Photocatalytic co-release spray hydrogel used after tumor operation

Abstract

The invention relates to the field of biomedicine, and discloses photocatalytic co-release spray hydrogel used after tumor surgery. Firstly, the C3N4 / Au nano photocatalyst is disclosed and is obtained by carrying out photocatalytic reduction reaction on g-C3N4 and an Au < 3 + > solution; secondly, the invention discloses a C3N4 / Au / SA hydrogel which is prepared by the following steps: mixing the C3N4 / Au with a CaCl2 aqueous solution, and uniformly dispersing to obtain a dispersion liquid A; dissolving sodium alginate SA in deionized water, and heating to obtain a solution B; mixing the dispersion liquid A and the solution B through spraying to obtain C3N4 / Au / SA hydrogel; the invention further discloses an application of the C3N4 / Au / SA hydrogel in drug combination with doxorubicin DOX (doxorubicin). According to the application, the convenience and controllability of treating tumors by using CO gas after operation are remarkably improved, and the aim of inhibiting postoperative recurrence and metastasis of malignant tumors by combining gas treatment and chemotherapy is fulfilled.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and more specifically, to a photocatalytic co-release spray hydrogel for post-tumor surgery. Background Art

[0002] Breast cancer (BC) is one of the most common malignant tumors threatening the health of women globally. The conventional treatment methods for breast cancer mainly include surgery, radiotherapy, and chemotherapy. Surgical treatment only removes visible tumor tissues and cannot eliminate the tiny tumors infiltrated into tissues. More than 80% of patients receive this treatment. Chemotherapy is often required after surgery, including the systemic use of chemical drugs to kill tumor cells, thereby inhibiting the recurrence and metastasis of tumors to a certain extent. However, chemotherapy drugs cannot accurately identify tumor cells and cause significant damage to normal cells while killing cancer cells. In addition, chemotherapy has problems such as strong side effects, poor targeting, and multi-drug resistance, severely limiting its therapeutic effect. Therefore, there is an urgent need to develop new, efficient, and low-toxic post-operative treatment strategies to improve the prognosis of breast cancer patients and enhance their quality of life.

[0003] In recent years, with the revelation of the physiological regulatory functions of gas molecules in cancer development, gas therapy has attracted increasing attention from researchers. Due to its low in vivo toxicity and high efficiency, it is considered a safe and effective "green" cancer treatment method. Endogenous gas molecules, such as nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S), play crucial roles in the physiological and pathological processes of the human body. They are involved in various biological processes, including cell signaling, regulation of vascular tone, and regulation of immune responses. Compared with traditional drug therapy, gas molecules have the advantages of small molecular weight, fast diffusion rate, and easy penetration of biological barriers, enabling them to quickly reach the target and achieve effective biological effects. Notably, CO can interfere with the antioxidant defense system of cancer cells, disrupt the redox balance within tumor cells, and increase their sensitivity to oxidative stress damage induced by chemotherapeutic drugs. Researchers have developed various carbon monoxide-releasing molecules (CORMs) to achieve slow or targeted carbon monoxide release in vivo. However, most CORMs have a short half-life and are rapidly metabolized and decomposed, making it difficult to maintain a stable concentration of CO and fully exert its therapeutic effect. Due to the enhanced greenhouse effect, the conversion and utilization of CO2 are becoming increasingly popular, and CO is one of the reduction products. Many studies are exploring highly efficient CO2 photocatalytic reduction materials, including metal nanoparticles, metal-organic frameworks (MOFs), and two-dimensional materials. Among them, g-C3N4 is a two-dimensional photocatalyst with good biocompatibility, a large specific surface area, and an appropriate bandgap structure. This enables it to respond in the visible light range, promote the separation and transfer of photo-generated charges, and be widely used in fields such as photocatalytic water splitting. Modifying g-C3N4 can change the selectivity and yield of its photocatalytic products. For example, loading gold or silver nanoparticles on the surface of g-C3N4 can expand its light absorption range, act as electron traps, effectively promote the separation of photo-generated charges, and inhibit charge recombination, thus significantly improving its photocatalytic activity for CO production. In addition, compared with the atmospheric environment (pCO2≈0.2 mmHg), tumor cells have a higher HCO3 - concentration and a lower pH value, resulting in a higher concentration of CO2 in tumor tissues (pCO2≈80 mmHg). Therefore, using photocatalysts to continuously and controllably generate CO in vivo can improve the effectiveness of chemotherapy while enhancing its safety.

[0004] Systemic drug delivery is not conducive to the enrichment of photocatalysts in the tumor area. For postoperative treatment of tumors, in situ drug delivery is increasingly showing its unique advantages. Compared with traditional methods, in situ drug delivery increases the concentration of CO at the tumor site while avoiding its widespread distribution throughout the body. This significantly reduces unnecessary damage to normal tissues and reduces systemic toxic reactions. As a polymer material with a three-dimensional network structure, hydrogel has good biocompatibility and can simulate the microenvironment of human tissue, providing a suitable environment for cell adhesion and proliferation. Its unique physicochemical properties enable the effective loading of various drugs, bioactive molecules and cells, and achieve controlled and sustained drug release. This prolongs the residence time of drugs at the tumor site, thereby improving the therapeutic effect. Sodium alginate (SA) is a natural polysaccharide extracted from brown algae such as kelp or sargassum, which has good biodegradability and biocompatibility. It gradually degrades into non-toxic small molecules in the body and then metabolizes and excretes. SA undergoes a rapid and mild cross-linking reaction with calcium ions to form a stable network hydrogel structure. This characteristic of in situ hydrogel formation is conducive to in situ drug delivery at the surgical site. The photocatalyst can be loaded into a calcium chloride solution and quickly mixed with the SA solution at the tumor resection site by spraying or other methods. The hydrogel formed by cross-linking SA with calcium ions can adhere tightly to the wound surface, acting as a physical barrier to effectively prevent the invasion of external bacteria and reduce the risk of postoperative infection. In addition, the loaded photocatalyst can continuously produce CO under light, penetrate tumor cells, and increase their sensitivity to chemotherapy. This sprayable hydrogel drug delivery system is simple and convenient to operate and can be prepared and applied immediately at the surgical site. It is a powerful tool for adjuvant therapy after tumor surgery.

[0005] Therefore, constructing a SA sprayable hydrogel composite system containing photocatalyst is of great significance for enhancing postoperative chemotherapy for breast cancer and preventing tumor recurrence. Summary of the invention

[0006] In order to enhance the effect of chemotherapy after breast cancer surgery, the present invention constructs a SA sprayable hydrogel composite system containing a photocatalyst to release carbon monoxide in situ at the tumor resection site. Carbon monoxide gas can enhance the effect of chemotherapy and remove residual tumor cells, thereby preventing tumor recurrence and metastasis.

[0007] First, modify C3N4 nanoparticles with gold nanoparticles to prepare a photocatalyst (C3N4 / Au), and then mix it with a calcium chloride solution. Spray with SA solution to form an in-situ hydrogel C3N4 / Au / SA. C3N4 / Au / SA adheres to the surface of the postoperative tumor wound. Under blue light irradiation, C3N4 / Au photocatalytically converts CO2 into CO, which diffuses into tumor cells. Subsequently, inject folic acid (FA)-modified DOX micelles (FA@DM) to deliver DOX into breast cancer cells overexpressing the FA receptor, synergistically enhancing the chemotherapy effect with CO (Scheme 1). This strategy significantly improves the convenience and controllability of using CO gas to treat tumors after surgery, achieving the goal of combined gas therapy and chemotherapy to inhibit the recurrence and metastasis of malignant tumors after surgery.

[0008] To achieve the above-mentioned invention purpose, the present application adopts the following technical solutions:

[0009] In the first aspect, the present application provides a C3N4 / Au nano-photocatalyst, which is obtained by subjecting a g-C3N4 and Au 3+ solution to a photocatalytic reduction reaction to deposit gold nanoparticles on the surface of g-C3N4.

[0010] In the second aspect, the present application provides a C3N4 / Au / SA hydrogel, which is prepared by the following steps:

[0011] Mix the C3N4 / Au described in the first aspect with an aqueous CaCl2 solution and disperse evenly to obtain dispersion A;

[0012] Dissolve sodium alginate SA in deionized water and heat to obtain solution B;

[0013] Mix dispersion A and solution B by spraying to obtain a C3N4 / Au / SA hydrogel.

[0014] Furthermore, dispersion A and solution B are mixed in a volume ratio of 1:2.

[0015] In the third aspect, the present application provides the use of the C3N4 / Au / SA hydrogel described in the second aspect in the preparation of a drug for enhancing the sensitivity of tumor cells to chemotherapeutic drugs.

[0016] Furthermore, the chemotherapeutic drug includes doxorubicin DOX.

[0017] In the fourth aspect, the present application provides the use of the C3N4 / Au / SA hydrogel described in the second aspect in combination with doxorubicin DOX.

[0018] In summary, the present application has the following beneficial effects:

[0019] In the research, the design of the C3N4 / Au nanophotocatalyst is one of the key innovations, which significantly improves the efficiency of photocatalytic carbon monoxide (CO) production. Integrating this catalyst into the SA hydrogel enables precise and controllable drug delivery at the postoperative site. In addition, the combination of carbon monoxide (CO) therapy with folic acid (FA)-modified doxorubicin (DOX) micelles (FA@DM) achieves a synergistic effect between gas therapy and chemotherapy, further enhancing the therapeutic effect. Our research demonstrates the potential of this composite hydrogel system in providing a new method and safe strategy for efficient and precise postoperative tumor treatment. Our in vitro and in vivo experimental results highlight the enhanced antitumor effect of CO-sensitized chemotherapy, as well as the significant inhibition of tumor growth and the promotion of tumor cell apoptosis. We believe that our research provides valuable insights for the development of innovative strategies for postoperative breast cancer treatment. The unique method of combining gas therapy with chemotherapy through the spray hydrogel system has the potential to improve the prognosis and quality of life of breast cancer patients. Description of the Drawings

[0020] Figure 1 : Schematic diagram of CO-sensitized chemotherapy induced in situ in tumors by the C3N4 / Au nanophotocatalyst spray;

[0021] Figure 2 : Synthesis and characterization of C3N4 / Au. A) Synthesis route of C3N4 / Au. B) TEM and HRTEM images of g-C3N4 and C3N4 / Au. C) Elemental mapping images of C3N4 / Au. D) UV-visible absorption spectra of g-C3N4 and C3N4 / Au. E) XRD patterns of g-C3N4 and C3N4 / Au. F) FTIR spectra of g-C3N4 and C3N4 / Au;

[0022] Figure 3 : Performance and mechanism of C3N4 / Au photocatalytic CO generation. A) Synthesis route of the CO probe LysoFP-NO2 and the CO-responsive reaction. B) Variation of the fluorescence spectrum of C3N4 / Au detected by LysoFP-NO2 with illumination time. C) Variation of the photocatalytic CO and methane evolution concentrations of g-C3N4 and C3N4 / Au with illumination time. D) Schematic diagram of the photocatalytic reduction mechanism of C3N4 / Au. E) ESR spectrum of C3N4 / Au after incubation with O2. F) ESR spectra of ·OH after incubation with C3N4 / Au before and after illumination. G) ESR spectra of O2 before and after incubation with C3N4 / Au; 1 O2

[0023] Figure 4: In vitro generation of CO. A) Fluorescence images of CO generation before and after light irradiation after incubation of g-C3N4 and C3N4 / Au with L929 cells. B) Flow cytometry detection of LysoFP-NO2 in L929 cells cultured with g-C3N4 and C3N4 / Au. C) Flow cytometry examination of LysoFP-NO2 in L929 cells cultured with C3N4 / Au and irradiated with light for different times;

[0024] Figure 5 : Cytotoxicity of DOX and DOX + C3N4 / Au + Light against 4T1 cells. B) Imaging of ROS production in 4T1 cells treated with C3N4 / Au and C3N4 / Au / SA. C) Flow cytometry detection of ROS production in 4T1 cells irradiated with C3N4 / Au. D) Flow cytometry study of early and late apoptosis of 4T1 cells irradiated with C3N4 / Au. E) Imaging and F) Flow cytometry study of mitochondrial membrane potential of 4T1 cells irradiated with C3N4 / Au;

[0025] Figure 6 : Tumor targeting of FA@DM. A) Cellular uptake of DOX and FA@DM by 4T1 cells. B) Fluorescence quantification of DOX in 4T1 cells by flow cytometry. C) NIR-II imaging of DOX retention in tumors at different times after intravenous injection. D) Tumor fluorescence intensity at different times. E) NIR-II imaging of the heart, liver, spleen, lungs, kidneys, and tumors 24 hours after injection of DM and FA@DM. F) Quantitative fluorescence intensity of each tissue;

[0026] Figure 7 : A) Schematic diagram of in vivo experiments. B) Tumor volume and C) tumor weight of patients in different groups 21 days after treatment. D) Tumor photographs of patients in different groups 21 days after treatment. E) Changes in body weight of mice during the treatment process. F) Images of tumor tissues stained with H&E, Ki67 immunohistochemistry, CD31 immunofluorescence, and Tunel in different groups 21 days after treatment. Detailed implementation manners

[0027] The technical solutions and effects of this application will be further described in detail below in combination with examples and drawings. It can be understood that the specific examples described here are only used to explain the invention-creation, rather than limiting the invention-creation.

[0028] I. Experimental materials

[0029] Melamine, chloroauric acid trihydrate, 3-nitro-1,8-naphthalic anhydride, 4-(2-aminoethyl)morpholine, dimethyl sulfoxide, 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 2-(tert-butoxycarbonyl)-2-methyl-3,4-dihydro-2H-pyrrole-1-oxide (BMPO) and TPC were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0030] 2,2,5,5-Tetramethyl-2,5-dihydro-1H-pyrrole-3-carboxamide (TPC) was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0031] Hydrochloric acid was purchased from Zhejiang Sanying Chemical Reagent Co., Ltd.

[0032] Anhydrous methanol and anhydrous ethanol were purchased from Hangzhou Gaojing Fine Chemical Co., Ltd.

[0033] DSPE-PEG2000-FA, DSPE-PEG2000-ICG and DSPE-PEG2000 were purchased from Shanghai Pengshuo Biotechnology Co., Ltd.

[0034] CCK-8 kit, Annexin V-FITC / PI detection kit, reactive oxygen species detection kit and mitochondrial membrane potential detection kit (JC-1) were purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0035] II. Experimental methods

[0036] 2.1 Material characterization

[0037] The transmission electron microscope (JEM-2100Plus, JEOL) was used to observe the microscopic morphology, lattice structure and elemental distribution of the materials.

[0038] The ultraviolet-visible spectrophotometer (U-3900H, Hitachi) was used to measure the absorption spectra of the materials in the ultraviolet-visible region.

[0039] The X-ray diffractometer (D8-Advance, Bruker) was used to analyze the crystal structure of the materials and obtain XRD patterns.

[0040] The X-ray photoelectron spectrometer (K-Alpha, Thermo Fisher) was used to characterize the surface chemical properties and elemental chemical states of the materials.

[0041] The Fourier transform infrared spectrometer (Nicolet iS50, Thermo Fisher) was used to perform infrared spectroscopy tests on the materials. Using the KBr pellet method, the sample was mixed and ground with KBr and then pressed into a pellet for measurement.

[0042] A nuclear magnetic resonance spectrometer (FTNMR Digital, Bruker) and a high-resolution mass spectrometer (JMS-T100LP AccuTOF, JEOL) were used to characterize the structure and molecular weight of the compounds.

[0043] 2.2 Synthesis of g-C3N4

[0044] 3 g of melamine was placed in an alumina crucible and heated to 550 °C in a muffle furnace at a heating rate of 3 °C / min and held for 4 hours. After cooling to room temperature, the powder was ground and dispersed in 100 mL of 5 mol / L hydrochloric acid aqueous solution and refluxed for 24 hours. g-C3N4 was obtained after filtration, washing with deionized water until neutral, and vacuum drying.

[0045] 2.3 Synthesis of C3N4 / Au

[0046] 70 mL of deionized water was mixed with 30 mL of anhydrous methanol, and 1 g of g-C3N4 and 1 mL of 25 mg / mL AuCl3 aqueous solution were added. The mixture was irradiated with a 300 W xenon lamp for 3 hours under stirring. The precipitate was separated by centrifugation, washed with deionized water, and freeze-dried to obtain C3N4 / Au.

[0047] 2.4 Preparation of C3N4 / Au / SA hydrogel

[0048] 20 mg of C3N4 / Au was mixed with 1 mL of 2 mol / L CaCl2 aqueous solution and dispersed evenly with an ultrasonic processor to obtain dispersion A. 20 mg of sodium alginate (SA) was dissolved in 1 mL of deionized water and heated to obtain solution B. Dispersion A and solution B were mixed by spraying at a volume ratio of 1:2 to obtain C3N4 / Au / SA hydrogel.

[0049] 2.5 Preparation of DM

[0050] 5 mg of doxorubicin (DOX) was mixed with 25 mg of DSPE-PEG2000 and dissolved in 2 mL of dimethyl sulfoxide. Then the mixture was dialyzed in deionized water for 24 hours to obtain DM.

[0051] 2.6 Preparation of FA@DM

[0052] 5 mg of doxorubicin (DOX), 5 mg of DSPE-PEG2000-FA, and 25 mg of DSPE-PEG2000 were mixed and dissolved in 2 mL of dimethyl sulfoxide. Then the mixture was dialyzed in deionized water for 24 hours to obtain FA@DM.

[0053] 2.7 Preparation of DM@ICG

[0054] 1 mg of doxorubicin (DOX), 0.25 mg of DSPE-PEG2000-ICG, and 5 mg of DSPE-PEG2000 were mixed and dissolved in 1 mL of dimethyl sulfoxide. Then the mixture was dialyzed in deionized water for 24 h to obtain DM@ICG.

[0055] 2.8 Preparation of FA@DM@ICG

[0056] 1 mg of doxorubicin (DOX), 1 mg of DSPE-PEG2000-FA, 0.25 mg of DSPE-PEG2000-ICG, and 5 mg of DSPE-PEG2000 were mixed and dissolved in 1 mL of dimethyl sulfoxide. Then the mixture was dialyzed in deionized water for 24 h to obtain FA@DM@ICG.

[0057] 2.9 Synthesis of LysoFP-NO2

[0058] An ethanol solution (30 mL) of 4-(2-aminoethyl)morpholine (0.156 g) was slowly added dropwise to an ethanol solution (50 mL) of 3-nitro-1,8-naphthalic anhydride (0.243 g), and then the mixture was refluxed for 3 h. After filtration, the solvent was removed by rotary evaporation and vacuum drying to obtain LysoFP-NO2.

[0059] 2.10 Detection of carbon monoxide (CO) generation

[0060] For fluorescence detection, LysoFP-NO2 was dissolved with the help of 1% dimethyl sulfoxide and added to an aqueous dispersion containing g-C3N4 (5 mg / mL) or C3N4 / Au (5 mg / mL) to a final concentration of 10 μmol / L. Then the dispersion was irradiated with a 410 nm LED lamp (100 mW / cm 2 )). The fluorescence emission spectrum was measured every 1 min using a fluorescence spectrophotometer (FluoroMax-4, HORIBA) with an excitation wavelength of 440 nm. For gas chromatography, 30 mg of g-C3N4 or C3N4 / Au was used as the catalyst. A photocatalytic system (Labsolar-6A, PerfectLight) was used to measure the photocatalytic products. A 300 W xenon lamp was used to simulate sunlight irradiation, and the light source was 4 cm away from the reaction system. Samples were collected and analyzed every 1 h to monitor the changes in the concentrations of carbon monoxide (CO) and methane (CH4) in the system, and analysis was performed using a gas chromatograph (GC2002, KE CHUANG).

[0061] 2.11 Detection of reactive oxygen species (ROS)

[0062] The reactive oxygen species (ROS) generated during the photocatalysis process were detected using an electron paramagnetic resonance (ESR) spectrometer (EMX PLUS, Bruker). DMPO was used as a spin trap for hydroxyl radicals (·OH), BMPO for superoxide anions (O2 - ), and TPC for singlet oxygen ( 1 O2). The system was irradiated with a 410 nm LED lamp for 5 minutes or not irradiated. The presence of ROS was determined by observing the appearance or disappearance of characteristic signal peaks in the ESR spectrum.

[0063] 2.12 Cell culture

[0064] 4T1 (triple-negative breast cancer cell line), COS-7, BMDCs, and BMDM cells were cultured using RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% antibiotics. All cell cultures were carried out in a humid environment (37 °C, 5% CO2).

[0065] 2.13 Cytotoxicity

[0066] 4T1 cells were seeded into 96-well plates at a density of 8×10 3 cells per well. After 24 hours, the wells were treated with different groups. After 24 hours of treatment, 10 μL of CCK-8 solution was added to each well, and the plate was incubated in the dark for 1 hour. The absorbance (OD value) was measured at 450 nm using a microplate reader.

[0067] 2.14 Detection of reactive oxygen species (ROS) in vitro

[0068] 4T1 cells were seeded into six-well plates at a density of 1.5×105 cells per well. After the cells adhered, drugs were added and incubated for 6 hours. Subsequently, DCFH-DA (5 μM) was added and stained in the dark for 30 minutes. For the light-irradiated group, photodynamic treatment (400 nm, 100 mW / cm 2 , 5 minutes) was performed. Then the cells were washed three times with PBS and imaged under a confocal microscope or collected for flow cytometry analysis.

[0069] 2.15 Mitochondrial membrane potential detection experiment

[0070] 4T1 cells were seeded into six-well plates at a density of 1.5×10 5 cells per well. After the cells adhered and reached an appropriate density, different treatments were performed and incubated for 6 hours. For the light-irradiated group, light treatment (400 nm, 100 mW / cm 2 , 5 minutes) was performed. Subsequently, the cells were incubated for another 24 hours. Then, JC-1 staining solution (2 μM) was added and stained in the dark for 20 minutes. Finally, the cells were collected for flow cytometry analysis or confocal microscopy imaging.

[0071] 2.16 Experimental animals

[0072] Female BALB / c mice aged 6 - 8 weeks and weighing approximately 20 g were purchased from Beijing SPF Biotechnology Co., Ltd. All experiments and animal operations were approved by the Ethics Committee of Zhoupu Hospital, Pudong New Area, Shanghai, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals.

[0073] 2.17 In vivo distribution imaging

[0074] A 4T1 tumor-bearing mouse model was constructed. When the tumor volume reached 150 mm 3 , the tumor-bearing mice were randomly divided into two groups. Mice were given DM@ICG or FA@DM@ICG via intravenous injection. The in vivo distribution of ICG at different time points was tracked and monitored using a second near-infrared (NIR-II) in vivo imaging system (excitation wavelength: 808 nm, emission filter: 900 nm). After 24 hours, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor tissues were collected for imaging analysis.

[0075] 2.18 In vivo antitumor experiment and immunofluorescence experiment

[0076] First, a 4T1 tumor-bearing mouse model was established. When the tumor volume reached 100 mm 3 , 80% of the tumor tissue was resected, and the mice were randomly divided into six groups: control group, AuCN / SA group, AuCN / SA + L group, FA@DM group, AuCN / SA + FA@DM group, and AuCN / SA + FA@DM + L group. The treatment conditions were as follows: the dose of FA@DM was 5 mg / kg, the dose of AuCN was 12 mg / kg, and the mice were irradiated with 18W LED light (400 nm, 100 mW / cm 2 ) for 5 minutes. During the treatment, the body weight of the mice was measured every two days. On the 21st day, the mice were sacrificed, and the heart, liver, spleen, lung, kidney, and tumor tissues were collected for further analysis. After 21 days of treatment, the tumor tissues were collected and processed by paraffin embedding. The paraffin was removed using xylene, followed by sectioning and immunohistochemical and immunofluorescence staining of the tissues. The results were observed and analyzed using a confocal laser scanning microscope (CLSM).

[0077] 2.19 Statistical analysis

[0078] The significant differences between the two groups were calculated by two-tailed Student's t-test. p < 0.05, 0.01, 0.001, and 0.0001 were considered statistically significant and were represented by *, **, ***, and ****, respectively.

[0079] III. Experimental results

[0080] Synthesis and Characterization of 3.1g-C3N4 and C3N4 / Au

[0081] The in-situ photodeposition method was used to load gold nanoparticles (AuNPs) onto the surface of g-C3N4, thereby improving the carrier migration ability of g-C3N4 and enhancing the selectivity for reducing CO2 to CO. As Figure 2 shown in A, g-C3N4 was successfully synthesized by the high-temperature polycondensation method using melamine as the raw material. Subsequently, gold nanoparticles were loaded onto the surface of g-C3N4 by the in-situ reduction method to prepare the C3N4 nanophotocatalyst (C3N4 / Au) loaded with AuNPs. As Figure 2 shown in B, the morphologies of g-C3N4 and C3N4 / Au were observed by transmission electron microscopy (TEM). The prepared g-C3N4 exhibited an irregular flake structure. After treatment with AuCl3 under light irradiation, obvious small AuNPs were observed on C3N4 / Au. In addition, high-resolution transmission electron microscopy (HRTEM) provided an enlarged image of the Au NPs, showing lattice fringes with a spacing of 0.235 nm on C3N4 / Au, corresponding to the (111) plane of Au. In addition, TEM elemental mapping precisely showed the uniform distribution of C, N, and Au elements in the C3N4 / Au composite material. The Au element was dispersed on the plane composed of C and N, indicating that AuNPs were uniformly distributed on the surface of g-C3N4, forming a stable composite structure. The atomic content of gold was 0.41%, further confirming the successful loading of gold nanoparticles.

[0082] g-C3N4 and C3N4 / Au were further characterized using a UV-visible spectrophotometer. From Figure 2 D, it can be seen that the C3N4 / Au composite material had a characteristic peak near 570 nm, and the absorbance increased relative to g-C3N4. This peak was attributed to the surface plasmon resonance absorption of gold nanoparticles, indicating that AuNPs improved the spectral response range and carrier separation efficiency of the nanophotocatalyst. The XRD results are as Figure 2 shown in E, which confirmed the crystal structures of g-C3N4 and C3N4 / Au. Both materials showed diffraction peaks of g-C3N4 at 2θ angles of 13.2° and 27.6°, corresponding to the (100) and (002) crystal planes. In addition, new diffraction peaks appeared at 38.3° and 44.4° for C3N4 / Au, corresponding to the (111) and (200) crystal planes of Au, further confirming that the loading of AuNPs did not destroy the crystal structure of g-C3N4. As Figure 2 shown in F, Fourier transform infrared spectroscopy (FTIR) analysis showed that g-C3N4 and C3N4 / Au had the same functional groups, at 810 cm -1The characteristic peak at [the specific position] is a typical absorption peak of the triazine heterocycle.

[0083] 3.2 Performance of C3N4 / Au in photocatalytic generation of CO and ROS

[0084] The CO fluorescence probe LysoFP-NO2 was used to measure the CO generation ability of C3N4 / Au. The synthesis route of LysoFP-NO2 is as Figure 3 shown in A. LysoFP-NO2 is a lysosome-targeted fluorescence probe based on 3-nitrophthalimide. CO can reduce the nitro group in LysoFP-NO2 to an amino-functionalized derivative with high fluorescence intensity, LysoFP-NH2. Using 1 1H nuclear magnetic resonance and high-resolution mass spectrometry (HRMS) to characterize LysoFP-NO2. 1 The number and chemical shift of all hydrogen atoms in the 1H NMR spectrum are consistent with the structure of LysoFP-NO2. The mass-to-charge ratio of the sample measured by HRMS is 356.12505, and the calculated molecular formula is C 18 H 17 N3O5, which is consistent with [LysoFP-NO2 + H] + . These results together confirm the successful synthesis of LysoFP-NO2.

[0085] The fluorescence change of the LysoFP-NO2 probe was used to evaluate the ability of C3N4 / Au to photocatalytically generate CO in solution. As Figure 3 shown in B, under the irradiation of 410 nm LED light, the fluorescence intensity of the probe increased with the increase of irradiation time, indicating the photocatalytic ability of C3N4 / Au to produce CO. To further verify the photocatalytic products, a xenon lamp was used to illuminate to simulate sunlight in a sealed system, and the composition and quantity of the generated gas were analyzed by gas chromatography. As Figure 3 shown in C, the main products of photocatalytic reduction of C3N4 / Au are CO and CH4, and the generation rate of CO is higher than that of CH4. Compared with g-C3N4, the generation rates of CO and CH4 of C3N4 / Au are significantly increased, indicating that the incorporation of AuNPs improves the photocatalytic efficiency of g-C3N4. The mechanism of photocatalytic reduction of CO2 by C3N4 / Au is as Figure 3 shown in D. Under 410 nm irradiation, g-C3N4 mainly promotes the photocatalytic decomposition of water into hydrogen and oxygen. At the same time, it shows the ability to reduce a small amount of carbon dioxide, resulting in the production of CO and CH4. The introduction of AuNPs not only broadens the visible light response range of g-C3N4, but also improves the carrier separation efficiency, increasing the selectivity and generation rate of CO.

[0086] In addition, the reactive oxygen species generated during the photocatalytic process were detected by electron spin resonance (ESR), thus verifying the mechanism of CO2 photocatalytic reduction. Figure 3 E-3G showed that the characteristic peak change of superoxide anion (O2 - ) was significant, there was a weak characteristic peak for hydroxyl radical (·OH), and a characteristic peak for singlet oxygen ( 1 O2). This indicates that during the photocatalytic process of C3N4 / Au, O2 - and ·OH may be involved in the reduction of CO2. Specifically, O2 - can transfer electrons to CO2 to form a reactive intermediate (CO2 - ), which is the main reactive substance for the further reaction to generate CO.

[0087] 3.3 In vitro CO generation by C3N4 / Au

[0088] The ability of C3N4 / Au to produce CO in vitro was further evaluated using the CO fluorescent probe LysoFP-NO2. As Figure 4 shown in A, no fluorescence signal was observed in either the C3N4 group or the C3N4 / Au group under non-irradiation conditions. However, under irradiation conditions, the C3N4 group showed a relatively weak green fluorescence signal, indicating that it could reduce carbon dioxide to CO under blue light excitation. In contrast, the green fluorescence of the C3N4 / Au group increased significantly under irradiation, indicating the generation of CO. The enhanced fluorescence further demonstrated the effectiveness of gold doping in improving the catalytic efficiency of C3N4 for the reduction of carbon dioxide to CO.

[0089] Then, the intracellular CO concentration in L929 cells was quantitatively analyzed using a flow cytometer. As Figure 4 shown in B, 0.18% of the cells in the control group contained CO, while 75.8% of the cells treated with light and 90.7% of the cells in the C3N4 / Au group, indicating that the CO generated by photocatalysis could freely diffuse into the cells. At the same time, the intracellular CO levels in L929 cells containing C3N4 / Au were detected at different irradiation times. As Figure 4 shown in C, it was found that the intracellular CO content increased with the increase in exposure time. After irradiating with a 410 nm LED lamp for 10 min, CO was detected in 77.76% of the cells. After irradiating for 20 min and 30 min, this proportion further increased to 89.75% and 92.35% respectively. These results indicate that C3N4 / Au can continuously generate CO under irradiation conditions and diffuse into the cells.

[0090] 3.4 In vitro chemo-sensitizing effect of CO generation

[0091] After confirming the ability of C3N4 / Au to catalyze the reduction of intracellular carbon dioxide to CO under blue light irradiation, we used the CCK-8 assay to investigate the effect of the synergistic action of CO gas treatment and DOX on the viability of 4T1 cells. As Figure 5 shown in A, with the concentration of C3N4 / Au maintained at 50 mg / mL, it can be observed that with the increase of FA, the concentration of @DM gradually decreases. In addition, at the same DOX concentration, 4T1 cells treated with CO showed higher sensitivity to DOX. Specifically, under the influence of CO, when the DOX concentration was 0.1 μg / mL, the viability of 4T1 cells was approximately 70%. In contrast, in the absence of CO stimulation, a DOX concentration of 0.75 μg / mL was required to achieve a similar reduction in tumor cell viability. These results further confirmed that CO can significantly enhance the sensitivity of tumor cells to DOX-induced cytotoxicity.

[0092] The next goal was to investigate the mechanism by which carbon monoxide (CO) enhances the sensitivity of tumor cells to chemotherapeutic drugs. Some researchers believe that CO affects cell behavior by promoting mitochondrial synthesis and increasing the production of reactive oxygen species (ROS). In tumor cells, the enhancement of mitochondrial function by CO may lead to an increase in ROS levels, which in turn induces apoptosis of tumor cells. To evaluate the production of intracellular ROS under CO stimulation, we used the ROS probe DCFH-DA. Figure 5 As shown in B, compared with the control group and the C3N4 / Au group, the C3N4 / Au+L group showed significant green fluorescence, indicating the production of ROS. This observation indicates that the CO produced by C3N4 / Au under blue light stimulation did stimulate the production of intracellular ROS. In addition, we also tested the ability of the hydrogel C3N4 / Au / SA to stimulate the production of ROS in cells. It was found that under light stimulation, C3N4 / Au / SA could also induce a significant increase in intracellular ROS. Flow cytometry was used to quantitatively analyze the production of intracellular ROS, as Figure 5 shown in C. After adding C3N4 / Au, only a very small number of cells (3.48%) had green fluorescence, while this proportion increased to 67.8% after light irradiation, and the fluorescence intensity was significantly enhanced. This further confirmed that C3N4 / Au can increase the level of intracellular oxidative stress after light irradiation.

[0093] PI / Annexin-V double staining and flow cytometry were used to further analyze the tumor cells treated under different conditions. Figure 5Figure D shows that compared with other groups, after treatment with C3N4 / Au, the early apoptosis rate (12.7%) and late apoptosis rate (37.1%) of tumor cells under light stimulation were significantly increased. These findings further confirmed the sensitivity of tumor cells to ROS-induced oxidative stress, leading to the induction of apoptosis and thus increasing the sensitivity to chemotherapeutic drugs.

[0094] The JC-1 mitochondrial membrane potential detection kit was used to detect the changes in mitochondrial membrane potential in tumor cells under different conditions. Figure 5 Figure E shows that both the control group and the C3N4 / Au group showed bright red fluorescence of j-aggregates, indicating a relatively high mitochondrial membrane potential. In contrast, the green fluorescence in the C3N4 / Au+L group was significantly enhanced. This indicates that CO generated by C3N4 / Au under blue light irradiation can reduce the mitochondrial membrane potential, promoting the transfer of JC-1 from the aggregated state to the monomer state. Subsequently, flow cytometry was used to verify the proportion of cells in the monomer state, which increased from 2.8% in the control group and 8.7% in the C3N4 / Au group to 16.6% in the C3N4 / Au+L group ( Figure 5 Figure F). In summary, C3N4 / Au can generate CO under the stimulation of blue light irradiation, leading to a decrease in the mitochondrial membrane potential of tumor cells. This decrease results in the generation of a large amount of ROS, triggering apoptosis of cells. Therefore, tumor cells become more sensitive to chemotherapeutic drugs.

[0095] 3.5 Tumor targeting effect of FA@DM in vitro and in vivo

[0096] Then, the uptake of FA@DM by 4T1 cells was observed using a confocal laser scanning microscope (CLSM). Figure 6 Figure A shows that the FA@DM group showed stronger red fluorescence than the DM group, indicating a greater uptake by tumor cells. This shows that FA@DM modified with FA enhanced the targeting ability to 4T1 tumor cells. At the same time, FA was used for pretreatment to block the FA receptors on the surface of 4T1 cells. In contrast, the red fluorescence in the FA pretreatment group (FA+FA@DM) was significantly weaker than that in the FA@DM group. This finding further demonstrated that FA receptors play a key role in the increased uptake of FA@DM by 4T1 cells and reaffirmed that the modification with FA significantly enhanced the tumor targeting of FA@DM. The targeting performance of FA@DM was characterized using flow cytometry, and the results are as Figure 6 shown in Figure B, further verifying the tumor targeting effect of FA.

[0097] In addition, FA@DM modified with ICG and DM were administered to mice via tail vein injection, and FA@DM@ICG and DM@ICG were obtained respectively. The in vivo distribution of these formulations was explored using a NIR-II in vivo imaging system. Figure 6C showed that the DM@ICG group exhibited weak fluorescence signals in the tumor region, but obvious signals were observed in the tumor regions of mice injected with FA@DM@ICG as early as 2 hours after injection, lasting up to 24 hours. Quantitative fluorescence data from the tumor regions showed that at the same time point, the fluorescence signals in the tumor regions of the FA@DM@ICG group were always higher than those of the DM@ICG group( Figure 6 D). After 24 hours of intravenous injection, fluorescence imaging of the major tissues showed that most of the micelles were metabolized through the liver and kidneys( Figure 6 E). Compared with the DM@ICG group without FA modification, the FA@DM@ICG group showed stronger fluorescence signals in the tumor tissues( Figure 6 F). These results further confirmed the in vivo tumor targeting effect of FA and provided a basis for the application of FA@DM in postoperative tumor drug administration.

[0098] 3.6 Antitumor effect of CO in vivo

[0099] A postoperative breast cancer model was constructed, and then the antitumor effect of C3N4 / Au / SA in generating CO in synergy with FA@DM during a 21-day treatment period was evaluated. As Figure 7 shown in A, an orthotopic model of murine breast cancer was initially established. When the tumor volume reached approximately 100 mm 3 , tumor resection was performed in situ, removing approximately 80% of the tumor volume. A solution containing calcium chloride and C3N4 / Au was sprayed on the surgical wound, and then an SA solution was added. Once the hydrogel formed, the wound was exposed to blue light for 5 minutes. Subsequently, intravenous administration was carried out on the 1st, 4th, and 7th days after surgery, ending the 21-day treatment period. As Figure 7 shown in B, after the 21-day treatment, we resected the tumor tissues of the mice and observed them. The antitumor effects of both the FA@DM group and the C3N4 / Au / SA+L group were slightly better than those of the control group. These results indicate that whether triggered by CO or induced by the chemotherapeutic drug DOX, apoptosis and cell death within the tumor can lead to a certain degree of tumor growth inhibition. The combination of C3N4 / Au / SA+L and FA@DM significantly enhanced the inhibitory effect on tumor growth. Similarly, after the 21-day treatment period, the tumor tissues of the mice were resected and weighed. The results showed that the tumor tissue weight of the FA@DM@C3N4 / Au / SA+L group was the lowest( Figure 7 C). In addition, the photos taken after resection of the tumor tissues of each group more directly showed that CO had a good antitumor effect when combined with chemotherapy( Figure 7 D).

[0100] As Figure 7As shown in E, during the entire 21-day treatment period, there were no significant changes in the body weights of the mice in each group. This indicates that neither the hydrogel C3N4 / Au / SA nor the chemotherapeutic micelles FA@DM showed obvious toxic side effects. After treatment, H&E staining was performed on the major organs (heart, liver, spleen, lung, kidney) of the mice in each group. The results showed no obvious tissue damage, further confirming this conclusion. After treatment, H&E and immunohistochemistry / fluorescence staining were performed on the tumor tissues of the mice in each group, and the results are as Figure 7 shown in F. H&E staining showed that the cell nuclei in the Control group and the C3N4 / Au / SA group were dense, dark purple, with abnormal proliferation and a large number of mitotic figures, suggesting that the proliferation rate of these tumor tissues was abnormally fast. While apoptotic and necrotic regions were observed in the other four groups of cells. Notably, there were obvious extensive necrotic regions in the FA@DM&C3N4 / Au / SA+L group. Correspondingly, the TUNEL staining sections of the tumor cells in the FA@DM&C3N4 / Au / SA+L group showed a large number of red fluorescences, representing cell apoptosis, further indicating that the combination of CO gas stimulation and FA@DM chemotherapy significantly enhanced tumor cell apoptosis. Immunohistochemical staining of Ki-67 was also performed on the tumor tissues. The results showed that the content of Ki-67 positive cells in the FA@DM&C3N4 / Au / SA+L group was significantly lower than that in the other groups, suggesting that the combination of CO and chemotherapy could significantly inhibit tumor cell proliferation. Another explanation is that CO may cause cell cycle arrest at certain stages, such as the G0 / G1 phase or the G2 / M phase, thereby increasing the cytotoxic effect of chemotherapeutic drugs on tumor cells. In addition, the comparison of the CD31 fluorescence staining results showed that CO could inhibit tumor angiogenesis, effectively limiting the access of tumor cells to nutrients, thereby restricting the tumor growth environment. When combined with chemotherapeutic drugs, it significantly improved its anti-tumor effect.

[0101] This specific embodiment is only an explanation of the present application, and it does not limit the present application. Those skilled in the art can make modifications without creative contributions to this embodiment according to needs after reading this specification, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. C3N4 / Au nano-photocatalyst, characterized in that: Composed of g-C3N4 and Au 3+ The solution undergoes a photocatalytic reduction reaction, causing gold nanoparticles to be deposited on the surface of g-C3N4.

2. C3N4 / Au / SA hydrogel, characterized in that Prepared by the following steps: The C3N4 / Au described in claim 1 is mixed with a CaCl2 aqueous solution and dispersed uniformly to obtain a dispersion A; Dissolve sodium alginate SA in deionized water and heat to obtain solution B; Dispersion A and solution B were mixed by spraying to obtain C3N4 / Au / SA hydrogel.

3. The C3N4 / Au / SA hydrogel according to claim 2, characterized in that Dispersion A and solution B were mixed at a volume ratio of 1:

2.

4. Use of the C3N4 / Au / SA hydrogel according to claim 2 in the preparation of a drug for enhancing the sensitivity of tumor cells to chemotherapeutic drugs.

5. The use according to claim 4, characterized in that The chemotherapy drug includes doxorubicin DOX.

6. Use of the C3N4 / Au / SA hydrogel according to claim 2 in combination with doxorubicin DOX.

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