Polymer hittorf red phosphorus heterostructure and application thereof in preparation of medicine for targeted treatment of renal clear cell carcinoma

Abstract

The application provides a polymer carbon nitride@Hittorf red phosphorus heterostructure and application thereof in preparation of a medicine for targeted treatment of renal clear cell carcinoma, and belongs to the technical field of nano medicines. The application designs a near-infrared light response Hittorf red phosphorus (HP) modified polymer carbon nitride heterostructure (PCN@HP) based on a simple chemical vapor deposition (CVD) method. Experimental research finds that the HP nanorods vertically grown on the PCN exhibit wide and strong light absorption in the near-infrared range. In-vivo and in-vitro experiments prove that under near-infrared light irradiation, when the concentration of the suspension is 100 μg / mL, the tumor inhibition rate reaches 100%. Notably, the composite has no damage to normal tissues and cells, but it has obvious targeted inhibition effect on deep ccRCC cells in the body. In addition, the composite material has good biocompatibility and spontaneous light effect, and can be completely metabolized in the body within 48 hours, and is a kind of nanoparticle with imaging and PDT / PTT synergistic treatment, and can be used for efficient tumor nano treatment.

Description

Technical Field

[0001] This invention belongs to the field of nanomedicine technology, and in particular relates to a polymer carbon nitride@Hittorf red phosphorus heterostructure and its application in the preparation of drugs for targeted treatment of clear cell renal cell carcinoma. Background Technology

[0002] Renal cell carcinoma (RCCs) is one of the ten most common cancers and the most common family of kidney tumors. Among all its subtypes, clear cell renal cell carcinoma (ccRCC) is the most common sporadic type of renal cell carcinoma, accounting for approximately 75% of all cases, and is frequently associated with malignant disease progression and poor treatment outcomes. Currently, although some advanced strategies have been developed for the treatment of RCC, such as immune checkpoint blockade therapy and combination therapy regimens, the inevitable toxicity and drug resistance of newly approved targeted drugs limit treatment options for advanced disease. At present, surgical resection or ablation remains the preferred treatment for localized ccRCC, but clinical treatment costs are high and patient prognosis is poor, thus urgently requiring research into targeted therapies.

[0003] The application of nanotechnology offers an effective option for improving tumor treatment outcomes. Photothermal and photodynamic therapies, due to their inherent advantages of low toxicity, minimal side effects, lack of drug resistance, and repeatability, are increasingly being used to treat various cancers. Although previous studies have reported on the treatment of clear cell renal cell carcinoma (ccRCC) based on photothermal and photodynamic therapy, their clinical application is limited by the high cost of required materials, difficulties in synthesis, insufficient depth of treatment for solid tumors, unclear metabolic status, or the potential toxicity of materials accumulating in vivo. However, the treatment prospects for ccRCC are not optimistic, making the development of new and more effective treatment methods urgent. Summary of the Invention

[0004] This invention provides a polymeric carbon nitride@Hittorf red phosphorus heterostructure and its application in the preparation of drugs for targeted therapy of clear cell renal cell carcinoma. This heterostructure exhibits broad and strong light absorption in the near-infrared range, and has a significant targeted inhibitory effect on deep ccRCC cells in vivo. Furthermore, this heterostructure has good biocompatibility and self-luminescence effect, and can be completely metabolized in vivo within 48 hours. It is a nanoparticle that combines imaging and PDT / PTT synergistic therapy, and can be used for highly efficient tumor nanotherapy.

[0005] To achieve the above objectives, the present invention provides the application of a polymeric carbon nitride@Hittorf red phosphorus heterostructure in the preparation of a drug for treating clear cell renal cell carcinoma.

[0006] Preferably, the polymeric carbon nitride@Hittorf red phosphorus heterostructure is a composite material prepared by vapor deposition, and its structural formula is PCN@HP.

[0007] Preferably, the composite material is prepared by the following method:

[0008] Polymer carbon nitride (PCN) was prepared by heating melamine to 550°C for 4 hours.

[0009] Grind PCN and amorphous red phosphorus powder together in a mortar at a ratio of 2:1.

[0010] The resulting powder was then transferred to a quartz ampoule and sealed with an oxygen-hydrogen flame under a low vacuum of -0.09 MPa.

[0011] The ampoules were heated to 550°C in an oven at 450°C at a rate of 2°C / min and held at that temperature for 4 hours. Then, they were cooled to 280°C at a rate of 1°C / min. When the temperature dropped to 280°C, the cooling rate was slowed down to 0.1°C / min and then slowly cooled to room temperature at this rate. The capsules were then crushed and washed with CS2, ethanol and distilled water, respectively, to obtain the PCN@HP heterostructure.

[0012] Preferably, in the heterostructure, HP is vertically anchored on PCN, with a length of 1-2 μm and a diameter of 40-200 nm.

[0013] Preferably, after irradiation with near-infrared light at 808 nm for 10 minutes, the PCN@HP suspension at a dose of 60 μg / mL showed an inhibition rate of over 50% against ccRCC tumors, and the tumor inhibition rate reached 100% when the suspension concentration was 100 μg / mL.

[0014] Compared with the prior art, the advantages and positive effects of the present invention are as follows:

[0015] This invention presents a near-infrared light-responsive Hittorf red phosphorus (HP)-modified polymer carbon nitride heterostructure (PCN@HP) designed using a simple chemical vapor deposition (CVD) method. Experimental studies revealed that HP nanorods vertically grown on PCN exhibit broad and strong light absorption in the near-infrared range. In vitro and in vivo experiments confirmed that, under near-infrared light irradiation, a tumor inhibition rate of 100% was achieved at a suspension concentration of 100 μg / mL. Notably, this composite material does not damage normal tissues and cells, but it exhibits significant targeted inhibitory effects on deep ccRCC cells in vivo. Furthermore, this composite material possesses good biocompatibility and self-luminescence properties, and is completely metabolized in vivo within 48 hours. It is a nanoparticle that combines imaging and PDT / PTT synergistic therapy, and can be used for highly efficient tumor nanotherapy. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the PCN@HP synthesis steps provided in an embodiment of the present invention;

[0017] Figure 2 Characterization of PCN@HP provided in embodiments of the present invention;

[0018] Figure 3 (a) The killing effect of PCN@HP on ccRCC: Under near-infrared light irradiation, the PCN@HP suspension at a dose of 60 μg / mL showed a tumor inhibition rate of over 50% against ccRCC, and at a suspension concentration of 100 μg / mL, the tumor inhibition rate reached 100%; (b) PCN@HP had no damaging effect on normal renal tubular epithelial cells HK-2; (c) Conventional antitumor drugs had the same killing effect on both ccRCC and HK-2 cells.

[0019] Figure 4 The left image shows PCN@HP treatment of ccRCC before the tumor; the right image shows a tumor with a volume of 1 cm. 3 Three hours after injection of PCN@HP into ccRCC mice, irradiation with near-infrared light for 10 minutes resulted in rapid flattening and shrinkage of the tumors, demonstrating a good tumor cell killing effect.

[0020] Figure 5 This is a metabolic diagram of PCN@HP injection provided in an embodiment of the present invention;

[0021] Figure 6 The graphs showing the changes in biochemical indicators of blood and organ samples after injection of PCN@HP suspension provided in this embodiment of the invention are as follows: (a) Scr; (b) BUN; (c) ALT; (d) AST. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Example 1: Synthesis of PCN@HP

[0024] like Figure 1 As shown, PCN@HP heterostructure composite materials were prepared using the classic chemical vapor deposition (CVD) method. The synthesis process is as follows:

[0025] The PCN preparation method is as follows:

[0026] Melamine was prepared in a muffle furnace by heating at a rate of 2 °C / min for 4 hours from room temperature to 550 °C.

[0027] PCN and amorphous red phosphorus powder (purchased from Alpha Corporation, commercial red phosphorus, purity 99.999%) were ground together in a mortar at a ratio of 2:1.

[0028] The obtained powder was then transferred to a quartz ampoule and sealed with an oxygen-hydrogen flame under low vacuum conditions (-0.09 MPa, with high-purity Ar added before evacuation).

[0029] The ampoules were heated to 550°C in a furnace at a rate of 2°C / min and held at that temperature for 4 hours. Then, they were cooled to 280°C at a rate of 1°C / min. When the temperature dropped to 280°C, the cooling rate was slowed down to 0.1°C / min and then slowly cooled to room temperature at this rate. The capsules were then crushed and rinsed with CS2, ethanol and distilled water, respectively, to obtain the PCN@HP heterostructure composite material.

[0030] Example 2 Characterization of PCN@HP mixture

[0031] The morphology and structure of the prepared samples were determined using X-ray diffraction (xRD), ultraviolet-visible spectroscopy, Raman spectroscopy, scanning electron microscopy (SEM, FEIMagellan 400), and transmission electron microscopy (TEM, JEOL JEM-2100F). The xRD conditions were CuKα radiation. 40kV, 30mA; Raman spectroscopy data acquisition excitation wavelength is 532nm; transmission electron microscope (TEM) and scanning electron microscope (SEM) voltage is 200kV.

[0032] like Figure 2 As shown in Figure a, the phase composition of PCN@HP was determined by XRD analysis. PCN@HP exhibited two distinct PCN diffraction peaks at 13.3° and 27.7°, and additional HP diffraction peaks at 15.4° and 34.2°; consistent with the XRD results, the Raman spectroscopy results (…) Figure 2 b) shows that PCN@HP exhibits characteristic peaks of both PCN and HP; the UV-Vis spectrum ( Figure 2 c) This shows that PCN@HP exhibits strong light absorption in the visible and NIR regions. SEM ( Figure 2 d) and TEM Figure 2 e, f) are used to evaluate the morphology and surface characteristics of the prepared samples. As shown in the figure, HP is vertically anchored on PCN with a length of 1-2 μm and a diameter of tens of nanometers. In addition, the

[110] crystal plane of HP is located in a lattice stripe with a spacing of 0.275 nm.

[0033] Example 3: Detection of the anti-tumor effect of PCN@HP

[0034] Human renal tubular epithelial cells (HK-2) and human ccRCC cells (786-O) were obtained from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. HK-2 cells were cultured in DMEM / F-12 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (100 U / mL penicillin, 0.1 mg / mL streptomycin), while 786-O cells were cultured in RPMI 1640 medium with the same concentration of fetal bovine serum and penicillin-streptomycin. Both cell types were cultured in a humidified environment at 37°C and 5% CO2. HK-2 and 786-O cells were cultured in 96-well plates at a density of 1 × 10⁻⁶ cells / well. 4 Cells were cultured at a density of 10 cells / well. Once the cell density reached 70%, the cells were treated with different concentrations (0, 10, 20, 60, 80, and 100 μg / mL) of PCN@HP and cultured for another 24 hours. Subsequently, the cells were irradiated with a near-infrared (NIR) laser at 808 nm for 10 minutes under dark conditions. Afterward, CCK-8 solution was added to the wells and the cells were incubated at 37°C for 1.5 hours, at which point relative cell viability was measured at 450 nm.

[0035] like Figure 3 As shown in Figure a, under near-infrared light irradiation, the PCN@HP suspension at a dose of 60 μg / mL exhibited an inhibition rate of over 50% against ccRCC tumors, and at a suspension concentration of 100 μg / mL, the tumor inhibition rate reached 100%. However, as... Figure 3 As shown in b, under the same conditions, the survival rate of HK-2 cells remained unchanged. It is noteworthy that, as... Figure 3 As shown in c, although DOX, as a widely used anti-tumor drug in clinical practice, also exhibits strong tumor-killing ability, it also caused significant death of HK-2 cells at the same concentration compared with PCH@HP, which highlights the superior safety and clinical application potential of PCN@HP.

[0036] Example 4: Detection of PCN@HP in vivo antitumor effect

[0037] Male nude mice (4-5 weeks old, 18-20g) were purchased from Beijing Vital River Co., Ltd. They were housed in individually ventilated cages at a room temperature of 21℃±2℃ and a relative humidity of 50%±15%. Food and water were readily available. After one week of acclimatization, 1×10⁻⁶ mice were introduced into the rearing tank. 6 786-O cells resuspended in RPMI 1640 medium were mixed with Matrigel at a 1:1 ratio and injected into the subcutaneous tissue of the axilla of male nude mice. When the tumor volume reached 100 mm², the mixture was injected. 3Mice were randomly divided into four groups (n=5 per group): PBS, NIR, PCN@HP, and PCN@HP-NIR groups. The corresponding fluid was injected intratumorally at a concentration of 1 mg / mL (100 μL) to treat the tumor. Three hours later, the tumor site was treated with an 808 nm laser (2.0 W / cm²). 2 Irradiate for 10 minutes and record the size of the mouse tumor.

[0038] like Figure 4 As shown, the tumor shrank immediately after PCN@HP-NIR treatment, indicating that PCN@HP-NIR has a strong tumor growth inhibition effect.

[0039] Example 5: In vivo metabolism of PCN@HP after injection

[0040] The biodistribution and metabolism of PCN@HP were investigated using the IVIS Spectrum system. Fluorescence imaging of major organs in mice was performed 30 min, 1 h, 2 h, 3 h, 4 h, 24 h, and 48 h after tail vein injection of 100 μL of PCN@HP suspension at a concentration of 1 mg / mL to record PCN@HP metabolism.

[0041] like Figure 5 As shown, PCN@HP administered intravenously is mainly metabolized through the intestines. Based on the changes in fluorescence intensity, it can be seen that excretion begins 2 hours after injection, peaks at 3 hours, and is completely metabolized at 48 hours. This indicates that PCN@HP nanoparticles do not pose a risk of accumulation in the body and have potential clinical application value.

[0042] Example 6: Biosafety Testing of PCN@HP

[0043] In vivo toxicity studies: Liver and kidney function were assessed in 50 C57BL6J mice (25 males and 25 females) via intraperitoneal injection of PCN@HP suspension (1 mg / mL, 200 μL). These mice were randomly assigned to five groups, each containing 5 males and 5 females. Ten untreated mice served as a control group. Blood and organ samples were collected at 1, 3, 7, 14, and 28 days post-injection of PCN@HP suspension for biochemical analysis.

[0044] like Figure 6 As shown in the ad, basic indicators of renal function (Scr, BUN) and key biomarkers of liver function (AST, ALT) did not change significantly over time compared with the control group. These findings suggest the biocompatibility of PCN@HP treatment and highlight its potential for clinical application.

Claims

1. Application of polymeric carbon nitride@Hittorf red phosphorus heterostructure in the preparation of drugs for targeted therapy of clear cell renal cell carcinoma.

2. The application according to claim 1, characterized in that, The polymer carbon nitride@Hittorf red phosphorus heterostructure is a composite material prepared by vapor deposition, and its structural formula is PCN@HP.

3. The application according to claim 2, characterized in that, The composite material was prepared by the following method: Polymer carbon nitride (PCN) was prepared by heating melamine to 550°C for 4 hours. Grind PCN and amorphous red phosphorus powder together in a mortar at a ratio of 2:

1. The resulting powder was then transferred to a quartz ampoule and sealed with an oxygen-hydrogen flame under a low vacuum of -0.09 MPa. The ampoules were heated to 550°C in an oven at 450°C at a rate of 2°C / min and held at that temperature for 4 hours. Then, they were cooled to 280°C at a rate of 1°C / min. When the temperature dropped to 280°C, the cooling rate was slowed down to 0.1°C / min and then slowly cooled to room temperature at this rate. The capsules were then crushed and washed with CS2, ethanol and distilled water, respectively, to obtain the PCN@HP heterostructure.

4. The application according to any one of claims 1-3, characterized in that, In the heterostructure, HP is vertically anchored on PCN, with a length of 1–2 μm and a diameter of 40–200 nm.

Images