Heavy metal-free photosensitizer as well as synthesis method and application thereof

Abstract

The invention discloses a heavy metal-free photosensitizer as well as a synthesis method and application thereof, and belongs to the technical field of cancer treatment medicines. The structural formula of the heavy-metal-free photosensitizer Cy-BF is shown in the specification, the heavy-metal-free photosensitizer Cy-BF has high phototoxicity, low dark toxicity and enhanced tumor targeting ability, ROS can be generated through NIR laser activation, and mitochondrial damage and immunogen cell death of tumor cells are induced. The Cy-BF can be used for preparing medicines for treating cancers, and the Cy-BF is combined with lithium carbonate to enhance the effect of photoimmunotherapy. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of cancer treatment drugs, and specifically relates to a heavy metal-free photosensitizer and a synthesis method thereof, and application thereof in the preparation of drugs for treating cancer. Background Art

[0002] Cancer is a common and fatal disease worldwide, posing a major challenge to public health due to its high morbidity and mortality. Phototherapy, as a non-invasive treatment method, has shown great potential in the field of tumor treatment. This method mainly includes photothermal therapy (PTT) and photodynamic therapy (PDT). In these therapies, photosensitizers (PSs) generate heat or reactive oxygen species (ROS) under the activation of specific laser wavelengths, effectively destroying tumor cells and tissues. However, the limitations of the tumor microenvironment often affect the efficacy of photosensitizers. In particular, lactic acid (LA) produced by metabolic reprogramming of tumor cells can inhibit T cell function and activation, thereby limiting the effectiveness of photoimmunotherapy (PIT). Summary of the invention

[0003] The purpose of the present invention is to overcome the defects and shortcomings of the prior art, provide a novel near-infrared light (NIR) activated heavy metal-free photosensitizer, provide a synthesis method of the heavy metal-free photosensitizer, and provide the use of the heavy metal-free photosensitizer in the preparation of drugs for treating cancer.

[0004] The purpose of the present invention is achieved through the following technical solutions:

[0005] A novel near-infrared light-activated heavy metal-free photosensitizer, named Cy-BF in the present invention, has the following structural formula:

[0006]

[0007] The synthesis method of the heavy metal-free photosensitizer Cy-BF, whose synthesis reaction formula is shown below, specifically comprises the following steps:

[0008]

[0009] (1) Synthesis of 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane:

[0010] Under the protection of inert gas, 2,4-pentanedione and boron trifluoride ether react in an organic solvent to obtain 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane. The organic solvent preferably includes dichloromethane, diethyl ether, ethylene glycol, etc., and the reaction temperature is preferably 10-40°C.

[0011] (2) Synthesis of 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt:

[0012] 1,1,2-trimethyl-1H-benzo[e]indole and iodomethane react in an organic solvent to obtain 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt. The organic solvent preferably includes acetonitrile, dichloromethane, ether, ethylene glycol, etc., and the reaction temperature is preferably 40-60°C.

[0013] (3) Synthesis of 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde:

[0014] POCl3 and 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt are reacted in an organic solvent, the resulting reaction solution is added to an ice-water mixture, and sodium hydroxide solution is added to adjust the pH to about 10-11 to obtain 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde. The organic solvent preferably includes dimethylformamide, ether, ethylene glycol, toluene, etc., and the reaction temperature is preferably 30-40°C.

[0015] (4) Synthesis of heavy metal-free photosensitizer Cy-BF:

[0016] 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane, 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde, and N,N'-dicyclohexylcarbodiimide (DCC) are refluxed in an organic solvent to obtain a heavy metal-free photosensitizer Cy-BF. The organic solvent is preferably a mixed solution of toluene, piperidine and acetonitrile, and the volume ratio of toluene, piperidine and acetonitrile is preferably 20-30:1-5:1-5.

[0017] Step (1) is preferably: under argon protection, 2,4-pentanedione and boron trifluoride ether are added to dichloromethane, and the reaction is carried out at room temperature. After the reaction is completed, 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane is obtained by extraction, drying and rotary evaporation.

[0018] Step (2) is preferably: adding 1,1,2-trimethyl-1H-benzo[e]indole and iodomethane to acetonitrile, reacting at 50°C, and after the reaction is completed, filtering, washing and recrystallizing to obtain 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt.

[0019] Step (3) is preferably: POCl3 and 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt are added to dimethylformamide at 0-5°C, the two solutions are mixed and reacted at 35°C, after the reaction is completed, the reaction solution is added to an ice-water mixture, and then sodium hydroxide solution is added to adjust the pH to about 10-11, and 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde is obtained by extraction, drying and rotary evaporation.

[0020] Step (4) is preferably: dissolving 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane, 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde, and N,N'-dicyclohexylcarbodiimide in a mixture of toluene, piperidine, and acetonitrile, and refluxing under nitrogen protection, and after the reaction is completed, purifying by column chromatography to obtain a heavy metal-free photosensitizer Cy-BF. The volume ratio of toluene to piperidine in the mixture is 25:1:1.

[0021] A bionic liposome encapsulating the heavy metal-free photosensitizer Cy-BF, wherein the bionic liposome is preferably a hybrid liposome obtained by mixing liposomes with platelet exosome vesicles.

[0022] The method for preparing the bionic liposomes encapsulating the above-mentioned heavy metal-free photosensitizer preferably includes the following steps: mixing the above-mentioned heavy metal-free photosensitizer Cy-BF, DSPE-PEG2000 and chloroform, obtaining Cy-BF-encapsulated liposomes (CL) by ultrasound and evaporation, and then mixing the liposomes with platelet exosome vesicles (PEV) to obtain Cy-BF-encapsulated bionic liposomes (CHL) by co-extrusion.

[0023] Application of the above-mentioned heavy metal-free photosensitizer or bionic liposome in the preparation of drugs for treating cancer.

[0024] Application of the heavy metal-free photosensitizer or bionic liposome and lithium carbonate in the preparation of drugs for treating cancer.

[0025] A drug for treating cancer, comprising the above-mentioned heavy metal-free photosensitizer or bionic liposome, and further comprising lithium carbonate.

[0026] Compared with the prior art, the present invention has the following advantages and effects:

[0027] (1) The present invention develops a novel heavy metal-free photosensitizer Cy-BF, which has high phototoxicity, low dark toxicity and enhanced tumor targeting ability.

[0028] (2) Cy-BF of the present invention can generate ROS through NIR laser activation, inducing mitochondrial damage and immunogenic cell death of tumor cells.

[0029] (3) The present invention uses lithium carbonate (LC) in combination to reuse lactic acid as an energy source for CD8+ T cells, thereby enhancing the effect of Cy-BF-mediated photoimmunotherapy.

[0030] (4) The present invention combines Cy-BF with lithium carbonate, which is not affected by exogenous lactic acid and provides a new strategy for reversing the lactic acid metabolism of tumor cells and T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 : Characterization of heavy metal-free photosensitizer Cy-BF and Cy-BF-loaded biomimetic liposomes (CHL).

[0032] Figure 2 :Verification of the photothermal effect and free radical generation effect of CHL.

[0033] Figure 3 : In vitro antitumor activity of CHL.

[0034] Figure 4 : In vitro immune response induction effect of CHL.

[0035] Figure 5 : Verification of the targeting effect and in vivo anti-tumor effect of CHL.

[0036] Figure 6 :Verification of the effect of CHL in activating immune response. DETAILED DESCRIPTION

[0037] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0038] Example 1: Synthesis and characterization of heavy metal-free photosensitizer Cy-BF

[0039] (1) Synthesis of 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane: Under argon protection, 2,4-pentanedione (10 g, 0.1 mol) and boron trifluoride ether (21.2 g, 0.15 mol) were added to 50 mL of dry dichloromethane. The reaction was carried out at room temperature for 8 hours. After the reaction was completed, 100 mL of saturated sodium bicarbonate aqueous solution was added and extracted with dichloromethane (100 mL) 2-3 times. The organic phase was dried over anhydrous sodium sulfate, and the organic solvent was removed by rotary evaporation to obtain a white solid 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane (13.9 g, yield 94%).

[0040] (2) Synthesis of 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt: 1,1,2-trimethyl-1H-benzo[e]indole (2.0 g, 9.6 mmol) and iodomethane (8.9 g, 63.0 mmol) were added to a reaction kettle, 10 mL of acetonitrile solvent was added, and the reaction was carried out at 50°C. After the reaction was allowed to proceed overnight, ethyl acetate (100 mL) was slowly added, and the precipitate was collected by filtration and washed with ethyl acetate. The crude product was collected and refluxed with ethyl acetate (100 mL) for 15 minutes. After cooling, the product was collected by filtration to obtain a gray solid 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt (3.7 g, yield 97%).

[0041] (3) Synthesis of 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde: Phosphorus oxychloride (5.25 g, 34 mmol) was added dropwise to dimethylformamide (10 mL) under ice bath conditions (5°C), maintaining the temperature at 5°C, and 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt (9.83 g, 28 mmol) was added dropwise to dimethylformamide (2.5 mL). After the addition was completed, the two solutions were mixed evenly and then maintained at 35°C for 40 minutes. The reaction solution was poured into an ice-water mixture (100 g) under stirring, and a 20% sodium hydroxide solution was added to adjust the pH to about 10-11, extracted three times with ether, dried over anhydrous sodium sulfate, and then dried with ether to obtain 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde (6.55 g, yield 92%).

[0042] (4) Synthesis of Cy-BF: 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane (60 mg, 0.406 mmol) was dissolved with 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde (0.501 g, 2.02 mmol) and N,N'-dicyclohexylcarbodiimide (DCC, 0.1 g, 0.49 mmol) in 50 mL of toluene, 2 mL of piperidine and 2 mL of acetonitrile. A water separator was installed and the mixture was refluxed for 7 hours under nitrogen protection. The color of the solution gradually turned red and eventually blue. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography (silica gel, n-hexane: dichloromethane = 1:5) to obtain blue powder Cy-BF (26.1 mg, yield 10.5%). 1H NMR (300MHz, DMSO-d6) δ8.33 (t, J=13.3Hz, 2H), 8.09 (d, J=8.6Hz, 2H), 7.90–7.80 (m, 4H), 7.56 (t, J=7.5Hz, 2H), 7 .38 (d, J=7.7Hz, 2H), 7.17 (d, J=8.8Hz, 2H), 5.85 (d, J=13.8Hz, 2H), 5.67–5.58 (m, 3H), 3.40 (s, 6H), 1.98 (s, 12H).

[0043] Characterization of Cy-BF: 1H NMR spectra were measured using a Bruker AVANCE 300M fully digital superconducting NMR spectrometer, using DMSO-d6 as solvent and tetramethylsilane (TMS; δ = 0 ppm) as an internal reference. TM The mass spectrometer system was operated in matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mode to obtain high-resolution mass spectra (HRMS). UV-visible spectra and fluorescence spectra were measured using a HORIBA Duetta spectrometer. The synthesis and characterization of Cy-BF were described above. Figure 1 The chemical structure diagram in A corresponds to that in FIG. 1 , which proves that the present invention successfully obtains the heavy metal-free photosensitizer Cy-BF.

[0044] Example 2: Preparation and characterization of biomimetic liposomes (CHL) loaded with Cy-BF

[0045] 1. Preparation of Cy-BF-loaded liposomes (CL):

[0046] (1) 20 mg of Cy-BF, 10 mL of DSPE-PEG2000 and 10 mL of chloroform were mixed and treated with ultrasound (12 W output) to obtain a clear solution.

[0047] (2) The above solution was quickly injected into 9 mL of water and subjected to vigorous ultrasonic treatment in water for 2 min to ensure that the liposomes were evenly dispersed and loaded with Cy-BF.

[0048] (3) The resulting mixture was placed in a fume hood and stirred for 12 hours to remove chloroform.

[0049] (4) Unencapsulated Cy-BF was removed by ultrafiltration (molecular weight cutoff 100 kDa) at 3000 g for 30 minutes.

[0050] (5) The obtained Cy-BF-loaded liposome (CL) suspension can be used for further characterization.

[0051] 2. Preparation of Cy-BF-loaded biomimetic liposomes (CHL):

[0052] (1) The Cy-BF-loaded liposomes (CL) prepared above were mixed with 1 mg of platelet exosomes (PEV).

[0053] (2) The mixture was repeatedly coextruded through a membrane with a pore size of 200 nm to form mixed liposomes (CHL).

[0054] (3) The obtained CHL particles were centrifuged and washed several times with PBS to remove unencapsulated PEVs.

[0055] (4) The final CHL particles obtained can be used for further characterization and analysis.

[0056] 3. Characterization of CL and CHL:

[0057] Figure 1 A is the molecular structure diagram of Cy-BF, showing its core structure and chemical composition, confirming that it is the target molecule for synthesis.

[0058] Figure 1 B is the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) distribution diagram of Cy-BF. HOMO covers the entire molecular framework, while LUMO is mainly concentrated in the central receptor region, showing the molecular orbital distribution and electronic transition characteristics.

[0059] Figure 1 C is a schematic diagram of the energy level transition of Cy-BF, showing the energy gap between the singlet state (S1) and the triplet state (T1, T2), emphasizing the ISC (intersystem transition) pathway from S1 to T2, and explaining its mechanism of enhancing the generation of reactive oxygen species (ROS).

[0060] Figure 1D is a comparison of the fluorescence spectra of Cy-BF in chloroform and CHL. The results show that the fluorescence of Cy-BF in CHL is significantly quenched, indicating that it effectively converts light energy into heat energy after aggregation, while enhancing the ISC ability.

[0061] Figure 1 E is a comparison of the absorption spectra of Cy-BF and CHL. The absorption peak of CHL is lower than that of Cy-BF, but the spectrum is wider and has double peaks, which may be due to the M-type aggregation of Cy-BF in CHL. There is no red shift or blue shift, indicating that there is no H-type or J-type aggregation.

[0062] Figure 1 F is a transmission electron microscopy (TEM) image of Cy-BF-entrapped liposomes (CL), showing the particle size and morphological characteristics of CL.

[0063] Figure 1 G is a transmission electron microscopy image of the biomimetic liposomes (CHL) encapsulating Cy-BF, showing the exosome membrane structure wrapped on the surface of CHL and the particle size difference of about 10 nm compared with CL.

[0064] Figure 1 H is a comparison of the particle size and zeta potential of CL, CHL, and PEV. The zeta potential of CHL is close to that of PEV, confirming that the exosome membrane has been successfully wrapped on the surface of CL.

[0065] Figure 1 I is the dimensional stability test result of CHL. CHL was dissolved in PBS solution and cell culture medium containing 10% fetal bovine serum, and its average particle size was counted by transmission electron microscopy within one week. The results show the ability of CHL to maintain good dimensional stability under different times and conditions.

[0066] Figure 1 J is the photostability test result of CHL, which shows that CHL exhibits good photostability under light conditions.

[0067] Figure 1 K is the detection of protein expression in PEV and CHL using Western blot, indicating the detection results of platelet membrane proteins (CD41 and P-selectin) expressed on the surface of CHL. The results confirmed that CHL successfully retained the functional membrane proteins of exosomes, among which P-selectin helped to target the CD44 receptor on the surface of tumor cells.

[0068] Example 3: Evaluation of CHL-mediated anticancer ability in vitro

[0069] ROS generation test: Electron spin resonance (ESR) spectroscopy was used to detect reactive oxygen species (ROS) generated by Cy-BF and CHL. In the experiment, SOSG, HPF, and DCF were used to estimate the generation of 1O2, ·OH, and total ROS under NIR laser irradiation (760nm, 0.5W / cm^2, 2 minutes). The concentration of Cy-BF was 0.02mg / mL.

[0070] MB degradation experiment detects hydroxyl radicals generated in CHL. The specific steps are as follows: CHL (Cy-BF final concentration 20 μg / mL) and MB (final concentration 5 μg / mL) are mixed in PBS buffer (pH 7.4). The mixed system is exposed to NIR laser irradiation (0.5 W / cm^2, 21 minutes). The absorbance of MB is measured every 3 minutes using a UV-visible spectrophotometer. Electron paramagnetic resonance (EPR) experiments are performed using a Bruker EMXplus EPR spectrometer.

[0071] Figure 2 A shows the temperature rise of different CHL concentrations. Figure 2 B shows the heating and cooling cycle of CHL with a concentration of 50 μg / mL. Figure 2 C is the cooling curve fitting of CHL. Figure 2 DF shows the generation of singlet oxygen (^1O_2), hydroxyl radical (·OH), and superoxide anion (·O_2^-) by Cy-BF and CHL respectively by electron spin resonance (ESR) spectroscopy. These results show that both Cy-BF and CHL can generate three major ROS: singlet oxygen (^1O_2), hydroxyl radical (·OH), and superoxide anion (·O_2^-). At the same concentration, the amount of ROS generated by CHL is significantly higher than that of Cy-BF, indicating that CHL has a stronger ROS generation ability. This result suggests that CHL can generate ROS more effectively in photodynamic therapy (PDT), which may lead to a stronger anti-tumor effect. Figure 2 GI demonstrated the use of different fluorescent dyes (SOSG, HPF, DCF) to detect the ROS generation of CHL under different illumination times. The results showed that CHL can continuously generate ROS under 760nm laser irradiation, and the amount of ROS generated increases with the increase of illumination time. This indicates that CHL can continuously generate ROS under longer laser irradiation, thereby improving the therapeutic effect.

[0072] Cell impact test: 4T1 cells (1.5×10^5 per well) were seeded in 12-well plates overnight. The cells were treated with the following four groups: (1) PBS+NIR (0.5W / cm^2, 10 minutes); (2) CHL; (3) CL+NIR; (4) CHL+NIR, Cy-BF concentration was 100μg / mL. After 24 hours of treatment, DCFH-DA, mitochondrial membrane potential detection kit (containing JC-1), and Annexin V-FITC / PI apoptosis kit were used according to the instructions to detect the generation of ROS, the effect on mitochondrial membrane potential, and the effect on cell apoptosis. A commercial kit (Beyotime) was used to measure the release of intracellular HMGB1, GSH, and LA content. Immunofluorescence staining of HMGB1 showed that the release of HMGB1 in the nucleus and the expression of CRT on the cell membrane increased in cells treated with CHL+NIR, indicating that the oxidative stress induced by CHL+NIR can lead to the immunogenic death of tumor cells. Figure 3 A shows the effect of CHL on ROS production in 4T1 cells. Compared with the PBS+NIR, CHL, and CL+NIR groups, the CHL+NIR treatment group resulted in a significant increase in ROS levels and changes in the mitochondrial membrane potential of the cells ( Figure 3 B) These changes indicate that CHL+NIR treatment caused mitochondrial damage in 4T1 cells. Figure 4 A shows the apoptosis of 4T1 cells after CHL+NIR treatment. Figure 4 B-4C demonstrates immunogenic cell death (ICD). Figure 4 B. Immunofluorescence staining shows the expression of HMGB1 (high mobility group protein B1) and calreticulin (CRT) as ICD markers. In cells treated with CHL and exposed to 760 nm laser irradiation, the nuclear green fluorescence was significantly reduced (indicating the release of HMGB1), which was consistent with the Figure 4 The statistical analysis data in C are consistent. The green fluorescence on the cell membrane increased significantly (indicating increased CRT expression). This phenomenon indicates that CHL+NIR-induced oxidative stress can lead to immunogenic cell death (ICD) of tumor cells, further enhancing the potential of CHL in tumor therapy.

[0073] ROS generation, ICD induction, and immunomodulatory gene expression analysis: Transcriptome sequencing results showed that CHL+NIR treatment significantly enriched signaling pathways related to ICD, ROS, and immunomodulation, further confirming the potential of CHL in immune sensitization therapy. Related differential gene expression and pathway analysis results can be found in Figure 4 D-4G. Figure 4D shows the differentially expressed genes in different treatment groups. As can be seen in the figure, the gene expression of 4T1 cells treated with CHL+NIR changed significantly compared with the PBS+NIR group. The differentially expressed genes showed significant enrichment in pathways related to immunogenic cell death (ICD), oxidative stress, and immune regulation. This further proves the potential of CHL in immunosensitization therapy. Figure 4 E The Venn diagram shows the number of genes transcribed in different treatment groups. By comparing the CHL+NIR group with other control groups, the figure shows the number of unique differentially expressed genes in each treatment group and their intersection, helping to identify genes that are differentially expressed under specific treatment conditions. Figure 4 The volcano plot in F shows the differentially expressed genes in different treatment groups. The X-axis represents the fold change in gene expression, and the Y-axis represents the significance of the gene expression change (p-value). As can be seen in the figure, there are multiple genes that are significantly up-regulated or down-regulated in the CHL+NIR treatment group, especially in the expression of genes related to oxidative stress, immune response, and cell death. Figure 4 G is the result of KEGG pathway enrichment analysis, showing the biological pathways enriched with differentially expressed genes. The analysis showed that after CHL+NIR treatment, the differentially expressed genes were significantly enriched in signaling pathways related to immunogenic cell death (ICD), ROS generation, and immune regulation, further confirming the potential of CHL in enhancing immune response and oxidative stress. The above results show that CHL+NIR treatment significantly changed the expression of multiple genes related to apoptosis and immunity, especially in genes related to immunogenic cell death and oxidative stress, further demonstrating the role of CHL in inducing immunogenic cell death (ICD) and enhancing immune response.

[0074] Example 4: Evaluation of the anticancer effect of CHL combined with LC in vivo

[0075] In vivo anti-tumor ability test: In 4T1 tumor model mice, CHL and LC were injected through the tail vein, and 760nm NIR laser irradiation was performed 12 hours later. The experimental groups included PBS+NIR, LC, CHL, CHL+NIR, CL+NIR+LC, and CHL+NIR+LC. The specific steps are as follows:

[0076] BALB / c mice were randomly divided into 6 groups (5 mice in each group): (1) PBS+NIR (0.5W / cm^2, 10 minutes); (2) LC; (3) CHL; (4) CHL+NIR; (5) CL+NIR+LC; (6) CHL+NIR+LC. Figure 5As shown in C, CHL or CL was administered via the tail vein, with a Cy-BF dose of 10 mg / kg, and phototherapy was performed on day 0 (12 hours after administration). LC was orally administered on days 2, 4, 6, 8, 10, and 12, each dose of 75 mg / kg. Mouse body weight and tumor volume were monitored every 3 days. Tumor length and width were measured using a caliper, and tumor volume was calculated according to the formula: tumor volume = tumor length × tumor width ^ 2 / 2. After 18 days of treatment, mice were killed, and five major organs (heart, liver, spleen, lung, and kidney) were harvested, washed with PBS, and fixed with formaldehyde for histological analysis. Tumor tissues were weighed and fixed with 4% neutral buffered formaldehyde, and routinely processed into paraffin sections with a section thickness of 4 μm. Stained with HE, CRT, TUNEL, Ki-67, and DCFH-DA, and finally examined using a fluorescence microscope (IX81, Olympus, Japan). The lactic acid content in tumor tissues was measured using a kit (Beyotime Biotech. Inc.). Tumor-infiltrating CD8+T cells were isolated using a CD8+T cell isolation kit (Thermo Fisher), and their mitochondria were detected by WB.

[0077] Tumor targeting ability assessment: The distribution of CHL in vivo was detected by fluorescence imaging system (IVIS), and the results showed that CHL has stronger tumor targeting ability than CL. For related fluorescence imaging results, see Figure 5 A and 5B. Figure 5 A and 5B demonstrate the in vivo tumor targeting ability of CL and CHL in 4T1 tumor-bearing mice. Figure 5 A is the external fluorescence imaging of the tumor and major organs of 4T1 tumor-bearing mice after CL and CHL injection at different time points. The results show that CHL has a stronger tumor targeting ability than CL, and CHL can accumulate more effectively at the tumor site, showing a more significant tumor localization effect. Figure 5 B shows the corresponding fluorescence intensity data, which further proves that the accumulation of CHL in tumors is significantly higher than that of CL. These results show that the targeting and drug delivery efficiency of CHL are superior to those of CL, which helps to improve its application potential in tumor treatment.

[0078] Tumor growth and weight measurement: Tumor volume was measured every 3 days during the treatment period and calculated by the formula. Figure 5 DF shows the survival curves, tumor volume growth, and tumor weight changes of mice in different treatment groups. Figure 5 D) shows that the combination of CHL-mediated phototherapy and LC treatment significantly improved the survival rate of mice, with the best survival effect compared to other treatment groups. Figure 5E), the tumor growth of the CHL+NIR group and the CHL+NIR+LC group was significantly inhibited, especially the CHL+NIR+LC group, whose tumor volume increased the slowest, indicating that the combined treatment has a significant advantage in inhibiting tumor growth. In addition, the measurement of tumor weight ( Figure 5 F) This was further verified by the fact that the tumor weight of mice in the CHL+NIR+LC group was significantly lower than that in the other groups, showing the powerful anti-tumor effect of this combined treatment regimen. These results indicate that CHL-mediated phototherapy combined with LC has a significant anti-tumor effect and provides a new strategy for cancer treatment.

[0079] Histological analysis and lactate content determination: Necrosis, ICD, apoptosis, and cell proliferation of tumor tissues were evaluated by HE staining and immunofluorescence staining. In addition, the lactate content in tumor tissues was determined, and the results showed that the lactate content in the CHL+NIR+LC group was significantly reduced, indicating that the combination therapy effectively inhibited the lactate production of tumor cells. Figure 5 G and Figure 5 H. Figure 5 G shows the relative content of lactic acid in tumor tissues of mice in different treatment groups. The results showed that the tumor lactic acid content in the CHL+NIR+LC combined treatment group was significantly lower than that in other treatment groups, indicating that the combined treatment significantly inhibited the lactic acid production of tumor cells and helped improve the tumor microenvironment. Figure 5 H shows the histological examination results of tumor sections, including HE staining, CRT immunofluorescence staining, and TUNEL staining. The results showed that the tumor sections of the CHL+NIR+LC treatment group showed extensive tumor necrosis, immunogenic cell death (ICD), and apoptosis. At the same time, Ki-67 staining showed a significant decrease in cell proliferation, indicating that this treatment regimen can effectively inhibit tumor growth. CD8+T cells were then extracted as Figure 5 As shown in I, the results of mitochondrial WB detection are as follows Figure 5 J, demonstrated the inhibition of mitochondrial activity by this treatment regimen.

[0080] Immune response evaluation: The number of CD8+T cells and the expression of IFN-γ and GZMB in tumor tissues were analyzed by flow cytometry. The results showed that the CHL+NIR+LC group effectively activated the T cell-mediated immune response. At the same time, the number of regulatory T cells (Tregs) in tumor tissues was significantly reduced. The results of immune response are shown in Figure 6 A-6F. Figure 6 A shows the changes in DC cell maturation, CD8+T cells, and CD8+T cell IFN-γ and GZMB expression after LC treatment. Figure 6 B shows the statistical results of DC mature cells. Figure 6C shows the statistical results of CD8+T cells. Figure 6 D shows the statistical results of IFN-γ positivity in CD8+ T cells. Figure 6 E shows the statistical results of GZMB positivity in CD8+ T cells. Figure 6 F shows the statistical results of Treg cells. IFN-γ, IL-6 and TNF-α in the blood were measured by ELISA. Figure 6 G, further showing the potential mechanism of LC in immunotherapy and the enhancement of immune cell function, confirming that LC can synergize with CHL+NIR treatment to enhance the anti-tumor response of the immune system.

[0081] The above embodiments are only used to help illustrate the present invention. The implementation methods of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A heavy metal-free photosensitizer, characterized in that: Cy-BF has the following structural formula:

2. The method for synthesizing a heavy metal-free photosensitizer according to claim 1, characterized in that: The following steps are involved: (1) Under the protection of an inert gas, 2,4-pentanedione and boron trifluoride ether react in an organic solvent to obtain 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane; (2) 1,1,2-trimethyl-1H-benzo[e]indole and iodomethane react in an organic solvent to obtain 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt; (3) POCl3 and 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt are reacted in an organic solvent, the resulting reaction solution is added to an ice-water mixture, and sodium hydroxide solution is added to adjust the pH to about 10-11 to obtain 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde; (4) 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane, 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde and N,N'-dicyclohexylcarbodiimide are refluxed in an organic solvent to obtain the heavy metal-free photosensitizer.

3. The method for synthesizing a heavy metal-free photosensitizer according to claim 2, characterized in that: In step (1), the organic solvent includes dichloromethane, ether, and ethylene glycol, and the reaction temperature is 10-40°C; And / or in step (2), the organic solvent includes acetonitrile, dichloromethane, ether, ethylene glycol, and the reaction temperature is 40-60°C; And / or in step (3), the organic solvent includes dimethylformamide, ether, ethylene glycol, toluene, and the reaction temperature is 30-40°C; And / or in step (4), the organic solvent is a mixture of toluene, piperidine and acetonitrile.

4. The method for synthesizing a heavy metal-free photosensitizer according to claim 2, characterized in that: The following steps are involved: (1) under argon protection, 2,4-pentanedione and boron trifluoride ether are added to dichloromethane, and the reaction is carried out at room temperature. After the reaction is completed, 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane is obtained by extraction, drying and rotary evaporation; (2) adding 1,1,2-trimethyl-1H-benzo[e]indole and iodomethane to acetonitrile, reacting at 50° C., and filtering, washing and recrystallizing to obtain 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt after the reaction is completed; (3) adding POCl3 and 1,1,2,3-tetramethyl-1H-benzo[e]indole-3-iodide salt to dimethylformamide at 0-5°C, mixing the two solutions and reacting them at 35°C, adding the reaction solution to an ice-water mixture after the reaction is completed, and then adding sodium hydroxide solution to adjust the pH to about 10-11, extracting, drying and rotary evaporating to obtain 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde; (4) 2,2-difluoro-4,6-dimethyl-1,3,2-dioxaborane, 2-(1,1,3-trimethyl-1,3-dihydro-2H-benzo[e]indole-2-ethyl)acetaldehyde, and N,N'-dicyclohexylcarbodiimide are dissolved in a mixture of toluene, piperidine, and acetonitrile, and refluxed under nitrogen protection. After the reaction is completed, the heavy metal-free photosensitizer Cy-BF is obtained by purification by column chromatography.

5. A biomimetic liposome encapsulating the heavy metal-free photosensitizer according to claim 1, characterized in that: The bionic liposome is a hybrid liposome obtained by mixing liposomes and platelet exosomes.

6. The method for preparing the biomimetic liposome according to claim 5, characterized in that: The method comprises the following steps: mixing the heavy metal-free photosensitizer according to claim 1, DSPE-PEG2000 and chloroform, obtaining liposomes encapsulating the heavy metal-free photosensitizer by ultrasound and evaporation, mixing the liposomes with platelet exosomes, and obtaining bionic liposomes encapsulating the heavy metal-free photosensitizer by co-extrusion.

7. Use of the heavy metal-free photosensitizer according to claim 1 or the biomimetic liposome according to claim 5 in the preparation of a drug for treating cancer.

8. Use of the heavy metal-free photosensitizer according to claim 1 or the biomimetic liposome according to claim 5 and lithium carbonate in the preparation of a drug for treating cancer.

9. A drug for treating cancer, characterized in that: The invention comprises the heavy metal-free photosensitizer according to claim 1 or the biomimetic liposome according to claim 5.

10. The drug for treating cancer according to claim 9, characterized in that: Also contains lithium carbonate.

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