce organic solvent residue. Next, the TSA@nanoparticle solution (ultrafiltration tube molecular weight cutoff 100 kDa) after ultrafiltration and dialysis at 5000 rpm was washed five times with distilled water to remove excess DSPE-PEG. 2000 The nanoparticle solution was collected in the ultrafiltration tube with an organic solvent to obtain a concentrated TSA@ nanoparticle solution. A 1 mg / mL (TPAPy) TSA@ nanoparticle solution was obtained and purified by filtration through a 0.45 μm filter membrane to remove large particles and unencapsulated material. The filtered solution was collected and stored at 4°C for later use.

[0072] 1.2.2 Preparation of T@Nanoparticles When preparing T@ nanoparticles, replace 1 mg TSA with 1 mg TPAPy, and follow the same steps as the TSA@ nanoparticle preparation method.

[0073] 1.2.3 Optical and AIE Property Analysis: The ultraviolet absorption of TPAPy was measured using a UV-Vis spectrophotometer, and the fluorescence emission spectrum of TPAPy was recorded using a fluorescence spectrometer to investigate its AIE (aggregation-induced emission) properties. Dimethyl sulfoxide (DMSO) was selected as a good solvent, and toluene as a poor solvent. Mixed solutions with different DMSO / toluene ratios (toluene content: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 95%) were prepared. Changes in fluorescence spectra under different mixing ratios were detected to confirm the AIE phenomenon.

[0074] Figure 6 The results in sections a to b show that TPAPy exhibits a typical AIE effect in both good and bad solvents.

[0075] 1.2.4 Particle size, morphology and optical characterization: The average particle size distribution of the prepared TSA@ was determined using a dynamic light scattering (DLS) instrument.

[0076] 1.2.5 Morphological observation by transmission electron microscopy: The morphology of TSA@ was observed and photographed under a transmission electron microscope.

[0077] 1.2.6 Detection of Reactive Oxygen Species (ROS): Detection of ROS generation in photosensitizer solutions using DCFH: Compound 2',7'-dichlorodihydrofluorescein (DCFH) was used as an indicator for detecting reactive oxygen species (ROS) in solution. When ROS is generated in the system, DCFH is oxidized to DCF, resulting in an increase in fluorescence intensity at 524 nm. 10 µM of photosensitizer (TSA@, TPAPy, or Ce6) was dispersed in 2 mL of PBS containing 50 µM DCFH. The resulting photosensitizer solution was placed in a cuvette and tested under white light (0.2 W / cm²). 2 Irradiation was performed. Fluorescence intensity from 480 nm to 650 nm was recorded using a fluorescence spectrometer.

[0078] Figure 6 The results from c~d show that TSA@ exhibits excellent ROS generation capability (see [reference]). Figure 6 (c); Compared to the traditional photosensitizer Ce6, the nano-prodrug photosensitizers TSA@ and TPAPy exhibit significantly stronger ROS generation capabilities (see c). Figure 6 (d).

[0079] Figure 6 The results of e~f indicate that TSA@ has a uniform morphology and is suitable for tumor-targeted delivery.

[0080] 1.2.7 Detection of drug encapsulation efficiency and drug loading The encapsulation efficiency (EE%) and drug loading (DLC%) of the photosensitizer nanoparticles were determined using a UV-Vis spectrophotometer to ascertain the encapsulation effect of T@ and TSA@ nanoparticles on TPAPy or TSA and their drug loading capacity. The experimental methods are as follows: 1. Establishment of Standard Curves: To accurately determine the content of TPAPy and TSA in T@ and TSA@ nanophotosensitizers, it is first necessary to establish concentration-UV absorption standard curves for the two molecules. The specific method is as follows: Prepare standard solutions of TPAPy or TSA at different concentrations: Weigh a certain amount of TPAPy or TSA and prepare a series of standard solutions of known concentrations with tetrahydrofuran (THF).

[0081] 2. UV-Vis spectrophotometer detection: The UV absorption spectra of TPAPy or TSA standard solutions of different concentrations were measured at the characteristic absorption wavelength using a UV-Vis spectrophotometer, and their maximum absorption peaks were recorded.

[0082] 3. Plot a standard curve: Plot a standard curve with solution concentration on the x-axis and the corresponding ultraviolet absorption intensity (OD value) on the y-axis. Use linear regression analysis to calculate the fitting equation (e.g., A = kC + b), where A is absorbance, C is concentration, and k and b are regression coefficients. Ensure the correlation coefficient (R²) of the fitted curve is accurate. 2 The value should be close to 1 to ensure the accuracy of the standard curve.

[0083] 4. Determination of drug content in nano-photosensitizers: Take 20 μL of concentrated nano-photosensitizer solution (concentration 1 mg / mL, based on TPAPy or TSA), add it to 2 mL of THF solution to disintegrate the nanoparticles and release the encapsulated TPAPy or TSA. Gently vortex or sonicate for 2 min to completely dissolve the drug in the THF solution.

[0084] 5. Ultraviolet Absorption Measurement: Place the dissolved nano-photosensitizer solution in a UV-Vis spectrophotometer to measure the absorbance. Read the absorbance value of the maximum absorption peak and substitute it into the standard curve equation to calculate the concentration of TPAPy or TSA in the solution.

[0085] 6. Calculate the actual concentration of TPAPy or TSA in the nano-photosensitizer: Calculate the concentration of TPAPy or TSA in the dissolved THF solution using the established standard curve equation. Multiply this concentration by 100 (since the sample volume is 20 μL and the dissolution volume is 2 mL) to obtain the TPAPy or TSA content in the original nano-photosensitizer concentrate (1 mg / mL).

[0086] 7. Calculate the encapsulation efficiency (EE%) and drug loading (DLC%): After obtaining the drug content in the nano-photosensitizer, the encapsulation efficiency (EE%) and drug loading (DLC%) of TPAPy or TSA can be further calculated. The calculation formula is as follows: EE% = (Drug loading mass) / (Initial drug loading mass) × 100%; DLC% = (Drug-loaded mass) / (Drug-loaded nanoparticle mass) × 100%.

[0087] Wherein: Drug loading mass refers to the actual TPAPy or TSA content measured in the photosensitizer nanoparticles. Initial drug loading mass refers to the actual mass of TPAPy or TSA added during the nanoparticle preparation process. The encapsulation efficiency of TPAPy or TSA in T@ and TSA@ nanoparticles can be measured by calculating EE%. The mass of drug-loaded nanoparticles needs to be measured by freeze-drying: a certain amount of nanoparticle solution is taken and freeze-dried using a freeze dryer (-80℃) until dry nanoparticle powder is obtained. The total mass of the freeze-dried nanoparticles is recorded. The DLC% can be calculated to measure the loading capacity of the nanoparticles for TPAPy or TSA.

[0088] The results showed that the encapsulation efficiency (EE%) of T@ and TSA@ was 82.73% and 71.29%, respectively, and the drug loading (DLC%) was 14.20% and 12.48%, respectively, indicating that TSA@ has good drug loading capacity and release control performance.

[0089] Next, the reduction-response release behavior of TSA@ was evaluated, simulating its release characteristics in the tumor cell microenvironment under different physiological conditions.

[0090] 1.2.8 Evaluation of in vitro drug release The experiment used dialysis diffusion method combined with ultraviolet-visible spectrophotometer to determine the release behavior of AZA from nanoparticles under simulated in vivo environmental conditions, in order to evaluate its release characteristics under different physiological environments.

[0091] 1. Sample Preparation and Dialysis Environment Setup: First, a TSA@nanoparticle drug solution was prepared. 2 mL of the TSA@nanoparticle drug solution (concentration 100 μg / mL) was precisely transferred and placed into a dialysis bag (molecular weight cutoff 5000 Da) to ensure that the drug within the TSA@nanoparticles would not diffuse directly into the external environment, but would be slowly released via membrane diffusion. The dialysis bag was then immersed in 15 mL of a simulated physiological environment solution to simulate the effects of different pH and reducing environments in vivo on drug release behavior. Two simulated physiological environment conditions were set: phosphate-buffered saline (PBS) at pH = 7.4, simulating the normal physiological environment, was used to study drug release behavior in the circulatory system (denoted as the TSA group). A phosphate-buffered saline solution at pH = 5.0 containing 10 mM GSH (glutathione) was used to simulate the tumor microenvironment (because tumor tissue is usually acidic and has a high GSH content), and was used to study drug release behavior in the environment surrounding cancer cells (denoted as the TSA+10 mM GSH group). The entire dialysis experiment was conducted in a 37°C constant temperature incubator to simulate in vivo temperature, and was kept oscillating at 100 rpm to ensure thorough mixing of the liquid outside the dialysis bag, thereby reducing the influence of drug concentration gradients in local areas and improving the accuracy of release data.

[0092] 2. Dialysis Sampling and UV Detection: To monitor drug release in real time, multiple time points were set for sampling and analysis. At the set time intervals, 3 mL of dialysis fluid was accurately taken using a pipette for testing. The absorbance of the extracted solution was immediately measured using a spectrophotometer, and the AZA concentration in the fluid at that moment was calculated. The drug concentration (Ct) in the solution was calculated using a pre-plotted AZA concentration-UV absorption standard curve. After each sampling, the same volume (3 mL) of fresh PBS buffer was immediately added to maintain a constant total volume of fluid outside the dialysis bag (15 mL), ensuring the stability of drug release behavior during the experiment. Each experimental condition was repeated three times, and the average value was calculated to ensure the accuracy and repeatability of the data. The cumulative percentage of drug release was calculated using the following formula: Drug release (%) = (Ct × V + Y) / M × 100. Where: V = total volume of dialysate (15 mL); Ct = drug concentration in dialysate at time t, calculated using UV-Vis detection; Y = total amount of drug collected before time t, i.e., the cumulative amount of drug from all previously sampled drugs; M = total amount of drug administered before dialysis, i.e., the initial AZA content in the nanoparticles. This formula takes into account the cumulative release, including not only the drug concentration in the dialysate at the current time point but also the accumulated amount of drug removed at all previous time points, to ensure data accuracy.

[0093] Figure 7The results showed that in a plasma-simulated environment (pH=7.4), TSA@ exhibited slight AZA release at a slow rate; however, in a tumor cell-simulated lysosomal environment (pH=5.0, 10 mM GSH), TSA@ showed significant AZA release, with nearly 75% AZA released within 24 hours. This indicates that under the highly reducing environment of tumor cells, highly water-soluble AZA can be rapidly released from the TSA@ nanophotosensitizer, thereby exerting its DNA demethylation and anti-tumor effects.

[0094] 1.3 Detection of ROS at the cellular level The production of reactive oxygen species (ROS) in mouse MB49 bladder cancer cells was detected using the DCFH-DA probe.

[0095] 1. Cell culture and experimental grouping: Cells were plated to a density of 7.5 × 10⁻⁶ cells / cm². 4 Cells were evenly seeded into confocal culture dishes at 37°C and 5% CO2 for 24 h to allow for full adhesion.

[0096] Experimental Groups: The experiment was divided into 6 groups to detect the ROS production levels under different treatment conditions: 1. Con. group (control group): Cells were cultured normally without any drug or light treatment, serving as a baseline control; 2. Light group (light control group): Cells were cultured and only exposed to light, without any drugs, to exclude the influence of light itself on ROS production; 3. AZA group (drug group, denoted as Aza. group): AZA (3 μg / mL) was added after cell culture to detect AZA-induced ROS production; 4. T@ group (nanophotosensitizer group): T@ nanoparticles (TPAPy=8 μg / mL) were added, but no light was applied to observe ROS generation under dark conditions; 5. T@+Light group (T@+light group): T@ (TPAPy=8 μg / mL) was added and the cells were exposed to light to observe light-induced ROS production; 6. TSA@+Light group (TSA@+light group): TSA@ (TPAPy=8 μg / mL) was added... The TSA@nanoparticles were subjected to light irradiation (μg / mL) to detect their ability to generate ROS under light irradiation.

[0097] 2. Nanophotosensitizer treatment: After culturing cells for 24 hours, carefully remove the old culture medium using a pipette to avoid interfering with the experimental results. Fresh drug-free 1640 basal medium was added to the Con. and Light groups, while the other groups were replaced with culture medium containing AZA (3 μg / mL), T@, or TSA@ (TPAPy=8 μg / mL), respectively.

[0098] 3. DCFH-DA Fluorescent Probe Incubation: PBS Washing: After culturing for 24 hours, wash cells 2-3 times with PBS buffer to remove unbound or residual photosensitizer nanoparticles and reduce background fluorescence interference. DCFH-DA Probe Incubation: Prepare 10 μM DCFH-DA working solution using serum-free 1640 as the solvent to avoid serum affecting probe hydrolysis. Gently add the DCFH-DA working solution and incubate at 37℃ in a CO2 incubator for 30 min to ensure uniform probe entry into the cells. After incubation, wash 3 times with PBS buffer to remove probes that have not entered the cells, thereby reducing background fluorescence signal.

[0099] 4. Illumination treatment and ROS induction: Under a white light irradiation system (light power density 200 mW / cm²), 2 Under the condition of ), the Light group, T@ + Light group, and TSA@ + Light group were subjected to light treatment, with white light irradiation (200 mW cm⁻¹). -2 The irradiation time was 3 minutes. The Con. group, AZA group, and T@ group were not irradiated to eliminate light interference. Immediately after irradiation, fluorescence imaging and flow cytometry were performed, and the results are as follows: Figure 8 and 9 As shown.

[0100] 5. Confocal fluorescence imaging: Observe the green fluorescence signal of DCF using a CLSM. Excitation wavelength (Ex): 488 nm, emission wavelength (Em): 530±20 nm, using a 40× or 60× objective lens.

[0101] 6. Flow Cytometry Detection: For each cell group, wash 2-3 times with PBS to remove unbound probes. Gently trypsinize the cells (approximately 1 min), then add complete culture medium to terminate the digestion. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and resuspend. Detection parameters: excitation wavelength 488 nm, emission wavelength 530±20 nm. Collect 20,000 cell events and record the mean fluorescence intensity (MFI). Perform statistical analysis using FlowJo to calculate the fluorescence intensity of each group. Compare the differences in ROS production among different experimental groups and perform statistical analysis.

[0102] Figure 8 The results showed that both T@ and TSA@ could significantly generate ROS after white light irradiation, oxidize DCFH to DCF and emit strong green fluorescence.

[0103] Figure 9The results showed that after light irradiation, the DCF fluorescence signal in each cell treated with T@ and TSA@ was significantly stronger than that in the Con. group, Light group, AZA group and T@ group, indicating that T@ and TSA@, these two nano photosensitizers, can effectively promote the generation of intracellular ROS after cell light irradiation.

[0104] Figures 8-9 The results demonstrated TSA@'s ability to generate ROS in vivo and in vitro, providing strong support for further research on ROS-mediated cell necrosis and apoptosis, where the AZA concentrations were 1.

[0105] 1.4 Cytotoxicity MTT assay to detect the killing effect of nano-photosensitizer on MB49 cells The experiment used the MTT assay to evaluate the dark toxicity and phototoxicity of nano-photosensitive agents (T@, TSA@) and AZA drugs at different concentrations on mouse MB49 bladder cancer cells. The dark toxicity assay aimed to assess the effects of different drugs on cells under dark conditions, while the phototoxicity assay was mainly used to detect whether the photosensitizer could significantly reduce cell viability under light conditions, thus reflecting its photodynamic activity (PDT effect).

[0106] 1. Dark toxicity assay: Cell culture and plating were performed. MB49 cells in the logarithmic growth phase were harvested, and cell density was calculated using a hemocytometer or automated cell counter. The cell density was then adjusted to 5 × 10⁶ cells / year. 3 Cells were evenly seeded into 96-well plates, with at least four replicates per group to ensure the stability of experimental data. The experiment was divided into AZA, T@, and TSA@ groups. Specifically, the plates were incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion and stable growth. After adhesion, the original culture medium was discarded, and medium containing different concentrations of AZA (0, 0.5, 1, 1.5, 3, and 6 μg / mL), T@, or TSA@ (T@ or TSA@ concentrations based on TPAPy, 0, 2, 4, 8, 16, and 32 μg / mL) was gently aspirated. Cells were incubated at 37°C with 5% CO2 for another 24 h to ensure sufficient interaction between the drug and cells. Serum-free DMEM + 0.5 mg / mL MTT was added, and the cells were cultured for another 4 h before MTT assay. Each experimental group was repeated in triplicate. The absorbance (OD value) was measured at 570 nm using a multi-functional microplate reader. The experimental data were recorded and the cell viability was calculated.

[0107] 2. Phototoxicity experiment: MB49 cells in logarithmic growth phase were taken and their density adjusted to 5 × 10⁻⁶. 3Cells were evenly seeded into 96-well plates, with 4 replicates per group. The experiment was divided into T@+L and TSA@+L groups. Specifically, the plates were incubated at 37°C with 5% CO2 for 24 hours to allow cell adhesion and stable growth. After cell adhesion, the old culture medium was removed and replaced with medium containing different concentrations of T@ or TSA@, based on the following TPAy concentrations: 0 μg / mL, 2 μg / mL, 4 μg / mL, 8 μg / mL, 16 μg / mL, and 32 μg / mL. Incubation was continued at 37°C with 5% CO2 for another 24 hours to ensure sufficient photosensitizer uptake by the cells. Light treatment: After 24 hours, cells were washed once with PBS to remove unbound photosensitizer. Complete culture medium (excluding MTT) containing 10% FBS was added to ensure the cells maintained physiological status during light exposure. Cells were then placed in a 200 mW / cm² incubator. 2 Under white light irradiation, cells were irradiated for 3 min to ensure sufficient excitation of T@ and TSA@. After irradiation, cells were placed back into the incubator for 24 h to observe the effect of photodynamic therapy on cell viability. After 24 h, the culture medium was aspirated, the cells were washed once with PBS, and serum-free DMEM + 0.5 mg / mL MTT was added. Cells were cultured for another 4 h, and MTT assays were performed. Each experimental group was repeated in triplicate. OD values ​​were measured at 570 nm using a multi-well microplate reader, and experimental data were recorded. Cell viability was calculated based on absorbance.

[0108] Figure 10 The results showed that T@ exhibited almost no cytotoxicity under light-free conditions, indicating that the photosensitizer itself had minimal effect on bladder cancer MB49 cells. TSA@ showed slight cytotoxicity at higher concentrations. However, after light exposure, both nano-photosensitizers (T@ and TSA@) significantly enhanced their tumor-killing effects, demonstrating their significant photodynamic therapy (PDT) efficacy. Notably, after light exposure, TSA@ showed significantly higher cytotoxicity against bladder cancer MB49 cells than T@, indicating that AZA treatment combined with PDT has a synergistic anti-tumor effect.

[0109] 1.5 AO staining and light microscopy confirmed the transformation of MB49 cells from apoptosis to necrotizing apoptosis. 1. Cell Culture and Grouping: First, MB49 cells in logarithmic growth phase were harvested, and the cell density was adjusted to 7.5 × 10⁻⁶. 4 Cells were evenly seeded into confocal culture dishes and placed in a cell culture incubator at 37°C and 5% CO2 for 24 hours to ensure full cell adhesion. Subsequently, the cells were divided into the following 6 groups according to the experimental design: Con. group (control group): Cells were not treated with any drugs or light and were cultured normally as a control. Light group (light control group): Cells were only treated with light after culture, without the addition of any drugs; AZA group (denoted as Aza. group): After adding 3 μg / mL AZA, cultured normally; T@ group (nano photosensitizer group): T@ nanoparticles (TPAPy=8μg / mL) were added, but no light exposure was performed; T@+Light group (T@+L group): After adding T@ (TPAPy=8μg / mL), the sample was subjected to light treatment; TSA@+Light group (TSA@+L group): After adding TSA@ (TPAPy=8μg / mL), the sample was subjected to light treatment.

[0110] 2. Nanoscale photosensitizer treatment: After culturing cells for 24 hours, carefully remove the old culture medium from the culture dish using a pipette. Replace the Con. group and Light group with drug-free 1640 basal medium and culture normally. For the other groups, replace the medium with one containing AZA (3 μg / mL), 8 μg / mL (based on TPAPy) T@, or 8 μg / mL (based on TPAPy) TSA@, according to the grouping requirements, to ensure that the treatment conditions of each group are consistent. Continue culturing for 24 hours to allow the nanoscale photosensitizer to fully interact with the cells.

[0111] 3. Light treatment: After 24 hours, wash the cells 2-3 times with PBS buffer to remove residual drugs or culture medium. Place the Light group, T@+Light group, and TSA@+Light group under a white light irradiation system (light power density: 200 mW / cm²). 2 ( ), Illuminate for 3 minutes, except for the Con. group, AZA group, and T@ group which do not undergo illumination treatment.

[0112] 4. AO staining and incubation: Wash cells 2-3 times with PBS buffer to remove residual drugs or culture medium, add 1 mL of AO staining buffer to cover the cells and mix well. Incubate at room temperature in the dark for 10-20 minutes.

[0113] 5. Observation and data recording: After incubation, gently wash the cells 1-2 times with PBS buffer to remove unbound AO dye, place them under a confocal laser scanning microscope, and observe the fluorescence using the following parameters: excitation wavelength: 488nm, emission wavelength: green fluorescence >530nm, red fluorescence >600nm.

[0114] 6. Simultaneously, observe the morphological changes of cells under an optical microscope, and observe the characteristics of apoptosis and necrotizing apoptosis, including chromatin condensation and nuclear fragmentation.

[0115] Figure 11The results showed that cells in the Con, Light, Aza, and T@ groups maintained a flattened, well-expanded morphology with a clear nucleocytoplasmic boundary. Red AO fluorescence was observed enriched around the entire nucleus in the cytoplasm, indicating normal cell condition. In the T@+L group, some cells exhibited nucleocytoplasmic condensation, irregularly stained nuclei (white arrows), and fragmented red cytoplasmic components (cyan arrows), suggesting apoptosis. In the TSA@+L group, some cells showed smooth, yellow-green spherical nuclei (white arrows), indicating necroptosis. The almost complete disappearance of red cytoplasmic fluorescence indicated thorough degradation of cytoplasmic RNA, consistent with typical characteristics of necroptosis. Due to the large number of cells undergoing necroptosis, washing during staining led to a decrease in cell density. Simultaneously, morphological changes in cells were observed under a light microscope. Cells in the Con, Light, Aza, and T@ groups showed normal morphology, intact cell membranes, and normal cell adhesion. However, cells in the T@+L group exhibited typical apoptotic morphology, such as cell shrinkage, reduced volume, small vesicles and irregular protrusions on the cell membrane surface, cell fragmentation, and the formation of apoptotic bodies. Cells in the TSA@+L group showed significant morphological changes, characterized by increased cell volume and swelling, more swollen cytoplasm, rounded and spherical cell shape, and reduced connections with neighboring cells, consistent with necrotizing apoptosis. AO staining and light microscopy provided a direct visual understanding of the changes in cell morphology and death patterns, indicating that MB49 bladder cancer cells underwent a transformation from apoptosis to necrotizing apoptosis after treatment with the nano-photosensitizer prodrug TSA@ and photodynamic therapy.

[0116] 1.6 Western blot analysis to detect the expression of p-RIPK3, p-MLKL and Cleaved-Caspase8 1. Cell seeding and grouping: MB49 cells were seeded at a ratio of 3 × 10⁻⁶ cells / year. 5 The holes are evenly distributed in a 6-hole plate and divided into the following six groups: Con. group (control group): serving as the baseline control, receiving no drugs or light treatment; Light group (light control group): Cells were only treated with light after culture, without the addition of any drugs; AZA group (denoted as Aza. group): treated with 2 μg / mL AZA; T@ group (nano photosensitizer group): T@ nanoparticles (TPAPy=8μg / mL) were added, but no light exposure was performed; T@+Light group (T@+L group): After adding T@ (TPAPy=8μg / mL), the sample was subjected to light treatment; TSA@+Light group (TSA@+L group): After adding TSA@ (TPAPy=8μg / mL), the sample was subjected to light treatment.

[0117] 2. Drug treatment: After the cells adhered and stabilized, the culture medium of the "Con. group" and "Light group" was replaced with blank culture medium; the other groups were replaced with culture medium containing AZA (2 μg / mL), T@ (TPAPy=8 μg / mL) or TSA@ (TPAPy=8 μg / mL) respectively, and incubated for another 24 hours.

[0118] 3. Light treatment: After 24 hours, discard the culture medium of all groups, wash three times with PBS buffer, and then replace with serum-free culture medium. Place the Light group, "T@+L group", and "TSA@+L group" in a white light irradiation system (200 mW / cm²). 2 Under light, irradiate for 3 minutes, and continue culturing for 24 hours after the light irradiation is completed.

[0119] 4. Protein extraction and detection: After 24 hours, the cells were washed once with PBS buffer, and then 150 μL of protein lysis buffer containing PMSF and protease inhibitors was added. The cells were then incubated on ice for lysis. Subsequent steps were performed according to the above method to detect changes in the protein expression of RIPK3, p-RIPK3, MLKL, p-MLKL, Cleaved-Caspase8, and total Caspase8.

[0120] According to section 1.6, after culturing cells in step 3 for 24 hours, 100 μL of lysis buffer was added to every 2 million cells, centrifuged for 10-15 minutes, and the supernatant was collected. The caspase-8 activity in the cells of each treatment group was detected using a caspase-8 activity assay kit.

[0121] 1.7 LDH Release Detection 1. Cell seeding and experimental grouping: 1. According to 5×10 3 To ensure a cell density of 80-90% confluence during the experiment, MB49 cells were evenly seeded into 96-well plates. 2. Cells were divided into the following three types of wells: simple culture medium wells (without cell detection of basic culture base signal); maximum enzyme activity control wells (used to detect the maximum amount of LDH released after complete cell lysis); experimental cell wells (divided into six experimental groups, with four replicates per group): Con. group (control group): cells were cultured normally without drug or light treatment; Light group (light control group): cells were exposed to light only, without drugs; AZA group (drug group): cells were incubated with AZA (2 μg / mL); T@ group (nanophotosensitizer group): cells were incubated with T@ (TPAPy = 8 μg / mL); T@+L group (T@+light group): cells were incubated with T@ (TPAPy = 8 μg / mL) and then exposed to light; TSA@+L group (TSA@+light group): cells were incubated with TSA@ (TPAPy = 8 μg / mL) and then exposed to light. 2. Cell Treatment: First, drug incubation: After cell adhesion, the Con. and Light groups were continued to be treated with blank medium, while the other groups were replaced with medium containing AZA (2 μg / mL), T@ (TPAPy=8 μg / mL), or TSA@ (TPAPy=8 μg / mL), respectively, and cultured for another 24 hours. Then, light treatment: After 24 hours, the medium was discarded, the cells were washed three times with PBS, and then replaced with serum-free medium. The Light, T@+L, and TSA@+L groups were placed in a 200 mW / cm² light source. 2 Under a white light irradiation system, the light was applied for 3 minutes.

[0122] 3. LDH release assay: (1) Cell lysis to determine maximum enzyme activity. (2) Collection of cell supernatant: At the end of the experiment, centrifuge the 96-well plate at 400g for 5 minutes to precipitate cell debris. Transfer 100μL of supernatant from each well to the corresponding well of a new 96-well plate, ensuring that no cell debris is carried. (3) LDH detection: Add 60μL of LDH detection working solution to each well and mix gently. Wrap the 96-well plate with aluminum foil to avoid light interference and incubate at room temperature on a horizontal shaker for 30 minutes to ensure that the reaction proceeds fully. After the reaction is complete, measure the absorbance at 490 nm using a microplate reader.

[0123] Figure 12 The results showed no significant difference in the expression level of total caspase 8 protein among the treatment groups. However, the expression of cleaved-caspase 8 was significantly increased in the T@+L group, indicating that cells in this group underwent significant apoptosis. This was consistent with the observations of AO staining and cell morphology under a light microscope, further supporting the conclusion that cell death in the T@+L group mainly occurred through the classical apoptosis pathway. Conversely, the expression of cleaved-caspase 8 was relatively low in the TSA@+L group, and the significant difference from the T@+L group suggests that the cell death mechanisms in the two groups may differ. Cells in the TSA@+L group may die more through necroptosis rather than the traditional apoptosis pathway. Meanwhile, cleaved-caspase 8 activity was almost non-existent in the Con, Light, Aza, and T@ groups, indicating that the cells in these groups were in good condition and no significant cell death or apoptosis occurred.

[0124] Figure 13 The results of study a showed that the T@+L group was significantly higher than that of the other groups, including TSA@+L, with a statistically significant difference.

[0125] Further investigation was conducted into the expression of RIPK3-MLKL pathway-related proteins in each group of cells to further determine the cell death mechanism in the TSA@+L group: such as Figure 12 As shown, RIPK3 protein expression was significantly upregulated in the Aza and TSA@+L groups, exhibiting a marked difference compared to other experimental groups. However, RIPK3 expression was almost undetectable in the Con, Light, Aza, and T@ groups. This result suggests that Aza may reactivate RIPK3 gene expression through its demethylation effect. This also indirectly confirms that RIPK3 gene expression in bladder cancer MB49 cells may be inhibited by hypermethylation. Furthermore, phosphorylated RIPK3 (p-RIPK3), a key signaling molecule activating the necroptosis pathway, and phosphorylated MLKL (p-MLKL), the final execution protein of necroptosis, were significantly expressed only in the TSA@+L group, and almost not expressed in the other groups. This indicates that TSA@+L treatment may effectively induce necroptosis, a result consistent with AO staining and cell morphology observations under a light microscope, further demonstrating that the main form of cell death in the TSA@+L group may be necroptosis.

[0126] Simultaneously, the release level of lactate dehydrogenase (LDH) in the cell supernatant of each group was measured. Figure 13 As shown in Figure b, the LDH release level in the TSA@+L group was significantly increased, being 6.9 times and 2 times higher than that in the control group and the T@+L group, respectively. This significantly elevated LDH level suggests that the cell membrane in the TSA@+L group ruptured, releasing cellular contents, further demonstrating that necrotizing apoptosis occurred in the cells.

[0127] All of the above results indicate that the TSA@nanophotosensitizer prodrug can effectively induce necrotizing apoptosis in bladder cancer MB49 cells under light activation.

[0128] 1.8 Detection of Cell-Level Immunogenic Cell Death (ICD) Related Indicators After confirming that TSA@-mediated PDT can effectively induce necrotizing apoptosis in MB49 cells, we further explored the changes in immunogenicity after the transition from apoptosis to necrotizing apoptosis, and detected key immunogenic cell death biomarkers, including ecto-CRT, HMGB1, HSP70, and ATP release.

[0129] 1.8.1 Detection of calreticulin eversion (ecto-CRT) by cellular immunofluorescence (IF) assay 1. Cell plating and grouping: Cells were plated at a density of 3 × 10⁶ cells / cm². 5Cells were seeded in 6-well plates and divided into 6 groups: "Con. group" (control group), "Light group" (light control group), "AZA group" (Aza. group), "T@ group" (nanophotosensitizer group), "T@+Light group" (T@+light group), and "TSA@+Light group" (TSA@+light group), with 4 replicates in each group. After seeding, the plates were placed in an incubator to allow for full adhesion.

[0130] 2. Treatment with drugs and nano-photosensitizers: After cell adhesion, except for the "Con. group" and "Light group", the other groups were replaced with culture medium containing AZA (2 μg / mL), T@ or TSA@ (TPAPy=8 μg / mL) for further culture. Blank culture medium was added to the "Con. group" and "Light group" as controls.

[0131] 3. Light treatment: After 24 hours, discard the culture medium, wash the cells three times with PBS, and add serum-free culture medium. Irradiate the "Light group," "T@+L group," and "TSA@+L group" with white light (200 mW / cm²). 2 (3 minutes), continue culturing after irradiation.

[0132] 4. Cell fixation and blocking: After 24 hours, wash the cells three times with PBS, then fix them with 4% paraformaldehyde fixative at room temperature for 15 minutes. After fixation, wash the cells three times with PBS, then block them with immunostaining blocking solution at room temperature for 30 minutes to prevent non-specific binding.

[0133] 5. Primary antibody incubation: After blocking, add diluted primary antibody (1:400) and incubate overnight at 4°C to allow the primary antibody to fully bind to the target protein.

[0134] 6. Secondary antibody incubation and nuclear staining: The next day, wash three times with PBS, add fluorescent secondary antibody (goat anti-rabbit Alexa Fluor 647, 1:400 dilution), and incubate at room temperature for 50 minutes. Then wash three times with PBS, add DAPI staining solution to stain the cell nuclei, and incubate at room temperature for 15 minutes.

[0135] 7. Observation and imaging: Cells were washed with PBS, and fluorescence signals were observed and captured using CLSM. The excitation wavelength was 633 nm and the emission wavelength was 671 nm to detect the expression of calreticulin outward turning (ecto-CRT).

[0136] 1.8.2 Western Blot analysis to detect HMGB1 and HSP70 release levels 1. Cell plating and treatment: Cells were plated at a density of 3 × 10⁶ cells / cm². 5The cells were seeded in 6-well plates, and the grouping and treatment methods were the same as in section 1.8.1 above. After seeding, the cells were placed in an incubator and cultured until they adhered to the plate, after which they were treated with drugs and nano-photosensitizers.

[0137] 2. Irradiation and Supernatant Collection: After light treatment, add 600 μL of serum-free culture medium to each well and continue culturing for 24 hours. After culturing, collect the cell supernatant and centrifuge at 1000 rpm for 3 minutes to remove dead cells and debris.

[0138] 3. Protein Denaturation: The supernatant was mixed with protein loading buffer and heated to 99°C to denature the proteins for subsequent Western Blot experiments. The changes in HMGB1 (25 kDa) and HSP70 (70 kDa) protein levels in the cell supernatant were detected using the aforementioned Western Blot method. The release of HMGB1 and HSP70 was analyzed by comparing the protein band intensities of different treatment groups.

[0139] 1.8.3 ATP release detection 1. According to the experimental design, MB49 cells were first cultured at a rate of 5 × 10⁻⁶. 4 Cells were seeded at a density of / wells in 12-well plates and divided into the following six experimental groups, with four replicates per group: Con. group (control group): cells were cultured normally without treatment; Light group (light-treated group): cells were treated with light only after culture; AZA group: cells were cultured with AZA (2 μg / mL); T@ group: cells were cultured with T@ (TPAPy = 8 μg / mL); T@+L group: cells were cultured with T@ and treated with light; TSA@+L group: cells were cultured with TSA@ (TPAPy = 8 μg / mL) and treated with light.

[0140] 2. After the cells have adhered and stabilized, except for the Con. group and the Light group, the culture medium of the other groups was replaced with a medium containing AZA (2 μg / mL), T@ (TPAPy=8 μg / mL) or TSA@ (TPAPy=8 μg / mL), and cultured for another 24 hours. Blank culture medium was added to the Con. group and the Light group.

[0141] 3. After 24 hours of culture, discard the culture medium, gently wash the cells three times with PBS, then add serum-free culture medium, and perform 200mW / cm² treatment on the Light group, T@+L group, and TSA@+L group. 2 Irradiate with white light for 3 minutes, and then continue culturing for 24 hours after the irradiation ends.

[0142] 4. Next, collect the supernatant from each group and centrifuge at 12000g for 5 minutes to remove dead cells. Freeze the supernatant on ice for later use.

[0143] 5. To detect ATP release, prepare a standard solution and plot a standard curve according to the instructions. Next, prepare the ATP detection working solution and add 100 μL / well of the working solution to a black 96-well plate. After incubating at room temperature for 5 minutes, add 20 μL of standard solution or sample solution to each well, mix well, and immediately perform detection using a chemiluminescence analyzer.

[0144] 6. Calculate the amount of ATP released in the supernatant of each group using the standard curve, and analyze and plot the results.

[0145] During ICD, ecto-CRT acts as an "eat me" signal, stimulating antigen-presenting cells (APCs) to phagocytose dead cells and promoting the presentation of tumor antigens, thereby inducing specific anti-tumor CD8. + T cell (CTL) responses. HMGB1 is an intranuclear protein released from the nucleus into the extracellular space during cell death, functioning as a damage-associated molecular pattern (DAMP). Released HMGB1 binds to pattern recognition receptors (PRRs) on the surface of myeloid cells, thereby activating dendritic cells (DCs), promoting their maturation, enhancing antigen presentation capabilities, and ultimately triggering an anti-tumor immune response. HSP70 is a stress protein whose release during ICD enhances antigen presentation and promotes the transmission of inflammatory signals. During cell death, ATP is released as a "search signal," recruiting dendritic cells (DCs) and macrophages to the tumor microenvironment and promoting phagocytosis and antigen presentation.

[0146] like Figure 14 As shown, bladder cancer MB49 cells showed significant ecto-CRT eversion after treatment with T@+L or TSA@+L, suggesting that bladder cancer MB49 cells are undergoing ICD process. The ecto-CRT eversion level in the TSA@+L group was significantly higher than that in the T@+L group.

[0147] Meanwhile, the levels of HMGB1 and HSP70 in the cell supernatant increased significantly, and the release level in the TSA@+L group was higher than that in the T@+L group (see...). Figure 15 (a) Compared with the T@+L group, the ATP release level in the TSA@+L group was significantly increased, reaching 1.8 times (see a). Figure 15 (b)

[0148] The above results indicate that TSA@ can effectively induce necrotizing apoptosis in bladder cancer MB49 cells under light activation. Compared with the T@+L group, the release level of ICD markers in the TSA@+L group was significantly increased. This suggests that the necrotizing apoptosis of bladder cancer MB49 cells induced by PDT after TSA@ treatment can enhance the immunogenicity of tumor cell death more than the immunogenic apoptosis induced by traditional PDT, and thus may promote the body's anti-tumor immune response.

[0149] 1.9 Detection of upregulation of PD-L1 receptor expression in MB49 cells after photodynamic therapy 1. Cell seeding and grouping: MB49 cells were seeded at a rate of 3 × 10⁶ cells / year. 5 Cells were evenly seeded into 6-well plates at a density of cells / well and divided into four experimental groups: "Con." group (control group), "Light" group (light control group), "TSA@" group (TSA@ treatment group), and "TSA@+Light" group (TSA@+light treatment group). After seeding, the cells were incubated in a 37°C, 5% CO2 incubator until they adhered stably.

[0150] 2. Treatment and incubation: "Con." group and "Light" group: replace with serum-free medium and continue culturing for 24 hours; "TSA@" group and "TSA@+Light" group: add 8 μg / mL TSA@ solution, replace with serum-free medium, and incubate for 24 hours.

[0151] 3. Light and subsequent cultivation: "Light" group and "TSA@+Light" group: After incubation, place in a 200 mW / cm² environment. 2 Under white light, the samples were illuminated for 3 minutes. After the illumination period, all experimental groups were replaced with serum-free culture medium and cultured again for 24 hours.

[0152] 4. PBS Washing: After culturing, discard the culture medium and wash the cells three times with PBS buffer to remove residual drugs. Replace with serum-free culture medium and continue culturing for 24 hours.

[0153] 5. Cell Collection and Detection: A portion of cells from each group was collected, lysed, and proteins extracted. PD-L1 expression was detected according to standard Western blot procedures. The remaining cells were collected and stained with PD-L1 flow cytometry antibody according to flow cytometry staining procedures; the excitation wavelength was 488 nm, and the emission wavelength was 633 nm. The average fluorescence intensity of PD-L1 was detected. FlowJo software was used for statistical analysis of the data, and the fluorescence intensity of different treatment groups was calculated and compared.

[0154] Figure 16The results in a~c showed that after PDT treatment, the PD-L1 expression level in the TSA@+Light group of MB49 cells was significantly higher than that in other groups.

[0155] Further verification showed that immunogenic necrotizing apoptosis (ICD-Necroptosis) can more effectively promote the body's anti-tumor immune response compared to immunogenic apoptosis (ICD-Apoptosis), and simulated the immune stimulation of dendritic cells (DCs) by tumor cells in in vitro experiments.

[0156] 1.10 In vitro maturation stimulation experiment of BMDCs cells 1. Cell plating and grouping: MB49 cells were plated at a density of 1×10⁻⁶ cells / mL. 5 The cells were laid in a number of wells on the upper layer of the Transwell chamber and divided into six experimental groups: "Con. group" (control group), "Light" group (light irradiation group), "AZA" group (Aza. group), "T@" group (nanoparticle group), "T@+Light" group (nanoparticle plus light irradiation group), and "TSA@+Light" group (TSA@ nanoparticle plus light irradiation group).

[0157] 2. Drug treatment: On the second day, after the cells adhered and stabilized, except for the control group and the light group, the culture medium of the other groups was replaced with a new culture medium containing AZA (2μg / mL), T@ or TSA@ (TPAPy=8μg / mL), and they continued to be cultured at 37℃. The control group and the light group were given blank culture medium without drugs.

[0158] 3. Light treatment: After 24 hours, discard the culture medium and gently wash the cells with PBS, then add complete culture medium. For the light group, T@+L group, and TSA@+L group, white light irradiation (200 mW / cm²) was performed. 2 Irradiation time: 3 minutes.

[0159] 4. Co-culture treatment: After the above treatment, immature dendritic cells (DCs) were placed in the lower layer of the Transwell chamber and co-cultured with the treated MB49 cells for 24 hours to simulate cell-cell interactions.

[0160] 5. Cell Collection and Staining: After 24 hours of co-culture, DCs were collected and transferred to flow cytometry tubes. They were labeled with CD80 antibody and stained at 4°C in the dark for 30 minutes. Simultaneously, the supernatant was collected for IL-12p70 cytokine detection.

[0161] 6. Detection and Analysis: The positive expression rate of cells in each group was analyzed by flow cytometry to assess the maturation level of dendritic cells (DCs). Simultaneously, cytokine assays were performed using an ELISA kit following the manufacturer's instructions.

[0162] Figure 17 The results showed that TSA@-induced immunogenic necroptosis in bladder cancer MB49 cells after photoactivation could effectively stimulate the maturation of bone marrow-derived dendritic cells (DCs) in vitro. PDT-pretreated MB49 cells (T@+L group) significantly promoted DC maturation, and CD80 expression levels were significantly increased compared to untreated MB49 cells co-cultured, suggesting that PDT-induced ICD enhances the antigen-presenting capacity of DCs. The DC maturation rate induced by TSA@+L group (immunogenic necroptosis) was significantly higher than that of T@+L group (immunogenic apoptosis), and the difference was statistically significant (P<0.05). This indicates that necroptotic cells are more effective at activating DCs than apoptotic cells, further supporting the view that immunogenic necroptosis is a stronger anti-tumor immune trigger, and that TSA@+L has a stronger therapeutic effect on bladder cancer through necroptosis.

[0163] Figure 18 The results showed that the TSA@+L group had the highest IL-12p70 secretion level compared with other groups, indicating that this group had a stronger maturation-promoting effect on DCs, resulting in a stronger IL-12p70 secretion.

[0164] In summary, necroptosis, compared to apoptosis, can induce more efficient maturation of dendritic cells (DCs), thereby promoting stronger antigen presentation and stimulating T cell activation, resulting in a more favorable anti-tumor immune response.

[0165] Example 2 Preparation of TSA@HH Nanophotosensitizer 2.1 Synthesis and Characterization of the C-HWFHRHH Peptide Sequence 1. Resin swelling: Weigh about 0.5g (0.5mmol) of dichloromethane resin and add it to the solid-phase synthesis reactor. Add sufficient dichloromethane (DCM) solvent and swell for 10 minutes to obtain DCM washing resin. Use a syringe to squeeze out the solvent (DCM washing resin) for later use.

[0166] 2. Coupling of the first amino acid: Dissolve 1 mmol of the first amino acid and 2 mmol of diisopropylethylamine (DIEA) in DCM, add to the reactor, and react at room temperature for 1.5 hours to obtain the reaction solution.

[0167] 3. Blocking active sites: After extruding the reaction solution, wash the resin with 10 mL of DCM each time, for a total of 5 washes. Then add 10 mL of blocking solution (the blocking solution is prepared by mixing methanol, DCM and DIEA in a volume ratio of 17:2:1, respectively), block at room temperature for 15-30 minutes, and then wash with DCM 5 times.

[0168] 4. Deprotection: Wash 5 times with DMF, then treat with a 20% piperidine / DMF mixture (the 20% piperidine / DMF is prepared by mixing piperidine and DMF at a volume ratio of 1:4) for 30 minutes to remove the Fmoc group and expose the amino group.

[0169] 5. Coupling of the second amino acid: After washing with DMF 5 times, 1 mmol of the second amino acid, 1 mmol of HBTU and 3 mmol of DIEA were dissolved in DMF, added to the reactor and reacted at room temperature for 2 hours.

[0170] 6. Repeat the cycle: Deprotection and coupling are performed sequentially according to steps 4 and 5, with each amino acid coupling time being 2 hours.

[0171] 7. Complete peptide chain synthesis: After the last amino acid at the N-terminus of the peptide chain is coupled, continue with the subsequent peptide chain cleavage and purification steps.

[0172] 8. Resin washing: Wash 5 times with DMF (10 mL DMF per wash). Wash 5 times with DCM (10 mL DCM per wash) to remove residual solvent and reagents.

[0173] 9. Peptide chain cleavage: First, prepare the cleavage reagent (TFA cleavage system is commonly used): Mix TFA, deionized water, triisopropylsilane (TIPS) and dithiothreitol (DTT, used to protect thiol groups) in a volume ratio of 90:5:2.5:2.5 to obtain the cleavage reagent. Then, take 10 mL of the cleavage reagent, add it to the solid phase reactor, and stir or let it stand at room temperature for 2-3 h to allow the peptide chain to be completely cleaved from the resin, thus obtaining the cleavage solution.

[0174] 10. Freeze-precipitation: Collect the cutting fluid into 50 mL of frozen acetone to precipitate the crude peptide. Centrifuge the crude peptide (4000 rpm, 10 min, 4℃), discard the supernatant, collect the precipitate, wash the precipitate 2-3 times with frozen acetone to remove residual TFA, and vacuum dry or blow dry with nitrogen to obtain crude peptide powder.

[0175] 11. Crude peptide dissolution: Use a mixed solvent (such as water / acetonitrile (ACN) / TFA (0.1%) mixed solvent) to dissolve the crude peptide, and sonicate to improve the solubility and obtain the dissolved crude peptide.

[0176] 12. Purification (RP-HPLC): The dissolved crude peptide was purified using reversed-phase high-performance liquid chromatography (RP-HPLC) with C0... 18 Chromatographic column, gradient elution (e.g., 0-60% ACN (containing 0.1% TFA), time 30-60 min). Collect the target peptide peak, confirm the molecular weight by LC-MS, and lyophilize the purified peptide solution to obtain pure peptide powder.

[0177] 13. Quality assessment: The purity of the peptide powder was determined by LC-MS (liquid chromatography-mass spectrometry) and HPLC, and the purity of the peptide powder was >95%.

[0178] The HWFHRHH (SEQ ID NO.1) can also be referred to as C-HWFHRHH or simply HH or C-HH, where C- represents the C-terminus. The structural formula of the C-HH (C-HWFHRHH) polypeptide is shown in [link to structural formula]. Figure 19 . Figures 20-21 The results show that C-HH was successfully synthesized in this invention.

[0179] 2.2 Preparation of TSA@HH Nanophotosensitizer (1) Surface modification with maleimide: To modify the surface of nanoparticles with peptides, nanoparticles with maleimide on the surface (TSA@-Mal) were prepared using the same nanoprecipitation method. The specific steps are as follows: During the preparation of TSA@-Mal, DSPE-PEG was used. 2000 Replace with DSPE-PEG 2000 -Mal is introduced to introduce maleimide groups on the surface of nanoparticles. The remaining steps are the same as the TSA@ preparation steps in section 1.2.1 of Example 1, to obtain TSA@-Mal.

[0180] The thiol group (-SH) of the cysteine ​​(Cys) side chain can undergo a Michael addition reaction with maleimide to form a stable thio-ether bond, which can be used for subsequent functionalization modification.

[0181] The polypeptide CHWFHRHH (abbreviated as HH or C-HH) prepared above was selected and characterized by LC-MS and high-resolution mass spectrometry to ensure its accuracy.

[0182] (2) Peptide coupling: Weigh 2.5 mg of HH peptide powder and dissolve it in 1 mL of TSA@-Mal (1 mg / mL, based on TPAPy) solution. Stir the reaction at room temperature for 12 h to covalently couple HH peptide with TSA@-Mal to obtain the coupling compound. Then, use 5000 rpm centrifugation to ultrafilter the coupling compound (the molecular weight cutoff of the ultrafiltration tube is 100 kDa) and wash it 5 times with distilled water to remove unbound peptides. Collect the TSA@HH nanoparticles in the ultrafiltration tube to obtain the modified TSA@HH nanophotosensitizer, named TSA@HH.

[0183] The morphology of TSA@HH was detected by TEM, and the results are shown in [Figure number missing]. Figure 22 .

[0184] Figure 22 The results showed that the morphology of TSA@HH was basically consistent with that of TSA@, indicating that HH modification did not significantly affect the physical properties of the nanocarrier.

[0185] 2.3 Evaluation of in vivo tissue distribution and tumor enrichment of photosensitizer nanoparticles 1. MB49 cell preparation: Collect MB49 cells in the exponential growth phase, centrifuge and discard the supernatant, then adjust the cell concentration to 2×10⁻⁶. 7 per mL.

[0186] 2. Subcutaneous tumor-bearing model construction: 100 μL of MB49 cell suspension (containing 2×10⁻⁶ cells) was drawn using an insulin syringe. 6 (100 cells) were injected subcutaneously into the back of C57BL / 6J mice to establish a subcutaneous MB49 bladder cancer tumor-bearing mouse model. The general condition of the mice and tumor growth were monitored regularly after injection, and the tumor volume was increased to approximately 100 mm². 3 Subsequent experiments will be conducted in due course.

[0187] 3. Injection of nano-photosensitizer: After the tumor volume reached the experimental requirements, the experiment was divided into the "Saline" group (control group), the "AZA" group, the "T@" group, and the "TSA@HH" group. In the Saline group, 200 μL of physiological saline was injected into mice via the tail vein. In the T@ group, 200 μL of 1 mg / mL T@ was injected into mice via the tail vein. In the TSA@HH group, 200 μL of 1 mg / mL nano-photosensitizer TSA@HH was injected into mice via the tail vein.

[0188] 4. Tissue Sample Collection and Fluorescence Imaging: Mice were sacrificed at different time points (0, 4, 8, 12, 24, and 36 hours after injection of the nano-photosensitizer), and vital organs (such as heart, liver, spleen, lung, and kidney) and tumor tissues were collected. The collected tissue samples were placed in a small animal in vivo imaging system for fluorescence imaging, and the fluorescence signal intensity of each tissue was recorded. Complete blood count and blood biochemical parameters were also measured in the mice.

[0189] 5. Data Analysis: By analyzing the fluorescence intensity values ​​and trends of various tissues at different time points, the distribution, metabolism, and tumor accumulation of the photosensitizer in vivo were evaluated. The focus was on the accumulation efficiency of the photosensitizer in tumor tissue and its metabolic clearance in major organs, providing experimental evidence for the optimization of subsequent photodynamic therapy.

[0190] Figure 23 The results showed that TSA@HH did not have a significant impact on the blood system or liver and kidney function, indicating that TSA@HH has good in vivo biocompatibility and biosafety, providing a solid safety foundation for subsequent in vivo anti-tumor research.

[0191] Figure 24 The results showed that TSA@HH was mainly enriched in tumor tissue and liver after intravenous injection, indicating that the photosensitizer nanoparticle has excellent tumor-targeting enrichment ability and is mainly metabolized by the liver. Notably, the enrichment level of the photosensitizer nanoparticle was highest at the tumor site 12 hours after intravenous injection, providing an important time window and guiding basis for the implementation of subsequent photodynamic therapy (PDT).

[0192] 2.4 In vivo study on subcutaneous tumor inhibition of bladder cancer According to the experimental design, a subcutaneous MB49 bladder cancer mouse model was constructed to evaluate the effects of different treatments of the nano-photosensitizer on tumor growth and survival in mice. The experiment included recording tumor growth curves, monitoring changes in mouse body weight during treatment, and observing the survival of mice in each group. The specific steps are as follows: 1. Mouse model construction: First, following standard operating procedures, 2×10⁻⁶ mg / L of the solution was subcutaneously injected into the back of C57BL / 6J mice. 6 MB49 bladder cancer cells were successfully used to construct a subcutaneous MB49 tumor-bearing mouse model of bladder cancer. After inoculation, the tumors in the mice gradually grew, with the initial tumor volume reaching approximately 75 mm². 3 This ensures that the experimental subjects are in an appropriate disease state.

[0193] 2. Grouping: On day 6 after tumor inoculation (i.e., day 1 of the experimental plan, when the tumor volume had reached the experimental requirements), the mice entered the treatment phase. At this time, all mice were randomly divided into five groups of 5 mice each, as follows: "Saline" group (control group), "AZA" group, "T@ +L" group, "TSA@ + L" group, and "TSA@HH+L" group. Each group received different treatment methods. The "Saline" group received only saline as a control, while the other groups received different drug treatments.

[0194] 3. Treatment Regimen: During the treatment phase, specifically days 1, 3, and 5 of the experimental plan, all mice received a single intravenous injection of different drug solutions. Specifically, the "AZA" group received 200 μL of 0.3 mg / mL AZA intravenously; the "T@ +L" group received 200 μL of 1 mg / mL (TPAPy based) T@ solution intravenously; the "TSA@ + L" group received 200 μL of 1 mg / mL (TPAPy based) TSA@ solution intravenously; and the "TSA@HH + ​​L" group received 200 μL of 1 mg / mL (TPAPy based) TSA@HH solution intravenously. For the photodynamic therapy (PDT) groups, namely the "T@ +L", "TSA@ + L", and "TSA@HH + ​​L" groups, 12 hours after drug injection, mice were exposed to white light radiation (400 mW / cm²). 2 (Irradiate for 10 minutes) to activate the nano photosensitizer.

[0195] 4. Tumor growth monitoring: During treatment, the tumor volume and body weight changes of mice in each group were monitored regularly. The formula for calculating tumor volume is: Tumor volume = 1 / 2 × L × W 2 Where L is the long axis of the tumor and W is the short axis of the tumor. In addition, the weight changes of the mice were recorded regularly to assess potential side effects during treatment. Tumor volume and mouse weight were measured every two days during the experiment.

[0196] 5. Endpoint and tissue collection: On day 16 of treatment, all mice were euthanized and tumor tissue was collected for subsequent pathological staining and immunofluorescence staining.

[0197] 6. Survival Observation: In addition to the tumor growth curve monitoring and weight change observation mentioned above, a survival rate observation experiment was also conducted. This experiment followed the same treatment methods as described above, with 10 mice in each group. The endpoint events of the experiment were: natural death of the mouse or the tumor volume reaching 2000 mmHg. 3 Mice were then euthanized appropriately. This observation allows for a more direct assessment of the impact of different treatments on mouse survival.

[0198] Figure 25The results showed that tumor growth was rapid in the "Saline" group, indicating that saline alone failed to effectively inhibit tumor growth. Tumor growth in the "AZA" group was not significantly different from that in the "saline" group, indicating that low-dose AZA alone did not produce a significant anti-tumor effect. Tumor growth in the "T@+L" group was significantly inhibited, but the anti-tumor effect was still not ideal compared to other treatment groups. The "TSA@+L" group showed a stronger tumor inhibition effect, indicating that the efficiency of tumor cell death and the therapeutic effect were significantly improved under the TSA@-mediated PDT-induced cell necrosis and apoptosis. The "TSA@HH+L" group exhibited the most significant tumor inhibition rate, significantly higher than the "TSA@+L" group. This result indicates that PDT-induced immunogenic necrosis and apoptosis combined with immune checkpoint inhibition (ICB) therapy based on TSA@ and TSA@HH can more effectively enhance the anti-tumor therapeutic effect.

[0199] Figure 26 The results of the study showed that, compared with the “Saline” group, the “AZA” group, and the “T@+L” group, the mice in the “TSA@+L” group and the “TSA@HH+L” group had a longer survival time. Among them, the mice in the “TSA@HH+L” group had the longest survival time, which further confirmed the powerful anti-tumor effect of the TSA@HH+L combination therapy. Figure 26 The results of study b showed that there was no significant difference in body weight change among the groups of mice throughout the treatment process, further indicating that the combined treatment based on TSA@HH and TSA@ had low toxicity to mice and did not cause significant adverse effects on their health.

[0200] During ICD, calreticulin (CRT) translocates from the endoplasmic reticulum to the cell membrane surface (ecto-CRT). As a "eat me" signal, it promotes antigen-presenting cells (APCs) to recognize and engulf tumor cells and further activate specific anti-tumor immune responses. To investigate the effects of different groups on the ability of immunogenic cell death (ICD) to induce ICD, immunofluorescence experiments were used to assess the membrane translocation level of ecto-CRT in tumor tissues.

[0201] Figure 27 The results showed that MB49 cells in the three groups of mice treated with PDT (“T@ +L” group, “TSA@ +L” group and “TSA@HH +L” group) all exhibited significant ecto-CRT. The ecto-CRT levels in the “TSA@ +L” group and “TSA@HH +L” group were significantly higher than those in the “T@ +L” group, indicating that TSA@-mediated immunogenic necrotizing apoptosis enhances CRT membrane translocation more effectively than T@-induced immunogenic apoptosis.

[0202] 2.5 In vivo antitumor immunological evaluation After completing the corresponding treatment experiments according to the experimental methods in Section 2.3, in order to evaluate the impact of different treatment groups on the maturation level of dendritic cells (DCs), tumor draining lymph nodes (TDLNs) of mice in each group after treatment were obtained, and the maturation rate of their dendritic cells (DCs) was analyzed by flow cytometry.

[0203] For lymphoid tissue: 1. Lymph node (TDLN) collection: Expose the lymph nodes draining from the groin, axilla, or corresponding tumor area of ​​the mouse, and gently dissect the lymph nodes to avoid excessive mechanical damage. Place them in centrifuge tubes or beakers containing PBS, and then transfer them to a sterile environment for grinding or homogenization.

[0204] 2. Grinding the lymph nodes: After collecting the lymph nodes, place them in a sterile glass homogenizer. Add 1 mL of PBS solution as a medium and gently grind the tissue to thoroughly break down the lymph nodes. Avoid creating large pieces of tissue during the grinding process.

[0205] 3. Filtration and Centrifugation: Filter the homogenized solution through a 70 μm cell sieve and collect the filtrate in a centrifuge tube. This step removes larger tissue fragments and obtains a more homogenous cell suspension. Centrifuge at 1200 rpm for 5 minutes and discard the supernatant. This effectively precipitates components such as lymphocytes.

[0206] 4. Resuspension and Cell Counting: Resuspend the cell pellet in PBS and adjust the concentration to 1×10⁻⁶. 7 Cells / mL.

[0207] 5. Staining: Add 100 μL of the resuspended lymph node single-cell suspension (1×10⁻⁶ cells) to each flow cytometry tube. 7 Add approximately 1 μL of the antibody mixture to the flow cytometer, and finally add 3 μL of the antibody mixture (containing CD11c, CD80, and CD86) to the flow cytometer. Mix gently to avoid damaging the cells due to excessive force.

[0208] 6. Incubation and washing: Place the cells in a 4°C dark environment and incubate for 30 minutes. After incubation, add 1 mL of PBS to stop staining, centrifuge (1200 rpm, 5 minutes), discard the supernatant, and resuspend in 200 μL of PBS.

[0209] Figure 28The results showed that the DC maturation rate in the "TSA@ + L" group was 42.4%, which was 2.7 times that of the "Saline" group and 1.3 times that of the "T@ + L" group. This indicates that TSA@-mediated immunogenic cell necrosis and apoptosis is more effective than T@-induced immunogenic cell apoptosis in promoting DC maturation, thereby enhancing tumor antigen presentation capacity, transforming immune "cold" tumors into immune "hot" tumors, and triggering a stronger anti-tumor immune response. The highest DC maturation rate was 53.7% in the "TSA@HH + ​​L" group, indicating that after binding with HH peptide, ICD-induced immunogenic necrosis and apoptosis can more effectively activate DCs and enhance the anti-tumor immune response.

[0210] 2.6 In vivo study on in situ tumor suppression of bladder cancer Establishment of a tumor-bearing mouse model: 1. Select male C57BL / 6J mice aged 6-8 weeks and acclimatize them for 1 week to ensure they are in good condition before the experiment. Fast them for 12 hours before the experiment, but allow them free access to water to reduce surgical complications.

[0211] 2. In situ tumor inoculation was performed using 2% isoflurane inhalation anesthesia to ensure complete anesthesia of mice while maintaining normal physiological functions.

[0212] 3. Under sterile conditions, mice were fixed in a supine position. The abdomen was prepared and the surgical area was disinfected with 75% ethanol. The skin and muscle layers were incised along the midline of the lower abdomen to gently expose the bladder. Using a 1mL cell syringe, 30μL of MB49-Luciferase cell suspension (1×10⁻⁶) was injected. 6 The bladder was slowly injected into the bladder wall after mixing 50% Matrigel matrix gel (prepared with RPMI-1640) with 30 μL of cells. The Matrigel was allowed to solidify (approximately 1-2 minutes), then the bladder was carefully repositioned. The muscles and skin were sutured layer by layer, the incision was applied with antibiotic ointment, and the mouse was awakened in a 37°C resuscitation incubator.

[0213] 4. On day 5 after modeling (day 1 of the experimental plan), mice were randomly divided into 5 groups (n=5 per group): "Saline" group, "AZA" group, "T@+L" group, "TSA@+L" group, and "TSA@HH+L" group. On day 1 and day 3 of the experiment, mice in each group received intravenous injections (tail vein) of different drugs: AZA group (AZA 0.3mg / mL, 200μL), PDT-related group (T@+L, TSA@+L, TSA@HH+L, TPAPy 1mg / mL, 200μL), and Saline group (equal volume of physiological saline 200μL).

[0214] 5. Twelve hours after drug injection, the PDT-related group underwent phototherapy: the skin and muscle layer were reopened at the original incision site to expose the bladder and tumor, and a white light source (400 mW / cm²) was used. 2 The bladder tumor was directly irradiated for 10 minutes. Mice in the Saline and AZA groups underwent the same surgical procedure to eliminate surgical interference factors.

[0215] 6. Starting from day 1 of the experimental plan, in vivo fluorescence detection was performed in mice every 3 days to record tumor growth. Procedure: 60 μL of luciferase substrate (30 mg / mL) was injected intraperitoneally. Five minutes later, imaging was performed using a small animal in vivo imaging system (IVIS) to record the tumor fluorescence intensity. Mouse weight was recorded every 3 days to assess systemic toxicity of the treatment and the overall health status of the mice.

[0216] 7. On day 13 of the experiment, the mice were euthanized, the bladder tumor tissue was removed, weighed and the tumor mass was recorded.

[0217] Figure 29 The results showed significant differences in the inhibitory effects on MB49 in situ tumor growth in bladder cancer among the different treatment groups, further validating the enhanced effect of TSA@-mediated tumor treatment. Tumor growth was rapid in the "Saline" group and the "AZA" group, with no significant difference, indicating that AZA alone failed to effectively inhibit tumor growth. Tumor growth was inhibited in the "T@+L" group, but the anti-tumor effect was still unsatisfactory. The tumor inhibition effects of the "TSA@+L" group and the "TSA@HH+L" group were significantly better than those of the "T@+L" group, with some mice showing loss of bioluminescent signal, indicating that necrosis and apoptosis can better promote antigen release and enhance anti-tumor immunity. The "TSA@HH+L" group exhibited the most significant tumor inhibition effect, with its bioluminescent signal lower than that of the "TSA@+L" group at both the early and final stages of treatment, indicating that PDT-induced necrosis and apoptosis combined with PD-L1 blockade can effectively reduce T cell exhaustion and enhance the immune killing ability of CTLs.

[0218] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A photosensitizer for treating tumors, characterized in that, The chemical structural formula of the photosensitizer drug is: It was named TSA.

2. A method for preparing the photosensitizer drug according to claim 1, characterized in that, Includes the following steps: TSA was obtained by using TPAPy and compound SS-AZA as reactants and sodium ascorbate and CuSO4 as catalysts via a "click" reaction. The chemical structural formula of TPAPy is: ； The chemical structural formula of the compound SS-AZA is as follows: The molar ratio of TPAPy, SS-AZA, sodium ascorbate and CuSO4 is (15~25):(15~25):(4~8):(2~4); the "click" reaction conditions are nitrogen protection, reaction at 20~25℃ for 20~26 hours.

3. The preparation method according to claim 2, characterized in that, The preparation method of TPAPy includes the following steps: condensing 4'-[bis(4-methoxyphenyl)amino]-[1,1'-biphenyl]-4-carboxaldehyde and 1-(4-azidobutyl)-4-methylpyridine-1-onium iodide, then performing ion exchange with saturated KPF6 aqueous solution, and purifying to obtain TPAPy.

4. The preparation method according to claim 3, characterized in that, The molar ratio of 4'-[bis(4-methoxyphenyl)amino]-[1,1'-biphenyl]-4-carboxaldehyde to 1-(4-azidobutyl)-4-methylpyridine-1-onium iodide is (0.5~1.5):(0.5~1.5); the condensation reaction is a reflux reaction for 40~50 h.

5. The preparation method according to claim 2, characterized in that, The preparation method of the compound SS-AZA includes the following steps: dissolving propyne-monoethylene glycol, N,N-diisopropylethylamine and 4-dimethylaminopyridine in anhydrous dichloromethane, then adding p-nitrophenyl chloroformate to carry out a first reaction to obtain a reaction stock solution, which is then concentrated and dissolved in N,N-dimethylformamide to obtain a reaction solution. Then, azacitidine, the reaction solution and triethylamine are mixed to carry out a second reaction to obtain a mixed solution. The mixed solution is then concentrated, extracted and purified to obtain the SS-AZA.

6. The preparation method according to claim 5, characterized in that, The molar ratio of propyne-monoethylene glycol, N,N-diisopropylethylamine, 4-dimethylaminopyridine, and p-nitrophenyl chloroformate is (0.1~0.5):(1~1.4):(0.01~0.05):(0.4~0.8); the addition ratio of azacitidine, reaction solution, and triethylamine is 1~1.5 mmol:5~10 mL:1~1.5 mmol.

7. A pharmaceutical composition for treating tumors, characterized in that, It includes an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is nanoparticles formed by the self-assembly of TSA and the amphiphilic block copolymer DSPE-PEG2000 and its derivatives as described in claim 1.

8. The pharmaceutical composition according to claim 7, characterized in that, The amphiphilic block copolymer DSPE-PEG2000 derivative is DSPE-PEG2000-Mal, and the surface of the nanoparticles is further coupled with a PD-L1 blocking peptide. The amino acid sequence of the PD-L1 blocking peptide is shown in SEQ ID NO.1, which is HWFHRHH.

9. The use of the photosensitizer drug TSA according to claim 1, or the TSA prepared by the preparation method according to any one of claims 2 to 6, or the pharmaceutical composition according to claim 7 or 8, in the preparation of a drug for photodynamic therapy of tumors, characterized in that, After being activated by light, the drug or drug composition induces tumor cells to die by changing from apoptosis to necrotizing apoptosis.

10. The application according to claim 9, characterized in that, The tumor cells are bladder cancer cells, and the photoactivation is the activation of the photodynamic drug by applying light within a 12-hour time window after drug administration.

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