A supramolecular hydrogel against osteosarcoma and inhibiting tumor angiogenesis and a preparation method thereof

Abstract

The application discloses an anti-osteosarcoma and anti-angiogenesis supramolecular hydrogel and a preparation method thereof. The supramolecular hydrogel is composed of chlorogenic acid, calcium chloride, rhein and sodium bicarbonate solution. The preparation method comprises the following steps: first, preparing chlorogenic acid and calcium chloride mother liquor respectively, and finally adding weighed rhein solution into the chlorogenic acid and calcium chloride mother liquor and ultrasonic dispersing, so as to obtain the chlorogenic acid-rhein-calcium chloride supramolecular hydrogel. The chlorogenic acid and calcium chloride are added to self-assemble into the supramolecular hydrogel with a small amount of rhein, so that the toxicity of a large dose of rhein hydrogel to normal cells is reduced. In the anti-tumor experiment, the supramolecular hydrogel can effectively kill osteosarcoma cells, inhibit new blood vessel formation, effectively prevent tumor growth and diffusion, and has the advantages of simple preparation method, low cost, strong practicability and good application prospect.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a supramolecular hydrogel that resists osteosarcoma and inhibits tumor angiogenesis, and its preparation method. Background Technology

[0002] Osteosarcoma is a typical malignant bone tumor, commonly seen in primary malignant bone tumors in adolescents under the age of 20. It has a high rate of local invasion and metastasis. Reports indicate that osteosarcoma has become the second leading cause of death among young cancer patients, especially in the lung metastasis stage. Clinical treatment for osteosarcoma primarily involves surgery and chemotherapy. However, surgical intervention usually cannot completely eliminate the tumor, leading to postoperative recurrence and metastatic tissue with a very high metastatic and recurrent nature. Furthermore, the severe systemic damage caused by radiotherapy and chemotherapy remains unresolved, potentially leading to disability or even death for osteosarcoma patients, thus inflicting a heavy blow and loss on society. Therefore, seeking an innovative and effective anti-tumor treatment method to reduce the toxic side effects of cancer treatment is an urgent problem to be solved.

[0003] In recent years, small molecules of traditional Chinese medicine extracted from natural herbs have been regarded as promising drug resources due to their broad pharmacological activities. In particular, supramolecular self-assembly has become a means of creating new substances and functional structures. Under certain conditions, self-assembly can form various functional structures such as gels, vesicles, micelles, and fibers. Compared with traditional polymer hydrogels, supramolecular hydrogels composed of natural herbs can serve as drug carriers and possess intrinsic pharmacological activity, avoiding the poor biocompatibility, biodegradability, and unexpected side effects caused by external carriers. However, designing hydrogels using small molecules from natural herbs requires high concentrations of these molecules. For example, the gelling concentration of rhein is 5 mg / mL, which inevitably leads to cytotoxicity at high self-assembly concentrations. Based on this situation, there is an urgent need to provide a method for preparing high-performance natural supramolecular hydrogels at relatively low concentrations. Therefore, this invention proposes a supramolecular hydrogel for anti-osteosarcoma and tumor angiogenesis inhibition, along with its preparation method, to address the problems existing in the prior art. Summary of the Invention

[0004] To address the aforementioned problems, the present invention aims to propose a supramolecular hydrogel for anti-osteosarcoma and inhibition of tumor angiogenesis, and a method for its preparation. This solves the problem in the prior art that the design of hydrogels using small molecules of natural herbs requires high concentrations of natural herbal molecules, which inevitably leads to cytotoxicity of the drug at high self-assembly concentrations.

[0005] To achieve the objectives of this invention, the invention is implemented through the following technical solution: a supramolecular hydrogel for anti-osteosarcoma and inhibition of tumor angiogenesis, comprising the following raw materials in parts by weight: 5 parts chlorogenic acid, 5 parts calcium chloride, 2 parts rhein, and 0.96 parts sodium bicarbonate, wherein the calcium chloride is anhydrous calcium chloride, the sodium bicarbonate solution has a mass concentration of 16.8 mg / mL, the calcium chloride mother liquor has a mass concentration of 5 mg / mL, and the rhein solution has a mass concentration of 2 mg / mL;

[0006] Weigh 5 mg of chlorogenic acid into a 1.5 mL centrifuge tube, add 100 µL of sodium bicarbonate solution (16.8 mg / mL), and let stand for 2-5 minutes to obtain a chlorogenic acid stock solution. Weigh 2 mg of rhein into a 1.5 mL centrifuge tube, add 860 µL of sodium bicarbonate solution (16.8 mg / mL), and simultaneously add 40 µL of the prepared chlorogenic acid stock solution. Weigh 5 mg of anhydrous calcium chloride and add 1 mL of sodium bicarbonate solution (16.8 mg / mL) to make the concentration 5 mg / mL. Add 100 µL of the calcium chloride stock solution to the prepared chlorogenic acid / rhein solution and continue ultrasonic dispersion for 3 minutes to obtain a chlorogenic acid@rhein-calcium chloride supramolecular hydrogel.

[0007] A method for preparing supramolecular hydrogels that inhibit osteosarcoma and tumor angiogenesis includes the following steps:

[0008] Step 1: Weigh 5 mg of chlorogenic acid and add it to a 1.5 ml centrifuge tube. Then weigh and add 100 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL. After standing, prepare chlorogenic acid stock solution for later use.

[0009] Step 2: Next, weigh 2 mg of rhein into a 1.5 ml centrifuge tube, then weigh and add 860 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL, and at the same time add 40 μL of chlorogenic acid stock solution to prepare chlorogenic acid@rhein solution for later use.

[0010] Step 3: Then weigh 5 mg of anhydrous calcium chloride into a 1.5 ml centrifuge tube, and then weigh and add 1000 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL to prepare calcium chloride mother liquor for later use. The mass concentration of the calcium chloride mother liquor is 5 mg / mL.

[0011] Step 4: Then, weigh 100 μL of calcium chloride mother liquor and add it to chlorogenic acid@rhein solution for continuous ultrasonic dispersion to obtain chlorogenic acid@rhein-calcium chloride supramolecular hydrogel. The ultrasonic dispersion time after adding chlorogenic acid@rhein solution to calcium chloride mother liquor is 3-5 min, the ultrasonic power is 80-100 W, and the ultrasonic temperature is 20-30℃.

[0012] A further improvement is that, in step one, the standing time after the sodium bicarbonate solution is added to the centrifuge tube is 2 to 5 minutes.

[0013] Application of supramolecular hydrogels that inhibit osteosarcoma and tumor angiogenesis in the preparation of drugs for treating osteosarcoma.

[0014] The beneficial effects of the present invention are as follows: The morphology of the supramolecular hydrogel observed under scanning electron microscopy is a fibrous structure. While retaining the drug activity of each component, it solves the shortcomings of poor bioavailability of chlorogenic acid and rhein, and further reduces the gelling concentration of rhein, so that it can self-assemble with chlorogenic acid and calcium chloride through non-covalent bonds to form a supramolecular gel at a lower concentration, which greatly reduces the toxicity of the drug to normal cells due to high concentration.

[0015] The chlorogenic acid@rhein-calcium chloride carrier-free supramolecular hydrogel prepared by this invention can alter the mitochondrial membrane potential of osteosarcoma cells, and can also destroy the actin of osteosarcoma cells and inhibit angiogenesis, thereby killing osteosarcoma cells and achieving the goal of effective treatment of osteosarcoma disease. Attached Figure Description

[0016] Figure 1 This is a scanning electron microscope (SEM) image of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0017] Figure 2 This is an X-ray photoelectron spectroscopy (XPS) image of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0018] Figure 3 This is a rheological analysis diagram of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0019] Figure 4 This is a gelation diagram of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0020] Figure 5 This is a gelation time diagram of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0021] Figure 6 This is a diagram showing the gelation process of different drug ratios of chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in an embodiment of the present invention.

[0022] Figure 7 This is a cell survival rate diagram of mouse osteosarcoma cells (A) and normal human liver cells (B) using chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention.

[0023] Figure 8 This is a hemolysis experiment diagram of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel in the embodiments of the present invention;

[0024] Figure 9 This is a fluorescence staining image of chlorogenic acid@rhein-calcium chloride supramolecular hydrogel altering the mitochondrial membrane potential of mouse osteosarcoma cells in an embodiment of the present invention.

[0025] Figure 10 This is a fluorescent staining image of chlorogenic acid@rhein-calcium chloride supramolecular hydrogel disrupting actin in mouse osteosarcoma cells in an embodiment of the present invention. Detailed Implementation

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. 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.

[0027] Example 1

[0028] This embodiment provides a supramolecular hydrogel that inhibits osteosarcoma and tumor angiogenesis, comprising the following raw materials in parts by weight: 5 parts chlorogenic acid, 5 parts anhydrous calcium chloride, 2 parts rhein, and 0.96 parts sodium bicarbonate with a mass concentration of 16.8 mg / mL. The calcium chloride is anhydrous calcium chloride, the sodium bicarbonate solution has a mass concentration of 16.8 mg / mL, the calcium chloride mother liquor has a mass concentration of 5 mg / mL, and the rhein solution has a mass concentration of 2 mg / mL.

[0029] A method for preparing supramolecular hydrogels that inhibit osteosarcoma and tumor angiogenesis includes the following steps:

[0030] Step 1: Weigh 5 mg of chlorogenic acid and add it to a 1.5 ml centrifuge tube. Then weigh and add 100 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL. Let it stand for 2-5 minutes to obtain chlorogenic acid stock solution for later use.

[0031] Step 2: Next, weigh 2 mg of rhein into a 1.5 ml centrifuge tube, then weigh and add 860 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL, and at the same time add 40 μL of chlorogenic acid stock solution to prepare chlorogenic acid@rhein solution for later use.

[0032] Step 3: Then weigh 5 mg of anhydrous calcium chloride into a 1.5 ml centrifuge tube, and then weigh and add 1000 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL to prepare a calcium chloride stock solution with a mass concentration of 5 mg / mL for later use.

[0033] Step 4: Then weigh 100 μL of calcium chloride mother liquor and add it to chlorogenic acid@rhein solution for continuous ultrasonic dispersion. The ultrasonic dispersion time is 3-5 min, the ultrasonic power is 80-100 W, and the ultrasonic temperature is 20-30℃ to obtain chlorogenic acid@rhein-calcium chloride supramolecular hydrogel.

[0034] A small amount of freeze-dried chlorogenic acid@rhein-calcium chloride supramolecular hydrogel was placed on a silicon wafer containing double-sided carbon conductive tape and then sputtered with gold. Figure 1 Scanning electron microscopy (SEM) image of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel prepared in this example. Figure 1 The morphology of the hydrogel can be observed to be a fibrous network structure.

[0035] The supramolecular hydrogel used in this embodiment to treat osteosarcoma and inhibit tumor angiogenesis is applied to the preparation of drugs for treating osteosarcoma.

[0036] Example 2

[0037] In this embodiment, the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel prepared in Example 1 was freeze-dried, and the energy, angle and other parameters of the X-ray beam were adjusted. The obtained photoelectron spectroscopy data were analyzed to obtain information on the elemental chemical state and surface composition.

[0038] XPS results are as follows Figure 2 As shown, C, O, Na, and Ca elements are present in the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel. The atomic contents of Na and Ca in the supramolecular hydrogel are approximately 10.33% and 0.36%, respectively.

[0039] Example 3

[0040] In this embodiment, to conduct rheological evaluation of the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel, a rotational rheometer was used to perform frequency scanning, strain scanning, and time scanning tests on the supramolecular hydrogel prepared in Example 1. Alternating strain scans were performed on the hydrogel at alternating strains of 1% and 100% with an alternating strain interval of 60 s.

[0041] like Figure 3 As shown in Part A, the strain-dependent oscillatory rheology of the supramolecular hydrogel exhibits excellent shear resistance. When the strain increases to 10%, the loss modulus (G”) and storage modulus (G’) intersect and then invert, indicating a transition from the gel state to the solution state (gel state: G’>G″, solution state: G’). <G″)。

[0042] like Figure 3Part B of the diagram shows the step strain test of the hydrogel. At low strain, the G′ value is higher than the G″ value (0.5%), while at high strain, the G′ value is lower than the G″ value (30%), indicating that this hydrogel has good self-healing properties.

[0043] like Figure 3 Part C in Figure 3 As shown in section D, the dynamic frequency sweep and dynamic time frequency sweep data show that the G′ value is much higher than the G″ value throughout the process, which means that the hydrogel has stable properties.

[0044] These results demonstrate that the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel possesses recoverable mechanical properties between high and low strain, exhibiting good self-healing ability and injectability.

[0045] Example 4

[0046] Weigh 2 mg of chlorogenic acid into a 1.5 mL centrifuge tube, add 1 mL of sodium bicarbonate solution (16.8 mg / mL), and sonicate for 3 minutes to obtain a chlorogenic acid solution.

[0047] Weigh 2 mg of chlorogenic acid into a 1.5 mL centrifuge tube, add 900 µL of sodium bicarbonate solution (16.8 mg / mL) and let stand for 2 minutes. Weigh 5 mg of anhydrous calcium chloride into a 1.5 mL centrifuge tube and prepare a 5 mg / mL calcium chloride stock solution using sodium bicarbonate solution (16.8 mg / mL). Add 100 µL of the calcium chloride stock solution to a solution containing 2 mg of chlorogenic acid and sonicate for 3 minutes to obtain a chlorogenic acid@calcium chloride solution.

[0048] Weigh 2 mg of rhein into a 1.5 mL centrifuge tube, and add 1 mL of sodium bicarbonate solution (

[0049] A 2 mg / mL rhein solution was prepared by ultrasonic dispersion for 3 minutes (16.8 mg / mL).

[0050] Weigh 2 mg of rhein into a 1.5 mL centrifuge tube, add 900 µL of sodium bicarbonate solution (16.8 mg / mL) to obtain rhein solution, take 100 µL of the 5 mg / mL calcium chloride solution from (2) and add it to the rhein solution, sonicate for 3 minutes to prepare rhein@calcium chloride solution.

[0051] Weigh 5 mg of chlorogenic acid into a 1.5 mL centrifuge tube, add 100 µL of sodium bicarbonate solution (16.8 mg / mL), mix well with a pipette and let stand for 2 minutes. Weigh 2 mg of rhein into a 1.5 mL centrifuge tube, add 960 µL of sodium bicarbonate solution (16.8 mg / mL), then add 40 µL of chlorogenic acid solution, and sonicate for 3 minutes to obtain a chlorogenic acid@rhein solution.

[0052] Weigh 5 mg of chlorogenic acid into a 1.5 mL centrifuge tube, add 100 µL of sodium bicarbonate solution (16.8 mg / mL), and let stand for 2-5 minutes to obtain chlorogenic acid stock solution. Weigh 2 mg of rhein into a 1.5 mL centrifuge tube, add 860 µL of sodium bicarbonate solution (16.8 mg / mL), and simultaneously add 40 µL of the prepared chlorogenic acid stock solution. Weigh 5 mg of anhydrous calcium chloride and add 1 mL of sodium bicarbonate solution (16.8 mg / mL) to make the concentration 5 mg / mL. Add 100 µL of calcium chloride stock solution to the prepared chlorogenic acid / rhein solution and continue ultrasonic dispersion for 3 minutes to obtain chlorogenic acid@rhein-calcium chloride supramolecular hydrogel.

[0053] Images of different component solutions and the appearance of chlorogenic acid@rhein-calcium chloride supramolecular hydrogel: From Figure 4 As can be seen, chlorogenic acid (2 mg / mL) and chlorogenic acid@calcium chloride (2 mg / mL@0.5 mg / mL) are in solution state and are difficult to form gel; 2 mg / mL rhein is in emulsion state; rhein@calcium chloride (2 mg / mL@0.5 mg / mL) has a small amount of gel formation, but the gel is loose and not dense; chlorogenic acid@rhein (2 mg / mL@2 mg / mL) is a transparent solution; chlorogenic acid@rhein-calcium chloride (2 mg / mL@2 mg / mL-0.5 mg / mL) is sonicated to obtain a supramolecular hydrogel.

[0054] The chlorogenic acid@rhein-calcium chloride supramolecular hydrogel prepared according to Example 1 above exhibits the following appearance after ultrasonication at different times: Figure 5 As shown, after 1 minute of ultrasonic dispersion, some of the gel did not form and flowed down the centrifuge tube wall; after 2 minutes of ultrasonic dispersion, almost a gel was formed, reaching the critical gel state; after 3 minutes of ultrasonic dispersion, a complete gel was formed, and the texture was uniformly dispersed, with no liquid flowing down. This indicates that the ultrasonic dispersion time directly affects the formation of the gel.

[0055] According to the preparation method in Example 1, following... Figure 6 The gel formation was observed by using different concentrations of the components shown. Figure 6 It can be observed that, with chlorogenic acid concentration as the variable, gelation begins at a concentration of 2 mg / mL; with the concentrations of chlorogenic acid (2 mg / mL) and calcium chloride (2 mg / mL) fixed, gelation is difficult when the concentration of rhein is between 0.5 and 1 mg / mL, but a reddish-brown gel can be formed at a concentration of 2 mg / mL. As the concentration of rhein gradually increases, the gel color changes to orange-yellow; with calcium chloride concentration as the variable, the critical gelation concentration is 0.4 mg / mL, and a complete gel is formed at a concentration of 0.5 mg / mL.

[0056] This example demonstrates the survival rate detection of mouse osteosarcoma cells (K7M2 cell line).

[0057] First, K7M2 cells in the logarithmic growth phase were digested with trypsin, and the cell suspension was collected and centrifuged (1000 rpm, 3 minutes). The supernatant was discarded, and the cells were resuspended in fresh complete culture medium and plated. At this point, the cell confluence in each well was 70%. After the cells adhered, the medium was changed, and different concentrations of hydrogel were added to make the final concentrations 50 µg / mL, 100 µg / mL, 200 µg / mL, and 300 µg / mL, respectively. After 12 h of treatment, the drug-containing culture medium was discarded, and 100 µL of cck8 solution (10%) was added to each well. The cells were incubated at 37 °C for 1.5 hours, and the absorbance was measured to calculate the viability of K7M2 cells.

[0058] Cytotoxicity assay of chlorogenic acid@rhein-calcium chloride supramolecular hydrogel

[0059] First, human normal hepatocytes (QSG-7701 cell line) in the logarithmic growth phase were digested with trypsin, and the cell suspension was collected and centrifuged (1000 rpm, 3 minutes). The supernatant was discarded, and the cells were resuspended in fresh complete culture medium and plated, ensuring that the cell confluence in each well was 70%. After the cells adhered, the medium was changed, and different concentrations of hydrogel were added to make the final concentrations 50 µg / mL, 100 µg / mL, 150 µg / mL, and 200 µg / mL, respectively. After treatment for 24 h, the drug-containing culture medium was discarded, and 100 µL of CCK8 solution (10%) was added to each well. The cells were incubated at 37 °C for 1.5 hours, and the absorbance was measured to calculate the QSG-7701 cell viability.

[0060] Figure 7 Part A of the graph shows the concentration-viability profile of K7M2 cells treated with hydrogel. The graph indicates that hydrogel significantly inhibits K7M2 cell growth with increasing concentration. At a hydrogel concentration of 50 µg / mL, the cell viability was approximately 47%; at a concentration of 300 µg / mL, the cell viability was approximately 1%.

[0061] Figure 7 Part B is a concentration-survival graph of QSG-7701 cells treated with hydrogel. As can be seen from the graph, as the concentration increases, the hydrogel does not cause significant cytotoxicity to QSG-7701 cells, indicating that chlorogenic acid@rhein-calcium chloride supramolecular hydrogel can significantly inhibit K7M2 cells without harming normal cells.

[0062] Example 5

[0063] This embodiment conducts a hemolysis experiment using chlorogenic acid@rhein-calcium chloride supramolecular hydrogel.

[0064] Blood was first collected from the eyes of healthy Balb / c mice. The fresh blood was added to anticoagulant tubes and centrifuged at 1000 rpm for 10 minutes, discarding the serum. Subsequently, red blood cells were collected by washing (0.9% NaCl) and centrifuging (1500 rpm, 5 minutes). The red blood cells were diluted to a concentration of 5% with PBS and added at a 1:1 volume ratio to 1.5 mL centrifuge tubes containing hydrogels of different concentrations. Finally, after incubation at 37°C for 1 hour, 200 µL of the supernatant from each group was transferred to a 96-well plate. Red blood cells in PBS and 2% Triton X-100 were used as negative and positive controls, respectively. The absorbance at 542 nm was measured, and the hemolysis rate was calculated. The calculation formula is as follows:

[0065] Hemolysis rate (%) = [(OD)] 样品 -OD 阴性 ) / (OD 阳性 -OD 阴性 )]×100%

[0066] from Figure 8 As shown in Part A, the 2% Triton X-100 treatment group was clear and showed no precipitation, indicating that 2% Triton X-100 can alter the osmotic pressure of red blood cells, thereby causing red blood cell rupture. In contrast, both the hydrogel and PBS groups showed precipitation of red blood cells, indicating that the hydrogel did not cause red blood cell rupture. To more clearly understand the hemolysis situation in the hydrogel groups, the hemolysis rate was calculated from the supernatant of each group, and the results are as follows: Figure 8 As shown in Part B, the hemolysis rate of the 2% Triton X-100 group was 100%, indicating that the red blood cells were completely destroyed. When the hydrogel concentration was 50 µg / mL, the hemolysis rate was about 0.5%, and when the concentration was increased to 200 µg / mL, the hemolysis rate was only 4%, indicating that the chlorogenic acid@rhein-calcium chloride supramolecular hydrogel had a very low effect on the osmotic pressure of red blood cells and had good biocompatibility.

[0067] Example 6

[0068] This embodiment involves monitoring the mitochondrial membrane potential of K7M2 cells.

[0069] First, K7M2 cells with 80% confluence were passaged into confocal dishes, with 1×10⁶ cells per dish. 5 Cells were cultured for 24 hours. They were then treated with single-component drugs and hydrogels, respectively, and cultured for another 12 hours. After washing twice with PBS and staining with JC-1 (a fluorescent lipophilic carbonyl cyanide dye), fluorescence images were obtained using confocal microscopy. Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) was used as a positive control. The final concentrations of CaCl2, chlorogenic acid, rhein, and hydrogel were 50 µg / mL, 200 µg / mL, 200 µg / mL, and 200 µg / mL, respectively.

[0070] Ca 2+ Overload plays a crucial role in apoptosis and cell death. For example... Figure 9 As shown, compared with the control group, the red fluorescence weakened while the green fluorescence increased after hydrogel treatment, similar to the positive control drug CCCP. This is due to Ca... 2+ Overload can directly induce strong oxidative stress in the mitochondria of K7M2 cells, leading to a decrease in membrane potential and consequently causing damage to the mitochondria.

[0071] Example 7

[0072] This embodiment demonstrates the regulation of the F-actin cytoskeleton by chlorogenic acid@rhein-calcium chloride supramolecular hydrogel.

[0073] First, K7M2 cells with 80% confluence were passaged into confocal dishes, with 1×10⁶ cells per dish. 5 Cells were cultured for 24 hours. They were then co-incubated with single-component drugs and hydrogels for 12 hours, washed twice with PBS, fixed with 4% paraformaldehyde for 15 minutes, and then permeated with 0.1% Triton X-100 for 15 minutes. The F-actin cytoskeleton was stained with rhodamine-labeled phalloidin at room temperature for 20 minutes, washed twice with PBS, and the nuclei were stained and localized using Hoechst. Images were acquired using a laser scanning confocal microscope.

[0074] Actin is involved in regulating tumor cell invasion and metastasis. From... Figure 10 The confocal images show that the actin fibers in the control group cells are strong and straight, while the number of actin fibers in the cells treated with 200 μg / mL hydrogel is significantly reduced compared to the control group. This indicates that chlorogenic acid@rhein-calcium chloride supramolecular hydrogel can effectively regulate the F-actin cytoskeleton of K7M2 cells, thereby reducing the risk of cancer metastasis.

[0075] Example 8

[0076] This embodiment demonstrates an experiment on chlorogenic acid@rhein-calcium chloride supramolecular hydrogel-mediated tube formation of vascular endothelial cells (HUVEC cell line), as detailed below:

[0077] (1) First, K7M2 cells were passaged to 24-well plates, 1×10⁻⁶ cells per well. 5 Cells were incubated with single-component drugs and hydrogels for 12 hours, respectively. The supernatant was discarded, and the cells were washed twice with PBS to remove the drug. Fresh complete culture medium and K7M2 were added and incubated for another 6 hours. The cell supernatant was then collected for later use.

[0078] (2) Add 200 μL of Matrigel basement membrane matrix to each well of a 24-well plate and incubate at 37°C for 1 hour. Then resuspend HUVEC cells in culture medium containing K7M2 cell supernatant and seed 3 × 10⁶ cells per well. 4 HUVEC cells were cultured in 24-well plates at 37°C for 4 hours, stained with calcein, and the tubular structures were photographed under a fluorescence microscope.

[0079] Tumor growth and metastasis are accompanied by the formation of new blood vessels (tumor angiogenesis). By inhibiting tumor angiogenesis, tumor growth and spread can be effectively prevented, indicating that chlorogenic acid@rhein-calcium chloride supramolecular hydrogel has the potential to inhibit angiogenesis in an in vitro tumor-vascular endothelial cell co-culture model.

[0080] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Supramolecular hydrogel against osteosarcoma and inhibiting tumor angiogenesis, characterized by, The method comprises the following raw materials by weight: 5 parts of chlorogenic acid, 5 parts of calcium chloride, 2 parts of rhein and 0.96 parts of sodium bicarbonate, wherein the calcium chloride is anhydrous calcium chloride, the mass concentration of the sodium bicarbonate solution is 16.8 mg / mL, the mass concentration of the calcium chloride mother liquor is 5 mg / mL, and the mass concentration of the rhein solution is 2 mg / mL. 5 mg of chlorogenic acid is weighed into a 1.5 mL centrifuge tube, 100 μL of sodium bicarbonate solution is added, and the mixture is allowed to stand for 2-5 minutes to obtain a chlorogenic acid mother liquor. 2 mg of rhein is weighed into a 1.5 mL centrifuge tube, 860 μL of sodium bicarbonate solution is added, and 40 μL of the prepared chlorogenic acid mother liquor is added. 5 mg of anhydrous calcium chloride is weighed into a 1 mL centrifuge tube, and 1 mL of sodium bicarbonate solution is added to make the concentration 5 mg / mL. 100 μL of the calcium chloride mother liquor is added to the prepared chlorogenic acid / rhein solution, and ultrasonic dispersion is continued for 3 minutes to obtain a chlorogenic acid / rhein-calcium chloride supramolecular hydrogel.

2. A method for preparing an antiosseous sarcoma and tumor angiogenesis inhibiting supramolecular hydrogel, characterized by, The method comprises the following steps: Step one: 5 mg of chlorogenic acid is weighed into a 1.5 mL centrifuge tube, 100 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL is added, and the mixture is allowed to stand to obtain a chlorogenic acid mother liquor for standby use. Step two: 2 mg of rhein is weighed into a 1.5 mL centrifuge tube, 860 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL is added, and 40 μL of the chlorogenic acid mother liquor is added to obtain a chlorogenic acid / rhein solution for standby use. Step three: 5 mg of anhydrous calcium chloride is weighed into a 1.5 mL centrifuge tube, and 1000 μL of sodium bicarbonate solution with a mass concentration of 16.8 mg / mL is added to obtain a calcium chloride mother liquor for standby use, wherein the mass concentration of the calcium chloride mother liquor is 5 mg / mL. Step four: 100 μL of the calcium chloride mother liquor is added to the chlorogenic acid / rhein solution for ultrasonic dispersion, and a chlorogenic acid / rhein-calcium chloride supramolecular hydrogel is obtained, wherein the ultrasonic dispersion time of the calcium chloride mother liquor after being added to the chlorogenic acid / rhein solution is 3-5 minutes, the ultrasonic power is 80-100 W, and the ultrasonic temperature is 20-30°C.

3. The method of preparing an anti-osteosarcoma and anti-angiogenic supramolecular hydrogel according to claim 2, characterized in that: In step one, the standing time of the sodium bicarbonate solution after being added to the centrifuge tube is 2-5 minutes.

4. Use of the supramolecular hydrogel for resisting osteosarcoma and inhibiting tumor angiogenesis according to claim 1 in the preparation of a drug for treating osteosarcoma.

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