Photoresponse type carbon nitride nano-iron death inducer as well as preparation method and application of photoresponse type carbon nitride nano-iron death inducer

Abstract

The invention relates to the technical field of anti-tumor, in particular to a photoresponse type carbon nitride nano-iron death inducer as well as a preparation method and application of the photoresponse type carbon nitride nano-iron death inducer. The photoresponse type carbon nitride nano-iron death inducer is synthesized by using cheap and easily available urea as a raw material through calcining and inert atmosphere annealing processes, the process is simple, and the cost is low. The core of the material is that nitrogen vacancies are introduced to the edges of the carbon nitride nanosheets, the band gap of the material is reduced from 2.87 eV to 2.44 eV, the light absorption range is subjected to red shift to 560-620 nm, and the photo-induced electron-hole separation efficiency is remarkably improved. Under illumination of a specific wavelength, OCN can efficiently generate active oxygen such as hydroxyl free radicals, and Fe is directly catalyzed through Fenton reaction to start lipid peroxidation, so that tumor cells are efficiently and specifically induced to generate ferroptosis. The inducer realizes time-space controllability of tumor treatment, has good biocompatibility, and provides a new strategy for ferroptosis-driven tumor treatment.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of anti-tumor, more particularly, it relates to a light-responsive carbon nitride nanometer iron death inducer and a preparation method and application thereof. BACKGROUND

[0002] Iron death is a new type of iron-dependent programmed cell death, the core mechanism of which is that the activity of glutathione peroxidase 4 (GPX4) in cells is inhibited or glutathione (GSH) is depleted, resulting in that lipid peroxide (LPO) cannot be cleared in time and accumulates in large quantities, eventually causing oxidative damage to the cell membrane system. Since iron death can effectively overcome the drug resistance of tumor cells to traditional apoptosis induction therapy, it has shown great application potential in the field of tumor treatment.

[0003] At present, the strategy for inducing iron death mainly relies on small molecule compounds such as iron death inducers, such as Erastin which inhibits cystine / glutamate antiporter (System Xc-) or RSL3 which directly inhibits the activity of GPX4. However, these mainstream small molecule iron death inducers face several serious challenges in clinical transformation: first, the synthesis and purification process is usually complex, resulting in high cost; secondly, and most importantly, the action mode lacks an external controllable trigger switch, and once administered, it will have a systemic effect, making it difficult to activate precisely at a specific time and on a specific lesion site, which not only limits the efficacy, but also may cause off-target toxicity and other safety problems.

[0004] Photodynamic therapy (PDT) takes advantage of the property of photosensitizers to produce reactive oxygen species (ROS) under specific wavelength light, which provides a possibility for time and space controllable tumor treatment. However, traditional PDT photosensitizers (such as porphyrins) have the following limitations: on the one hand, their light absorption range is often narrow, limiting their penetration depth in biological tissues; on the other hand, the coupling efficiency of the ROS (such as singlet oxygen) they produce with the core biochemical pathway of iron death (i.e. lipid peroxidation catalyzed by Fenton reaction) is low, making it difficult to efficiently and specifically initiate the iron death signal chain, as shown in Figure 6 As shown in the figure, the traditional PDT material (Ti3C2@BSA) has the problems of insufficient ROS production and weak coupling with the iron death mechanism. Therefore, it is essential to develop a new platform that can efficiently combine the photodynamic effect with the iron death pathway.

[0005] In recent years, non-metallic semiconductor material graphite phase carbon nitride (g-C3N4) has attracted attention in the biomedical field due to its good biocompatibility and chemical stability. Among them, the synthesis route with urea as the precursor has the significant advantage that the raw material is cheap and easy to obtain. However, the urea-derived carbon nitride prepared by conventional methods (such as original urea-derived carbon nitride YCN, and the product WCN of YCN treated by air atmosphere heat) has inherent defects as a photosensitizer: its band gap is wide, resulting in limited light absorption capacity, and the photo-generated electron-hole pairs are quickly combined, resulting in a low ROS yield, which cannot meet the needs of efficient induction of ferroptosis.

[0006] In summary, there is an urgent need in the art to develop a new ferroptosis inducer that needs to meet the following conditions: (1) It has the economic advantages of easy-to-obtain raw materials and simple synthesis; (2) It has light-triggered characteristics and can achieve precise spatiotemporal control; (3) It can generate ROS that is highly adapted to the ferroptosis mechanism, thereby efficiently and specifically inducing ferroptosis. SUMMARY

[0007] In order to overcome the shortcomings of the prior art, the purpose of the present application is to provide a light-responsive carbon nitride nanometer ferroptosis inducer and its preparation method and application, which are used to solve the technical problems of the existing ferroptosis inducers, such as complex synthesis process, high cost, lack of spatiotemporal controllability, low induction efficiency, and weak coupling with the core mechanism of ferroptosis.

[0008] To achieve the above technical purposes, the present application adopts the following technical solutions: The present application provides a preparation method and application of a light-responsive carbon nitride nanometer ferroptosis inducer (OCN) using urea as a raw material. OCN can efficiently trigger ROS production under xenon lamp irradiation (equipped with a 420 nm filter, working current 20A, irradiation time 10 min), induce tumor cell ferroptosis, and significantly up-regulate heat shock protein (HSP) expression. Intratumoral injection of OCN in mice can promote the cross-activation process of antigen-presenting cells, enhance the effector function of CD8⁺ and CD4⁺ T cells, and achieve enhanced anti-tumor immune response in vivo.

[0009] The specific research and development process of the light-responsive carbon nitride nanometer ferroptosis inducer (OCN) is as follows: 1. Construction and characterization of ferroptosis inducer OCN (1) Preparation of OCN Yellow carbon nitride precursor (YCN) was prepared by calcination of urea at high temperature in air atmosphere. The precursor was further heat treated in air to obtain white carbon nitride (WCN). The yellow carbon nitride precursor (YCN) was then ground and annealed at high temperature under inert argon atmosphere to convert into the target product orange carbon nitride (OCN). To prepare the corresponding nanosheets, the above three materials (WCN, YCN, OCN) were first treated by ultrasonic crushing and then by centrifugation to obtain nanosheets.

[0010] (2) Physical and photocatalytic performance characterization External morphology characterization: High-resolution transmission electron microscopy (HR-TEM) and atomic force microscopy (AFM) were used to observe the overall morphology and thickness of each sample.

[0011] Structure and charge separation ability analysis: X-ray diffraction (XRD), Fourier infrared spectroscopy (FTIR), and Raman spectroscopy analysis were used to evaluate the nitrogen vacancy structure of OCN and its promotion of photogenerated carrier separation and migration.

[0012] Photocatalytic ability detection: UV-VIS spectroscopy, Mott-Schottky plots, transient photocurrent measurements, and electron spin resonance (ESR) signals were used to evaluate the energy band structure, photogenerated charge dynamics, and free radical generation ability of OCN.

[0013] (3) Detection of cell internalization of OCN Cell internalization of OCN: Fluorescein FITC conjugated materials were used, and flow cytometry was used to detect the internalization of OCN by cancer cells with prolonged incubation time.

[0014] 2. Evaluation of OCN's in vitro induction of ROS accumulation under xenon lamp irradiation and its killing effect on tumor cells After incubating tumor cells with OCN for 2h, xenon lamp irradiation was performed (equipped with a 420nm filter, working current 20A, irradiation time 10min). After 1h of light irradiation, ROS production was detected using a ROS probe, and the killing effect on tumor cells was detected using AM-PI live and dead staining.

[0015] 3. Evaluation of OCN's in vitro induction of ferroptosis under xenon lamp irradiation After pre-incubating various types of death inhibitors with tumor cells, and then incubating with OCN for 2h, xenon lamp irradiation was performed (equipped with a 420nm filter, working current 20A, irradiation time 10min). After 2h of light irradiation, CCK8 was used to detect cell viability.

[0016] After incubating tumor cells with OCN for 2h, xenon lamp irradiation was performed (equipped with a 420nm filter, working current 20A, irradiation time 10min). After 1h of light irradiation, Fe 2+Detection probes, lipid peroxidation probes, and JC-1 kits were used to detect iron death-related biological indicators, including intracellular free iron levels, lipid peroxidation levels, and changes in mitochondrial membrane potential. Meanwhile, total RNA was extracted from the cells and subjected to qPCR analysis to detect the expression of iron death-related genes.

[0017] 4. Evaluation of the in vivo anti-tumor effect of OCN under xenon lamp irradiation On the 10th day after tumor inoculation, the tumor-bearing mice were subjected to intratumoral injection of OCN (2 mg / kg). Two hours after administration, the injection site was irradiated with a xenon lamp (420 nm filter, operating current 20 A, irradiation time 10 min). The tumor volume and mouse body weight were continuously monitored after treatment, and the tumor growth curve was plotted.

[0018] 5. Evaluation of tumor microenvironment characteristics after in vivo treatment of OCN under xenon lamp irradiation Under the same administration and irradiation conditions as described in 4 above, the mice were sacrificed 12 h after irradiation and the tumor tissue was collected for Bulk RNA-seq, mass cytometry flow cytometry analysis, and conventional flow cytometry detection. The recruitment of dendritic cells (DCs) in the tumor microenvironment, as well as the infiltration levels of CD8⁺ T cells, CD4⁺ T cells, and Tregs, were evaluated.

[0019] 6. Evaluation of biological safety In vitro biocompatibility: Human umbilical vein endothelial cells HUVEC, mouse breast cancer cells 4T1, mouse skin melanoma cells B16F10, and mouse colon cancer cells MC38 were incubated with OCN for 8 h, and the cell activity was detected using a CCK8 kit.

[0020] Compared with the prior art, the present application has the following beneficial effects: (1) Raw material and process advantages: Urea is used as the nitrogen and carbon source, and OCN is prepared through a three-step process of "calcination-grinding-argon annealing". The high nitrogen content and controllable pyrolysis behavior of urea help to form a nitrogen-rich skeleton; annealing under argon conditions can introduce stable nitrogen vacancies in the edge region of the material, while avoiding excessive damage to the bulk structure, thereby maintaining good layer stability.

[0021] (2) Band structure optimization: The formation of edge nitrogen vacancies creates a defect level, reducing the band gap from 2.87 eV for YCN to 2.44 eV, and red-shifting the absorption spectrum to 560-620 nm, effectively improving the tissue penetration ability; (3) ROS generation efficiency optimization: The amorphous nanosheet structure exposes abundant edge defects, which helps to separate photo-generated electrons and holes, and promotes the simultaneous generation of various active species such as hydroxyl radicals (·OH), singlet oxygen (¹O2), etc. Among them, ·OH can directly catalyze Fe²⁺ to start lipid peroxidation through Fenton reaction, which is more suitable for ferroptosis mechanism (experimental results show that the total ROS yield is increased by 2.2 times compared with YCN, Figure 5 ).

[0022] (4) Biological effect verification: Under xenon lamp irradiation, OCN effectively induced tumor death, and the reversal rate of ferroptosis inhibitor was significantly higher than that of apoptosis / necrosis. It is manifested as the increase of lipid peroxidation level, the down-regulation of mitochondrial membrane potential, etc. At the same time, the mild photothermal effect of the material promotes the up-regulation of heat shock protein (HSP) and MHC-I related biosynthesis pathway, enhances CD8⁺T cell infiltration, and forms a synergistic anti-tumor effect extending from "local ferroptosis ablation" to "systemic immune activation". Animal experiments did not observe the decrease of mouse body weight or pathological damage of major organs, indicating that the material has good biological safety. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 It is a schematic diagram of the structure of OCN in Example 1, a high-resolution transmission electron microscope image, and an atomic force microscope image.

[0024] Figure 2 It is an XRD, FTIR and Raman diagram of OCN in Example 1.

[0025] Figure 3 It is a UV-VIS spectrum, a Mott-Schottky diagram, a transient photocurrent measurement, and an electron spin resonance (ESR) diagram of OCN in Example 1.

[0026] Figure 4 It is a flow cytometry detection of cell internalization of OCN in Example 1.

[0027] Figure 5 It is a ROS generation level fluorescence microscope and flow cytometry detection of the Ctrl group, the WCN group, the YCN group, the OCN group, the xenon lamp irradiation group (Lamp), the WCN+Lamp group, the YCN+Lamp group, and the OCN+Lamp group in Example 2.

[0028] Figure 6 It is a ROS generation level fluorescence microscope detection of the ordinary photosensitizer Ti3C2@BSA group in Example 2.

[0029] Figure 7 It is an AM-PI live and dead staining detection of tumor cell killing function in Example 2.

[0030] Figure 8Figure for OCN-mediated tumor cell death mechanism detection in Example 3.

[0031] Figure 9 Figure for lipid ROS detection of ferroptosis-related indicators in Example 3.

[0032] Figure 10 Figure for Fe detection of ferroptosis-related indicators in Example 3. 2+ Figure for detection.

[0033] Figure 11 Figure for mitochondrial membrane potential detection of ferroptosis-related indicators in Example 3.

[0034] Figure 12 Figure for ferroptosis-related gene expression detection in Example 3.

[0035] Figure 13 Figure for tumor inhibition curve of OCN in vivo inhibition of 4T1 mouse breast cancer in Example 4.

[0036] Figure 14 Figure for tumor diameter comparison of OCN in vivo inhibition of 4T1 mouse breast cancer in Example 4.

[0037] Figure 15 Figure for Bulk RNA-seq results in Example 5.

[0038] Figure 16 Figure for mass cytometry flow cytometry analysis results in Example 5.

[0039] Figure 17 Figure for the recruitment of DCs, infiltration of CD8+ T cells, CD4+ T cells and Tregs in tumors after OCN injection in Example 5.

[0040] Figure 18 Figure for four in vitro cytotoxicity detection results of OCN in Example 6.

[0041] Figure 19 Figure for the change in body weight of mice during in vivo treatment of OCN in Example 6.

[0042] Figure 20 Figure for H&E staining histopathology sections of the main organs (heart, liver, spleen, lung, kidney) of mice after in vivo treatment of OCN in Example 6.

[0043] Figure 21 Figure for in vitro hemolysis experiment results of OCN in Example 6. DETAILED DESCRIPTION

[0044] In order to make the objects, characteristics and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application are described in detail below in combination with the drawings. Several embodiments of the present application are shown in the drawings. However, the present application can be realized in many different forms and is not limited to the embodiments described herein.

[0045] The specific conditions not specified in the following examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not specified by the manufacturer, but are conventional products that can be obtained by commercial purchase. Unless otherwise specified, the present application uses the existing technology in the field.

[0046] Example 1: Construction and characterization of light-responsive carbon nitride nanometer iron death inducer (OCN) (1) Preparation of OCN First, by calcining urea with a purity of more than 99% at 550 ℃ in air atmosphere for 3 hours, yellow carbon nitride precursor (YCN) was prepared. Then, YCN was heated at 530 ℃ in air for 2 hours to obtain white carbon nitride (WCN). Finally, YCN was ground to a powder with a mesh size of 200-350, and then annealed in an argon atmosphere at 680 ℃ for 10 minutes and cooled to room temperature to obtain the target product, orange carbon nitride (OCN). The heat treatment conditions in an inert atmosphere promote the conversion of C-NH2 groups in the edge sites to C-H structures (as shown in A of Figure 1 ), forming stable edge nitrogen vacancies without destroying the overall carbon-nitrogen network, thereby constructing a defect-rich ultrathin structure (as shown in the red dotted circle part of A of Figure 1 ).

[0047] 10 mg of each of the above three materials (WCN, YCN, OCN) was dissolved in ddH2O, ultrasonically pulverized for 20 minutes (pulse mode: working 2.2 seconds / intermittent 6.6 seconds; amplitude 80%) to further reduce the particle size, and then centrifuged at 13000 rpm to collect the exfoliated nanosheets in the supernatant and freeze-dried.

[0048] (2) Physical and photocatalytic performance characterization High-resolution transmission electron microscopy (TEM) and atomic force microscopy (AFM) were used to observe the overall morphology and thickness of the exfoliated nanosheets, as shown in B of Figure 1 , OCN is an ultrathin nanosheet with a wrinkled morphology, Figure 1 C shows that its thickness is about 1.3 nm. This ultrathin structure is beneficial to the transmission of photo-generated charges between the sheets, thereby improving the electron dynamics performance in the photocatalytic process.

[0049] The structural characteristics and photocatalytic properties of OCN were analyzed by XRD, FTIR and Raman spectroscopy, and compared with those of WCN and YCN. Figure 2 The XRD results of A in the study showed that the (002) diffraction peak of OCN was relatively wider and lower in intensity, indicating a decrease in interlayer order and a higher degree of structural disorder. This structural feature usually helps to enhance surface reaction sites and improve charge separation efficiency. In FTIR (… Figure 2 In (B) of the spectrum, the NHx characteristic peak of OCN is significantly weakened, indicating that some edge -NH2 groups are removed during annealing, thus supporting the formation of nitrogen vacancies. Raman spectroscopy ( Figure 2 C) indicates that despite undergoing heat treatment and crushing and peeling, the main carbon nitride skeleton of the material remains stable, and the nitrogen vacancies formed in the edge regions do not disrupt the main chain structure.

[0050] The photocatalytic performance of OCN was systematically evaluated using UV-VIS spectroscopy, Mott-Schottky assay, transient photocurrent measurement, and electron spin resonance (ESR) analysis. UV-VIS absorption spectroscopy (…) Figure 3 As shown in A), due to the higher nitrogen vacancy density at the material edges, the band gap of OCN narrows from 2.87 eV for YCN and 3.03 eV for WCN to 2.44 eV, and the absorption spectrum red-shifts to 560-620 nm (yellow-orange-red), enhancing its absorption capacity in the visible light region. Mott-Schottky curves ( Figure 3 (B) indicates that all three materials exhibit n-type semiconductor behavior. Based on UV-VIS and Mott-Schottky analysis results, a band structure model of the materials is constructed ( Figure 3 In the C group, OCN exhibits a downward shift in the valence band maximum (VBM), indicating stronger oxidation ability and greater facilitating of the directional migration and separation of photogenerated charges.

[0051] Transient photocurrent testing ( Figure 3 Figure D further confirms that the photocurrent response generated by OCN under illumination is higher than that of YCN and WCN, indicating that its photogenerated carrier separation efficiency is higher. The ESR detection results (E and F in Figure 3) show that compared with the free radical signal under illumination of WCN and YCN, OCN generates a stronger ESR response under illumination, verifying its efficient photocatalytic ROS generation capability.

[0052] (3) Detection of cell internalization of OCN Cellular internalization of OCN: Using the fluorescein FITC conjugated material WCN / YCN / OCN, flow cytometry was used to detect the internalization of 300 μg / mL of WCN / YCN / OCN by 4T1 cells after xenon lamp irradiation (equipped with a 420 nm filter, working current 20 A, irradiation time 10 min). The results are shown in Fig. 1A, and as the incubation time is prolonged, the cells can effectively endocytose the three materials WCN / YCN / OCN. Figure 4

[0053] Example 2: OCN mediated tumor killing effect The ROS level in the cells was detected using a ROS detection kit, and the 4T1 cells were divided into groups for treatment (Ctrl group, WCN group, YCN group, OCN group, xenon lamp irradiation group (Lamp), WCN+Lamp group, YCN+Lamp group, OCN+Lamp group). The Ctrl group was the conventional 4T1 cell culture without additional treatment. The WCN / YCN / OCN group was specifically 300 μg / mL of WCN / YCN / OCN added to the 4T1 cells, which was incubated in the cell incubator for 2 h before detection. The Lamp group was irradiated by a xenon lamp, specifically: the xenon lamp was equipped with a 420 nm filter, the working current was 20 A, and the 4T1 cells were irradiated for 10 min before being incubated in the cell incubator for 2 h before detection. The WCN+Lamp / YCN+Lamp / OCN+Lamp was specifically: the 4T1 cells were pre-treated by co-incubation with WCN / YCN / OCN for 2 h, then irradiated by the Lamp for 10 min, and incubated in the cell incubator for 1 h before detection. The results of fluorescence microscopy are shown in Fig. 2A, which shows that under 420 nm xenon lamp irradiation, the ROS production rate of the OCN+Lamp group is the highest, showing the strongest green fluorescence intensity. As a comparison, under 808 nm near-infrared light irradiation, no obvious ROS signal was detected for the same concentration (300 μg / mL) of the photosensitizer Ti3C2@BSA (as shown in Fig. 2B, no green fluorescence was observed), which indicates that even under different excitation light wavelengths, the ROS production efficiency of OCN in the visible light region is better than that of the traditional material in the near-infrared region. As shown in Fig. 2B, under the same operation, the flow cytometry quantitative detection of the digested cells showed that the mean fluorescence intensity (MFI) of the OCN+Lamp group was 12500, which was significantly higher than that of the YCN+Lamp group (5700), indicating that the ROS production level was about 2.2 times that of the YCN+Lamp group. The killing effect of OCN on tumor cells was detected using the AM-PI live and dead staining kit, and the groups and experimental procedures were the same as above. As shown in Fig. 2C, more cells died in the OCN+Lamp group, indicating the excellent tumor killing effect of OCN under the xenon lamp. Figure 5 Figure 6 Figure 5 Figure 7 ​​​​​

[0054] Example 3: OCN induces ferroptosis in tumor cells Pre-treatment of 4T1 cells with 10 mM ferroptosis inhibitor DFO, 5 mM ferroptosis inhibitor Fer1, 10 mM apoptosis inhibitor ZVAD-FMK and 10 mM necrosis inhibitor Necrosulfonamide, respectively, in the culture system. Subsequently, 300 pg / ml OCN was added for 2 h pre-incubation, and irradiation was performed under a xenon lamp light source (equipped with a 420 nm filter, working current 20 A, irradiation time 10 min). Cell viability was detected by CCK-8 and AM-PI 2 h after light irradiation. As shown in Figure 8 , the addition of ferroptosis inhibitors DFO and Fer-1 can significantly weaken the cell death effect induced by OCN under xenon lamp irradiation, while the protective effect of apoptosis and necrosis inhibitors is weak, suggesting that OCN mainly mediates tumor cell damage through the ferroptosis pathway.

[0055] 4T1 cells were divided into groups for treatment (Ctrl group, WCN group, YCN group, OCN group, xenon lamp light irradiation group (Lamp), WCN+Lamp group, YCN+Lamp group, OCN+Lamp group). The Ctrl group was the conventional 4T1 cell culture without additional treatment. The WCN / YCN / OCN group was specifically 300 pg / mL WCN / YCN / OCN added to 4T1 cells, and the cells were incubated in the cell incubator for 2 h before detection. The Lamp group was treated with xenon lamp irradiation, specifically: the xenon lamp was equipped with a 420 nm filter, the working current was 20 A, and the 4T1 cells were irradiated for 10 min, then incubated in the cell incubator for 2 h before detection. The WCN+Lamp / YCN+Lamp / OCN+Lamp group was specifically: 4T1 cells were pre-treated with WCN / YCN / OCN for 2 h, then irradiated with Lamp for 10 min, and then incubated in the cell incubator for 1 h before detection. The cells were incubated with 1 mM Fe 2+ probe FerroOrange, 5 mM lipid ROS probe C11-BODIPY probe, and then photographed under a fluorescence microscope. The JC-1 kit was used to detect the changes in mitochondrial membrane potential, and RNA was extracted for qPCR to detect the expression of ferroptosis-related genes. As shown in Figure 9 , 10 , the lipid ROS level and intracellular Fe 2+ level in the OCN+Lamp group were significantly higher than those in the control group. JC-1 staining showed that in the LAMP+OCN group, the mitochondrial membrane potential of 4T1 cells decreased the most, showing an increase in the ratio of JC-1 monomer to polymer (results as shown in Figure 11OCN+Lamp group (p<0.05, Fig. 6B). qPCR analysis also showed that the expression of ferroptosis-driven gene (Ptgs2) was significantly up-regulated in OCN+Lamp group compared with other groups (p<0.05, Fig. 6C). In contrast, the expression of ferroptosis-inhibited genes (Nfe2l2, Slc7a11, Hifla and Ezh2) was significantly down-regulated in OCN+Lamp group compared with other groups (p<0.05, Fig. 6D). These results collectively support that OCN can effectively induce ferroptosis under xenon lamp irradiation, accompanied by disruption of iron homeostasis, enhanced lipid peroxidation and mitochondrial dysfunction. Figure 12 These results collectively support that OCN can effectively induce ferroptosis under xenon lamp irradiation, accompanied by disruption of iron homeostasis, enhanced lipid peroxidation and mitochondrial dysfunction.

[0056] Example 4: Evaluation of in vivo anti-tumor effect of OCN under xenon lamp irradiation BALB / c mice (6 weeks old, male) were randomly divided into four groups: Ctrl group (n=7), Ctrl+Lamp group (n=8), OCN group (n=7), OCN+Lamp group (n=7). Each mouse was subcutaneously inoculated with tumor cells (1x10 6 cells / mouse) on the back. Intervention was started on the 10th day after modeling, in which the Ctrl group was subcutaneously injected with normal saline; the mice in the Ctrl+Lamp group were subcutaneously injected with normal saline at the injection site and subjected to xenon lamp irradiation (420 nm filter, working current 20 A, irradiation time 10 min); the mice in the OCN group were subjected to intratumoral injection of 2 mg / kg OCN (NS dissolved OCN); the mice in the OCN+Lamp group were subjected to intratumoral injection of 2 mg / kg OCN, and the injection site was subjected to xenon lamp irradiation (420 nm filter, working current 20 A, irradiation time 10 min) 2 h after administration. The change in tumor volume was recorded using a vernier caliper, and calculated according to the following formula: Volume (mm 3 ) = width 2 x length x 0.5. The experimental results are shown in Figures 13-14 As shown, the OCN+Lamp group showed a stronger tumor growth inhibition effect compared with the control groups, suggesting that OCN has good in vivo anti-tumor potential under xenon lamp irradiation.

[0057] Example 5: Evaluation of in vivo anti-tumor effect of OCN under xenon lamp irradiation BALB / c mice (6 weeks old, male) were randomly divided into two groups (n =3), namely the Ctrl group and the OCN+Lamp group. Each mouse was subcutaneously inoculated with tumor cells (1x10 6cells / mouse). Intervention was initiated at day 10 post modeling, where Ctrl group was injected with saline subcutaneously, OCN+Lamp group received 2 mg / kg OCN intratumorally, and 2 h later the injection site was irradiated with xenon lamp (equipped with 420 nm filter, working current 20 A, irradiation for 10 min). Mice were sacrificed 12 h after the end of irradiation, tumor tissues were collected and sent for Bulk RNA-seq and mass cytometry analysis. Bulk RNA-seq and GO enrichment analysis results are shown in FIG. 6A and FIG. 6B. Figure 15 As shown, the expression level of ferroptosis-related genes in OCN+Lamp group was higher than that in Ctrl group, while the functional pathways such as protein refolding of heat shock protein family, positive regulation of MHC class I molecule biosynthesis, and regulation of leukocyte migration showed a significant enrichment trend. Previous studies have reported that the activation of heat shock protein family can enhance inflammation-related signaling and increase the expression level of MHC class I molecules, which may further promote antigen-specific recognition and enhance T cell-mediated tumor killing effect.

[0058] Mass cytometry detection results are shown in FIG. 7A and FIG. 7B. Figure 16 As shown, 23 cell subgroups with different immune characteristics were identified by high-dimensional cell phenotype analysis. t-SNE analysis showed that the tumor immune microenvironment of OCN+Lamp group changed significantly, with the proportion of C08 and C09 cell clusters increasing, suggesting that CD4 + T cells and CD8 + T cells were in an activated state, reflecting that their functions in proliferation, differentiation, and survival were enhanced. The ratio of cDC1 to cDC2 also increased, suggesting that the antigen presentation ability increased, further promoting the activation of CD8 + T cells. At the same time, the proportion of C23 cell cluster representing M2 type immunosuppressive macrophages decreased, showing that the tumor microenvironment was transformed towards an immune activated state.

[0059] According to the foregoing grouping and processing scheme, the mice were sacrificed and the tumor tissues were taken, digested, ground, filtered and washed after cracking red treatment, and the tumor single cell suspension was obtained by centrifugation at 400 g for 5 min, and then divided into two parts. One part of the tumor single cell suspension was added with PE / Cy7-labeled anti-mouse CD11b antibody and PE-labeled anti-mouse CD11c antibody, and stained for 30 min for evaluation of the recruitment of dendritic cells (DCs). The other part was used for T cell-related analysis: FITC-labeled CD45 antibody, PE / Cy7-labeled CD3 antibody, PerCP-Cy5.5-labeled CD8 antibody, BV711-labeled CD4 antibody and APC-labeled CD25 antibody were mixed and added to the single cell suspension, incubated for 40 min, then treated with a membrane breaking reagent for 1 h and washed, and then PE-conjugated anti-mouse Foxp3 flow cytometry antibody was added to the tumor single cell suspension after membrane breaking to stain for 30 min to detect the proportion of Tregs. The experimental results are shown in FIG. 9. Figure 17 It is shown that the level of DC recruitment in the tumor tissue of the OCN+Lamp group is increased, the infiltration of CD8 + T cells and CD4 + T cells is increased, and the proportion of Tregs is decreased, showing overall immune microenvironment characteristics more conducive to anti-tumor response.

[0060] Example 6: In vitro and in vivo toxicity evaluation of OCN Human umbilical vein endothelial cells HUVEC and three murine tumor cell lines 4T1, MC38, B16F10 were inoculated in 96-well plates, and after the cells grew to about 80% confluence, different concentrations of WCN, YCN or OCN (0, 25, 50, 75, 100, 200, 300, 400, 500 μg / mL) were added to the corresponding wells. After incubation at 37°C for 8 h, the cell viability was detected using a CCK-8 kit. As shown in FIG. 18, the material concentration of 300 μg / mL maintained a high cell viability in each cell line, indicating good in vitro biocompatibility. During the in vivo treatment in Example 4, the body weight of the mice remained stable (as shown in FIG. 19), and no significant fluctuations were observed, indicating that the material was well tolerated at the treatment dose. Figure 19

[0061] After the experiment, the heart, liver, spleen, lung and kidney of the mice in Example 4 were taken for pathological examination. The results of H&E staining are shown in FIG. 20. Figure 20 ​As shown, the material processing group is consistent with the normal tissue structure, and no obvious damage is observed, indicating that OCN has good safety in vivo. Fresh mouse peripheral blood was used to evaluate the hemolysis risk of the material. After centrifugation of mouse peripheral blood at 1500 rpm for 10 min, red blood cells were collected and prepared into a 2% red blood cell suspension with 1xPBS. The positive control group was 0.5 mL ddH2O mixed with 0.5 mL 2% red blood cell suspension; the negative control group was 0.5 mL PBS mixed with 0.5 mL red blood cell suspension; the experimental group mixed different concentrations of OCN (25, 50, 75, 100, 200, 300 μg / mL) 0.5 mL with 0.5 mL red blood cell suspension, and after 4 h incubation, it was added dropwise to the glass slide for optical microscope observation. As shown in FIG. 6A, the red blood cells in the negative control group were normal and no hemolysis was observed. As shown in FIG. 6B, the red blood cells in the 300 μg / mL OCN treatment group were similar to the negative control, and no hemolytic changes were observed, indicating that the material has good blood compatibility. Figure 21 As shown in FIG. 6B, the red blood cells in the 300 μg / mL OCN treatment group were similar to the negative control, and no hemolytic changes were observed, indicating that the material has good blood compatibility.

[0062] In summary, the present application provides a carbon nitride nanometer iron death inducer with light response characteristics and its preparation method and application, which is used to solve the technical problems of complex synthesis process of small molecule inducer, easy to produce off-target toxicity and tolerance in existing iron death induction technology, limited light absorption range of traditional photosensitizer, insufficient ROS quantum yield, wide band gap of conventional carbon nitride material, limited active site, and limited ROS generation efficiency and immune synergistic effect. The edge nitrogen vacancy modified carbon nitride (OCN) based on urea precursor constructed in the present application realizes wide spectrum light absorption up to 420-620 nm through band gap regulation (band gap reduced to 2.44eV) and amorphous structure design, and can efficiently generate ROS. OCN promotes the progress of lipid peroxidation, activates the heat shock protein pathway and drives the transformation of tumor immune microenvironment to immune activation state, thereby improving the efficiency of iron death induction and anti-tumor immune response. In addition, the preparation route based on urea precursor has the advantages of low cost, easy source, stable process and scalability, so that the light response type iron death inducer has good repeatability and translation potential, providing a standardized, low-cost and clinically translatable solution for iron death driven tumor treatment.

[0063] The above description is only the preferred embodiment of the present application, and the protection scope of the present application is not limited to the above examples. Any technical solutions falling within the concept of the present application shall be within the protection scope of the present application. It should be noted that, for ordinary skilled persons in the art, some improvements and refinements without departing from the principles of the present application shall also be considered as the protection scope of the present application.

Claims

1. A light-responsive carbon nitride nanoscale iron death inducer, characterized in that, The light-responsive carbon nitride nanosheet iron death inducer is a carbon nitride nanosheet with an edge nitrogen vacancy structure, which is prepared from a urea precursor by calcination, grinding and annealing treatment in an inert atmosphere, and can generate reactive oxygen species under 420-620 nm wavelength light to induce tumor cell ferroptosis.

2. The photoresponsive carbon nitride nanoparticle iron death inducer according to claim 1, characterized in that, The edge nitrogen vacancy structure makes the band gap of the carbon nitride nanosheet 2.40-2.50 eV. 3.The photo-responsive g-C3N4@FeN nanocomposite-induced death agent of claim 1, wherein, The thickness of the carbon nitride nanosheet is 1.0-1.5 nm.

4. The method for preparing a light-responsive carbon nitride nanoscale iron oxide death inducer according to any one of claims 1-3, characterized in that, The method comprises the following steps: performing first calcination with urea as a precursor to obtain a yellow carbon nitride precursor; introducing edge nitrogen vacancies by grinding and annealing treatment of the yellow carbon nitride precursor under inert gas protection; and performing ultrasonic crushing and centrifugal treatment on the annealed product to obtain a carbon nitride nanosheet.

5. The production method according to claim 4, characterized by, The calcination temperature is 550-650℃, and the time is 2-4 hours; the annealing temperature is 680-780℃, and the time is 1-10 minutes.

6. Use of the light-responsive carbon nitride nanosheet iron death inducer according to any one of claims 1-3 in the preparation of an antitumor drug.

7. Use according to claim 6, characterized in that, The antitumor drug is an injection or a topical gel.

8. A pharmaceutical composition, characterized by, The method comprises the light-responsive carbon nitride nanosheet iron death inducer according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition of claim 8, wherein, The pharmaceutical composition is irradiated by a light source with a wavelength of 420 nm and an intensity of 20A after administration to activate its iron death induction function. The light-responsive carbon nitride nanosheet iron death inducer according to any one of claims 1-3 and a pharmaceutically acceptable carrier.

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