Sustained release lbq657 hydrogel for treating fibrosis and methods of making same

Abstract

The application discloses a sustained-release LBQ657 hydrogel capable of treating fibrosis and a preparation method thereof, and belongs to the technical field of material synthesis and biological medicine. The sustained-release LBQ657 hydrogel comprises chitosan, beta-glycerophosphoric acid sodium, distilled water and LBQ657. The w / v concentration of the chitosan solution used for configuration of the hydrogel is 3.3%, the w / v concentration of the beta-glycerophosphoric acid sodium aqueous solution is 56%, and the volume ratio of the chitosan solution to the beta-glycerophosphoric acid sodium solution is 4:1-10:1. The sustained-release LBQ657 hydrogel can quickly form a stable gel and is well adhered to the esophagus after being covered on the surface of the esophagus, can provide a protection for at least 7 days under physiological conditions, can significantly relieve TGF-beta induced fibrosis, and can provide a new idea for esophageal fibrosis treatment by covering the wound and long-term drug release.

Description

Technical Field

[0001] This invention belongs to the fields of materials synthesis and biomedicine, specifically relating to a sustained-release LBQ657 hydrogel for treating fibrosis and its preparation method. Background Technology

[0002] Esophageal cancer originates from the esophageal mucosal epithelium and is one of the most common malignant tumors of the digestive tract. Early and effective diagnosis and treatment of esophageal cancer are crucial measures to reduce its mortality rate. For superficial esophageal cancer that has invaded the submucosa, endoscopic submucosal dissection (ESD) is an important method for clinical diagnosis and treatment. However, post-ESD esophageal stricture is a major challenge that seriously affects patient prognosis and quality of life. Reducing fibrosis of esophageal fibroblasts in the early stages is a key strategy to prevent stricture formation in this hollow organ.

[0003] Transient receptor potential (TRPM7) is an important target for alleviating pathological fibrosis. As a bifunctional protein with both kinase and ion channel structures, TRPM7 is an important calcium channel. 2+ One of the channels, especially for Ca in fibroblasts 2+ Influx plays a crucial regulatory role. Existing research data confirms that TRPM channels can regulate hypoxia-induced cardiac fibrosis. Recent studies have found that LBQ657, a metabolite of sacubitril / valsartan—a recommended drug for treating heart failure—can bind to the TRPM7 ion channel protein, thereby effectively inhibiting TRPM7-mediated calcium channel blockade on cardiac fibroblasts. 2+ Influx of toxins reduces fibroblast activation. Therefore, this study hypothesizes that the small molecule compound LBQ657 reduces muscle layer fibrosis after ESD for superficial esophageal cancer by inhibiting the function of TRPM7 channels in fibroblasts. Several studies have reported that muscle layer fibrosis and esophageal scarring are the main causes of esophageal stricture during esophageal mucosal healing after ESD. Fibrosis is a type of scar; after surgical healing, the scar tissue contracts, ultimately causing luminal narrowing due to the unique structure of the esophagus. Currently, there is no effective treatment to block or reverse the development of fibrosis. Summary of the Invention

[0004] The purpose of this invention is to provide a sustained-release LBQ657 hydrogel for treating fibrosis and its preparation method. After being applied to the surface of the esophagus, it quickly forms a stable gel and adheres well to the esophagus. After ESD, it can be administered via endoscopic spraying to directly spray the drug onto the esophageal wound, providing long-term sustained drug release while protecting the wound.

[0005] The objective of this invention is achieved through the following technical solution:

[0006] A sustained-release LBQ657 hydrogel for treating fibrosis comprises chitosan, sodium β-glycerophosphate, distilled water, and LBQ657 ((2R,4S)-5-(biphenyl-4-yl)-4-((3-carboxypropionyl)amino)-2-methylvaleric acid), with the following structural formula I:

[0007]

[0008] The ratio of the chitosan solution to the sodium β-glycerophosphate solution is 4:1 to 10:1.

[0009] Preferably, the ratio of chitosan solution to sodium β-glycerophosphate solution is 8:1.

[0010] The present invention also provides a method for preparing the above-mentioned sustained-release LBQ657 hydrogel for treating fibrosis, comprising the following steps:

[0011] Step S1: Mix sodium β-glycerophosphate (β-GP) and distilled water to prepare a β-GP aqueous solution with a w / v concentration of 56%;

[0012] Step S2: Mix concentrated hydrochloric acid and distilled water to prepare a 0.1M dilute hydrochloric acid solution;

[0013] Step S3: Add chitosan (CS) to the dilute hydrochloric acid prepared in step S2, heat and stir to fully dissolve the chitosan, and autoclave to obtain a CS solution with a w / v concentration of 3.3%;

[0014] Step S4: Add LBQ657 to the chitosan solution formed in step S3 in an ice bath and stir to ensure that the drug LBQ657 is evenly dispersed in the system;

[0015] Step S5: Add the β-GP aqueous solution prepared in step S1 to the solution obtained in step S4, mix well, and let stand at 37°C for 5 minutes to obtain sustained-release LBQ657 hydrogel.

[0016] Preferably, the concentration of LBQ657 in step S4 is 100uM-250uM.

[0017] The present invention also provides the use of the sustained-release LBQ657 hydrogel in the preparation of medicaments for treating fibrosis-related diseases.

[0018] Compared with the prior art, the beneficial effects of the present invention are:

[0019] 1. The sustained-release LBQ657 hydrogel of this invention, after covering the esophageal surface, rapidly forms a stable gel and adheres well to the esophagus, alleviating TGF-β-induced fibrosis. By providing protection for at least 7 days under physiological conditions, it can be directly sprayed onto the esophageal wound via endoscopic spraying after ESD, allowing the esophageal wound to form a stable gel in a timely manner and achieve long-term sustained drug release, providing a new approach for the treatment of esophageal fibrosis.

[0020] 2. The sustained-release LBQ657 hydrogel prepared by the present invention has a hemolysis rate of less than 3% in its extract, exhibits good biocompatibility, and has no potential cytotoxicity. Attached Figure Description

[0021] Figure 1 The solidification process of the sustained-release LBQ657 hydrogel for treating fibrosis of the present invention at 37°C with a mixing ratio of 4:1 for CS solution and β-GP solution;

[0022] Figure 2 The solidification process of the LBQ657 hydrogel for treating fibrosis of the present invention at 37°C with a mixing ratio of 6:1 for CS solution and β-GP solution;

[0023] Figure 3 The solidification process of the LBQ657 hydrogel for treating fibrosis of the present invention at 37°C with a mixing ratio of 8:1 for CS solution and β-GP solution;

[0024] Figure 4 The solidification process of the LBQ657 hydrogel for treating fibrosis of the present invention at 37°C with a mixing ratio of 9:1 for CS solution and β-GP solution;

[0025] Figure 5 The solidification process of the fibrotic sustained-release LBQ657 hydrogel of the present invention at 37°C with a mixing ratio of 10:1 for CS solution and β-GP solution;

[0026] Figure 6 Viscosity diagrams of hydrogels containing different concentrations of LBQ657 with a CS solution and β-GP solution mixed in a ratio of 8:1 prepared in this invention.

[0027] Figure 7 Electron microscopy results of hydrogels containing different concentrations of LBQ657 prepared for this invention, with a CS solution and β-GP solution mixed in a ratio of 8:1;

[0028] Figure 8 Infrared spectra of hydrogels containing different concentrations of LBQ657 prepared for this invention, with a CS solution and β-GP solution mixing ratio of 8:1;

[0029] Figure 9 Swelling curves of hydrogels containing different concentrations of LBQ657 prepared for this invention, with a mixing ratio of CS solution and β-GP solution of 8:1;

[0030] Figure 10 Degradation curves of hydrogels containing different concentrations of LBQ657 prepared for this invention, with a mixing ratio of CS solution and β-GP solution of 8:1;

[0031] Figure 11 pH curves of hydrogels containing different concentrations of LBQ657 prepared for this invention with a CS solution and β-GP solution mixing ratio of 8:1 during the degradation process;

[0032] Figure 12 This is a biocompatibility diagram of hydrogel extracts containing different concentrations of LBQ657, prepared according to the solubility test of CS solution and β-GP solution at a mixing ratio of 8:1 based on the solubility test in this embodiment of the invention.

[0033] Figure 13 A schematic diagram showing the mRNA expression results of hydrogels containing different concentrations of LBQ657 prepared for this invention, with a CS solution and β-GP solution mixed in a ratio of 8:1, alleviating mouse embryonic fibroblast (MEF) cell fibrosis.

[0034] Figure 14 A schematic diagram showing the protein expression results of hydrogels containing different concentrations of LBQ657 that alleviate MEF cell fibrosis, prepared according to the present invention with a CS solution and β-GP solution mixed in a ratio of 8:1;

[0035] Figure 15 A schematic diagram showing the mRNA expression results of hydrogels containing different concentrations of LBQ657 that alleviated primary mouse esophageal endothelial cell fibrosis, prepared according to the present invention with a CS solution and β-GP solution mixed in a ratio of 8:1 and a mixture of different concentrations of LBQ657.

[0036] Figure 16 A schematic diagram showing the protein expression results of hydrogels containing different concentrations of LBQ657 that alleviate primary mouse esophageal endothelial cell fibrosis, prepared according to the present invention with a CS solution and β-GP solution mixed in a ratio of 8:1. Detailed Implementation

[0037] To enable those skilled in the art to better understand the content of this invention, the embodiments of this invention are described in detail below. These embodiments are implemented based on the technical solution of this invention, and provide detailed implementation methods and specific operation processes. However, the content of this invention is not limited to the following examples.

[0038] The main sources of the primary materials and reagents used in the following examples are as follows:

[0039] Chitosan: Aladdin

[0040] β-glycerophosphate sodium: GlpBio

[0041] LBQ657: Sigma-Aldrich

[0042] HCl: Nanjing Chemical Reagent Co., Ltd.

[0043] DMEM: Gibco

[0044] FBS: Excell

[0045] MEFs: ATCC

[0046] This invention provides a sustained-release LBQ657 hydrogel for treating fibrosis. After being applied to the surface of the esophagus, it quickly forms a stable gel and adheres well to the esophagus. Its loose and porous structure allows the drug to diffuse within the carrier, thereby releasing the drug.

[0047] Example 1: Preparation method of sustained-release LBQ657 hydrogel with different components

[0048] Dissolve 5.6g of β-GP powder in 10ml of distilled water, stirring until completely dissolved to obtain a clear, transparent liquid. Store at 4℃ for later use. Dissolve 3.3g of CS powder in 100ml of 0.1mol / L hydrochloric acid solution, stirring magnetically at 50-60℃ until completely dissolved to obtain a pale yellow turbid solution. Autoclave the pale yellow turbid solution at 121℃ for 15 minutes. Transfer the solution to an EP tube, centrifuge at 4000 rpm for 15 minutes, and store the supernatant at 4℃ for later use. Vortex the 4℃ CS solution continuously, adding 1-2.5ul of 50mM LBQ657 solution, vortexing until well mixed. Then add the β-GP solution dropwise, vortexing until well mixed. The ratio of CS solution to β-GP solution is 4:1-10:1.

[0049] 1) Determine gelation time using the inverted test tube method.

[0050] The CS / GP solutions prepared in Example 1 were added to test tubes at mixing ratios of 4:1, 6:1, 8:1, 9:1 and 10:1, respectively. The test tubes were heated to 37°C in a water bath. The test tubes were inverted at certain time intervals. The time it took for the solution to change from a liquid state to a gel state was called the gel time. Figures 1-5The solidification process of the sustained-release LBQ657 hydrogel is shown in the figure. The time statistics show that the hydrogel with a mixing ratio of 8:1 at 37°C has good liquid flowability in about 1 minute and can form a non-flowing solid hydrogel in about 2 minutes. It can quickly form a stable gel after covering the esophageal surface.

[0051] 2) The rheological properties of the hydrogel were detected using a rotational rheometer.

[0052] Dissolve 5.6g of β-GP powder in 10ml of distilled water, stirring until completely dissolved to obtain a clear, transparent liquid. Store at 4℃ for later use. Dissolve 3.3g of CS powder in 100ml of 0.1mol / L hydrochloric acid solution, stirring magnetically at 50-60℃ until completely dissolved to obtain a pale yellow turbid solution. Autoclave the pale yellow turbid solution at 121℃ for 15 minutes. Transfer the solution to an EP tube, centrifuge at 4000 rpm for 15 minutes, and store the supernatant at 4℃ for later use. Take 444ul of the 4℃ CS solution and vortex continuously. Add 1-2.5ul of 50mM LBQ657 solution, vortexing until well mixed. Add 56ul of β-GP solution dropwise, vortexing until well mixed, and then crosslink at 37℃ for 5 minutes.

[0053] The viscoelasticity of the hydrogel was determined under dynamic time-oscillation mode using 25 mm parallel plates (500 μm spacing) at 37 °C, angular frequency 1.0 rad / s, and strain 1%. The results are as follows: Figure 6 As shown, the horizontal axis represents shear rate, and the vertical axis represents viscosity. The results indicate that LBQ657 does not alter the rheological properties of the hydrogel. The sustained-release LBQ657 hydrogel maintains a low shear viscosity at the given drug concentration, indicating good anti-settling, anti-water separation, anti-sagging, and storage stability. Therefore, it can be inferred that this hydrogel can adhere well to the esophagus without issues such as settling or denaturation.

[0054] 3) Observe the microstructure of the hydrogel using a scanning electron microscope (SEM).

[0055] In this test, the preparation of the sustained-release LBQ657 hydrogel was the same as that used in the process described above for testing the rheological properties of the hydrogel.

[0056] The hydrogel solution was heated to 37°C to form a gel, fixed with 2.5% glutaraldehyde, dehydrated in ascending ethanol solutions, and then freeze-dried in a vacuum freeze dryer for 72 hours. The dried hydrogel was longitudinally cut and adhered to the surface of a conductive adhesive, then sputter-coated with gold before analysis. Electron micrographs of the three concentrations of hydrogel were taken at 1,000 and 10,000x magnification. The results are as follows. Figure 7As shown, the gel exhibits a loose and porous structure, allowing drugs to diffuse within the carrier and be released.

[0057] 4) Infrared spectral waveform test

[0058] In this test, the preparation of the sustained-release LBQ657 hydrogel was the same as that used in the process described above for detecting the rheological properties of the hydrogel. After obtaining the hydrogel, it was concentrated using a vacuum centrifuge to form lyophilized tablets, which were then ground into powder as dry samples. Tests were performed using a disc containing 0.2 mg of the dry sample and 10 mg of KBr. Fourier transform infrared (FTIR) spectra were obtained at room temperature from 4000 to 400 cm⁻¹. -1 Within the range, 2cm per point -1 The data acquisition rate was recorded. The test results are as follows: Figure 8 The addition of the drug did not affect the infrared spectrum waveform of the hydrogel, proving that the molecular structure and functional groups in the system remained unchanged.

[0059] 5) Swelling rate test

[0060] In this test, the preparation of the sustained-release LBQ657 hydrogel was the same as that used in the process of detecting the rheological properties of the hydrogel described above; after the hydrogel was prepared, it was lyophilized into freeze-dried sheets using a vacuum centrifuge.

[0061] The swelling rate of the hydrogel lyophilized tablets containing different concentrations of LBQ657 prepared above was measured in physiological saline at 37℃ for 5, 10, 30, and 60 minutes: Swelling rate = (weight of swollen gel - weight of dried gel) / weight of dried gel. The swelling curves are shown below. Figure 9 The freeze-dried hydrogel reached a swelling rate of about 150% within 1 hour under physiological conditions, indicating that the hydrogel can achieve rapid swelling and remain stable, thus covering the wound surface and protecting the wound from drug release.

[0062] 6) Degradation rate and pH value of physiological saline during degradation process.

[0063] Dissolve 5.6g of β-GP powder in 10ml of distilled water, stirring until completely dissolved to obtain a clear, transparent liquid. Store at 4℃ for later use. Dissolve 3.3g of CS powder in 100ml of 0.1mol / L hydrochloric acid solution, stirring magnetically at 50-60℃ until completely dissolved to obtain a pale yellow turbid solution. Autoclave the pale yellow turbid solution at 121℃ for 15 minutes. Transfer the solution to an EP tube, centrifuge at 4000 rpm for 15 minutes, and store the supernatant at 4℃ for later use. Take 1776ul of the 4℃ CS solution and vortex continuously. Add 4-10ul of 50mM LBQ657 solution, vortexing until well mixed. Add 224ul of β-GP solution dropwise, vortexing until well mixed, and then crosslink at 37℃ for 5 minutes.

[0064] 8 ml of physiological saline was added to the prepared hydrogel, and it was stored at 37°C. The degradation rate and the pH value of the physiological saline during the degradation process were measured at 1, 3, 5, and 7 days. The results are as follows: Figure 10-11 As shown, the hydrogel degrades by approximately 30% within 7 days, with the pH value decreasing from around 6.5 to 6.4. This indicates that the hydrogel can provide at least one week of protection for esophageal wounds, and its degradation does not affect the pH value of the physiological environment.

[0065] 7) Biocompatibility testing of hydrogel extract

[0066] Dissolve 5.6g of β-GP powder in 10ml of distilled water and stir until completely dissolved to obtain a clear, transparent liquid. Store at 4℃ for later use. Dissolve 3.3g of CS powder in 100ml of 0.1mol / L hydrochloric acid solution and stir magnetically at 50-60℃ until completely dissolved to obtain a pale yellow turbid solution. Autoclave the pale yellow turbid solution at 121℃ for 15 minutes. Transfer the solution to EP tubes and centrifuge at 4000 rpm for 15 minutes. Collect the supernatant and store at 4℃ for later use. Take 444ul of the 4℃ CS solution and vortex continuously. Add 1-2.5ul of 50mM LBQ657 solution and vortex until well mixed. Add 56ul of β-GP solution dropwise and vortex until well mixed. Spread the mixture into 6-well plates and crosslink at 37℃ for 5 minutes. Add 2ml of DMEM medium to each well and incubate at 37℃ for 24 hours. Collect the DMEM.

[0067] The hemolysis test was performed on the hydrogel according to GB / T 14233.2-2005. The results are as follows: Figure 12 As shown, the hemolysis rate of the hydrogel extract is less than 3%, which meets the national standard. These results indicate that the hydrogel possesses excellent biocompatibility.

[0068] 8) Cytotoxicity test

[0069] The preparation of the sustained-release LBQ657 hydrogel in this test was the same as the preparation components used in the above-mentioned process for testing the biocompatibility of hydrogels.

[0070] The cytotoxicity of the hydrogel was tested using the CCK8 assay. The effect of the hydrogel on MEF cell proliferation was also tested. MEF cells were cultured in prepared DMEM for 24 hours. Figure 12 As shown, the proliferation rate of MEF cells was greater than 100% at a drug concentration of 100 μM and greater than 80% at a drug concentration of 250 μM. According to the standard described in GB / T 16886.5, a test result >70% indicates no potential cytotoxicity.

[0071] 9) The effect of sustained-release LBQ657 hydrogel on TGF-β-induced fibrosis was detected using MEF and primary mouse esophageal endothelial cells.

[0072] Cells were treated with the DMEM extract prepared in the biocompatibility test of the above hydrogel extract for 12 hours, followed by stimulation with 10 ng / ml TGF-β for 24 hours to induce cell fibrosis. The extract of the hydrogel without LBQ657 was used as a control, and the mRNA transcription and protein expression levels of the cells were analyzed. The results showed that after LBQ657 treatment, the mRNA expression levels of fibrosis marker genes Collagen I and α-SMA in fibroblasts and primary esophageal endothelial cells were significantly decreased, as were the protein expression levels of fibrosis marker proteins MMP9 and α-SMA, indicating that the addition of LBQ657 inhibited cell fibrosis. Figure 13-14 The results of this invention, showing the effects of hydrogels containing different concentrations of LBQ657 on mitigating MEF cell fibrosis, are illustrated. Figure 15-16 The results of this invention, which uses hydrogels containing different concentrations of LBQ657, show the effects of alleviating fibrosis in primary mouse esophageal endothelial cells.

[0073] Test results show that the sustained-release LBQ657 hydrogel obtained in this embodiment of the invention has the effect of alleviating TGF-β-induced fibrosis. After covering the esophageal surface, it quickly forms a stable gel and adheres well to the esophagus, thereby releasing the drug, alleviating the fibrosis process, and providing at least 7 days of protection under physiological conditions.

[0074] The above description is only a preferred embodiment of the present invention and is not limited to the above implementation method. Any equivalent modifications, substitutions and improvements made by those skilled in the art based on the content disclosed in the present invention should be included in the protection scope of the claims.

Claims

1. The application of a sustained-release LBQ657 hydrogel in the preparation of a drug for treating esophageal fibrosis, characterized in that, The sustained-release LBQ657 hydrogel comprises chitosan, sodium β-glycerophosphate, distilled water, and LBQ657. The chitosan solution used in the hydrogel preparation has a w / v concentration of 3.3%, and the sodium β-glycerophosphate aqueous solution has a w / v concentration of 56%. The volume ratio of chitosan solution to sodium β-glycerophosphate solution is 4:1-10:

1. Dilute hydrochloric acid is used as the solvent system for the chitosan solution. After ESD, the drug is administered via endoscopic spraying, directly spraying the drug onto the esophageal wound.

2. The application according to claim 1, characterized in that, The ratio of chitosan solution to sodium β-glycerophosphate solution is 8:

1.

3. The application according to claim 1, characterized in that, The preparation method of sustained-release LBQ657 hydrogel includes the following steps: Step S1: Mix sodium β-glycerophosphate and distilled water to prepare a sodium β-glycerophosphate solution with a w / v concentration of 56%; Step S2: Mix concentrated hydrochloric acid and distilled water to prepare a 0.1M dilute hydrochloric acid solution; Step S3: Add chitosan to the dilute hydrochloric acid prepared in step S2, heat and stir to fully dissolve the chitosan, and sterilize by autoclaving to obtain a chitosan solution with a w / v concentration of 3.3%; Step S4: Add LBQ657 to the chitosan solution formed in step S3 in an ice bath and stir to ensure that the drug LBQ657 is evenly dispersed in the system; Step S5: Add the sodium β-glycerophosphate solution prepared in step S1 to the solution obtained in step S4, mix well, and let stand at 37°C for 5 minutes to obtain sustained-release LBQ657 hydrogel.

4. The application according to claim 3, characterized in that, In step S4, the concentration of LBQ657 is 100uM-250uM.

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