A hydrogel system, its preparation method and use

Abstract

The application provides a hydrogel system and a preparation method and application thereof. The hydrogel system provided by the application comprises oxidized hyaluronic acid, chitosan, perfluorotributylamine and a vascular growth factor, and can provide oxygen for islet cells, promote in-situ islet vascular reconstruction, inhibit islet cell apoptosis and promote insulin secretion, thereby providing a new strategy for islet transplantation and even treatment of diseases caused by insulin deficiency or insufficiency.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a hydrogel system, its preparation method, and its application. Background Technology

[0002] Diabetes severely impacts patients' quality of life. Type 1 diabetes (T1D) is caused by autoimmune attack leading to the destruction or loss of pancreatic function and absolute insulin deficiency. Islet transplantation is an emerging treatment method for T1D in recent years.

[0003] However, both immune attack and hypoxia after transplantation can damage the transplanted islets, ultimately leading to transplantation failure. To address these two issues, establishing an immune-tolerant microenvironment and ensuring adequate oxygen supply at the graft site is a good strategy. Good blood supply can provide the necessary oxygen and nutrients, and post-transplant vascular revascularization is crucial to ensuring the long-term survival and function of transplanted islet cells.

[0004] Hydrogels are gels with a three-dimensional network structure formed by the physical or chemical cross-linking of polymers, and they have been widely used in the field of bioengineering in recent years. Hydrogels can not only encapsulate cells to mimic the cellular microenvironment, but also have the function of sustained-release therapeutic drugs. Encapsulating islet grafts with natural or synthetic materials forms an immune-isolating barrier on the graft surface, which can reduce the host's immune rejection response. In addition, islet encapsulation materials can also serve as carriers for oxygen-carrying drugs or angiogenesis-promoting drugs.

[0005] However, the encapsulation of islet cells in hydrogels restricts oxygen diffusion, making them susceptible to hypoxia and necrosis. Currently used hydrogel materials for islet transplantation include collagen hydrogels, alginate hydrogels, polyethylene glycol (PEG), and polylactic-co-glycolic acid (PLGA) hydrogels, among others. These materials each have drawbacks, such as rapid degradation potentially triggering immune responses; lack of bioactivity; and lack of cell adhesion sites, often requiring modification with bioactive molecules. Furthermore, they do not address the early hypoxia problem of islet cells or the need for in-situ islet vascular reconstruction. Traditional oxygen-carrying materials, such as hydrogen peroxide, can generate free radicals that damage cells; hemoglobin suffers from immunogenicity and stability issues, resulting in a very short oxygen-carrying time. Summary of the Invention

[0006] In view of this, in order to overcome the shortcomings of the prior art, the present invention is proposed.

[0007] The first aspect of the present invention provides a hydrogel system comprising oxidized hyaluronic acid, chitosan, perfluorotributylamine and angiogenic factor.

[0008] In this invention, oxidized hyaluronic acid and chitosan are physically cross-linked into a gel through a Schiff base reaction and an amine-aldehyde condensation reaction between natural polysaccharides.

[0009] Furthermore, the chitosan includes, but is not limited to, carboxymethyl chitosan, hydroxyethyl chitosan, quaternized chitosan, thiolized chitosan, acylated chitosan, phosphorylated chitosan, glycosylated chitosan, and polyethylene glycol-modified chitosan.

[0010] Furthermore, the chitosan is selected from carboxymethyl chitosan.

[0011] Furthermore, the perfluorotributylamine is an oxygen-loaded perfluorotributylamine.

[0012] Furthermore, the mass ratio of chitosan to perfluorotributylamine is 1:20-3:7.

[0013] Furthermore, the mass ratio of chitosan to perfluorotributylamine is 2:7.

[0014] Furthermore, the mass ratio of chitosan to oxidized hyaluronic acid is 1:2-3:1.

[0015] Furthermore, the mass ratio of chitosan to oxidized hyaluronic acid is 2:1.

[0016] Furthermore, the mass concentration of the chitosan is 0.67%-2%.

[0017] Furthermore, the mass concentration of the chitosan is 1.33%.

[0018] Furthermore, the mass concentration of the angiogenesis factor is 33.3-66.7 ng / ml.

[0019] A second aspect of the present invention provides a pharmaceutical composition comprising the hydrogel system described in the first aspect of the present invention.

[0020] Furthermore, the pharmaceutical composition also includes pancreatic islet cells.

[0021] Furthermore, the pharmaceutical composition includes a pharmaceutically acceptable carrier and / or excipient.

[0022] In this invention, "pharmaceutically acceptable carriers and / or excipients" include, but are not limited to, diluents, binders, surfactants, humectants, adsorbents, lubricants, disintegrants, emulsifiers, bioavailability enhancers, suspending agents, sweeteners, flavoring agents, colorants, excipients, preservatives, solubilizers, dispersants, and / or wetting agents. The diluents include, but are not limited to, lactose, sodium chloride, glucose, urea, starch, and water; the binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, gum arabic, gelatin, methylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginate and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose; the surfactants include, but are not limited to, polyethylene oxide sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, and hexadecyl alcohol; the humectants include, but are not limited to, glycerol and starch; the adsorbents include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, and soap clay; and the lubricants include, but are not limited to, zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate, magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearate fumarate, polyoxyethylene monostearate, monolauric sucrose ester, sodium lauryl sulfate, magnesium lauryl sulfate, and magnesium lauryl sulfate.

[0023] In this invention, the pharmaceutical composition is administered in an injectable dosage form.

[0024] Furthermore, the injectable dosage forms include, but are not limited to, various injectable drugs such as intravenous injections, intramuscular injections, subcutaneous injections, intradermal injections, or intracavitary injections.

[0025] Furthermore, the injectable dosage form is selected from intravenous injections.

[0026] Furthermore, the vein in question is the portal vein.

[0027] In this invention, the pharmaceutical composition and at least one additional therapeutic agent or therapy may be administered sequentially, simultaneously, and / or alternately, wherein the at least one additional therapeutic agent or therapy includes, but is not limited to, insulin therapy, diet management, exercise intervention, adjunctive drug therapy, and emerging therapeutic therapies.

[0028] Furthermore, the insulin therapy includes, but is not limited to, the administration of ultra-short-acting, short-acting, intermediate-acting, and long-acting insulin.

[0029] Furthermore, the ultra-short-acting insulin includes, but is not limited to, insulin aspart and insulin lispro, which are used for pre-meal injections to control postprandial blood glucose.

[0030] Furthermore, the short-acting insulin includes, but is not limited to, regular insulin, which is used for pre-meal injection or intravenous injection (such as in diabetic ketoacidosis).

[0031] Furthermore, the intermediate-acting insulin includes, but is not limited to, neutral protamine zinc insulin, which is used for basal insulin supplementation.

[0032] Furthermore, the long-acting insulin includes, but is not limited to, insulin glargine, insulin detemir, and insulin degludec, which are used for basal insulin supplementation.

[0033] Furthermore, the insulin administration methods include, but are not limited to, subcutaneous injection, insulin pump administration, and inhalation administration.

[0034] Furthermore, the adjunctive drug therapy includes, but is not limited to, the administration of pramlintide and SGLT-2 inhibitors.

[0035] Furthermore, the emerging treatments include, but are not limited to, stem cell therapy and immunotherapy.

[0036] In this invention, the pharmaceutical composition is administered at a therapeutically effective dose. The term “therapeutically effective dose” refers to the level or amount of a target agent that will not produce significant negative or adverse side effects: (1) delay or prevent the onset of diseases or complications caused by insulin deficiency or insufficiency; (2) slow or prevent the progression, aggravation or worsening of diseases or complications caused by insulin deficiency or insufficiency; (3) improve the symptoms of diseases or complications caused by insulin deficiency or insufficiency; (4) reduce the severity or incidence of diseases or complications caused by insulin deficiency or insufficiency; and (5) cure diseases or complications caused by insulin deficiency or insufficiency.

[0037] A third aspect of the present invention provides a method for preparing the hydrogel system described in the first aspect of the present invention, the method comprising mixing a vascular endothelial growth factor-oxidized hyaluronic acid solution with a chitosan-perfluorotributylamine solution.

[0038] Furthermore, the volume ratio of the vascular endothelial growth factor-oxidized hyaluronic acid solution to the chitosan-perfluorotributylamine solution is 3:5-3:10.

[0039] Furthermore, the volume ratio of the vascular endothelial growth factor-oxidized hyaluronic acid solution to the chitosan-perfluorotributylamine solution is 1:2.

[0040] Furthermore, the mass concentration of oxidized hyaluronic acid in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 2%-4%.

[0041] Furthermore, the mass concentration of oxidized hyaluronic acid in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 2%.

[0042] Furthermore, the mass concentration of vascular endothelial growth factor in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 100-200 ng / ml.

[0043] Furthermore, the mass concentration of perfluorotributylamine in the chitosan-perfluorotributylamine solution is 7%-20%.

[0044] Furthermore, the mass concentration of perfluorotributylamine in the chitosan-perfluorotributylamine solution is 7%.

[0045] Furthermore, the chitosan-perfluorotributylamine solution contains 1%-3% by mass.

[0046] Furthermore, the chitosan-perfluorotributylamine solution contains 2% chitosan by mass.

[0047] Furthermore, the method also includes a method for preparing a blood vessel growth factor-oxidized hyaluronic acid solution, wherein the preparation method includes dissolving blood vessel growth factor in an oxidized hyaluronic acid solution.

[0048] Furthermore, the method also includes a method for preparing an oxidized hyaluronic acid solution, the preparation method comprising dissolving oxidized hyaluronic acid in a PBS solution.

[0049] Furthermore, the pH value of the PBS solution is 7.4.

[0050] Furthermore, the method also includes a method for synthesizing oxidized hyaluronic acid, the synthesis method comprising dissolving hyaluronic acid in deionized water, adding NaIO4 solution, adding ethylene glycol, and dialyzing.

[0051] Furthermore, the mass ratio of the hyaluronic acid to the NaIO4 is 1.43:1 to 2.4:1.

[0052] Furthermore, the volume ratio of the deionized water to the NaIO4 solution is 5:1-6:1.

[0053] Further, after adding NaIO4 solution, stir in the dark until the reaction is complete.

[0054] Further, lyophilization is performed after dialysis.

[0055] Furthermore, the method also includes a method for preparing a chitosan-perfluorotributylamine solution, wherein the preparation method includes dissolving chitosan in a perfluorotributylamine nanoemulsion.

[0056] Furthermore, the method also includes a method for synthesizing perfluorotributylamine nanoemulsion, the synthesis method comprising the following steps: dissolving lecithin, cholesterol and DSPE-PEG2000 in dichloromethane, removing the dichloromethane, diluting the mixture with PBS solution, sonicating in an ice bath, mixing the mixture with perfluorotributylamine, and sonicating to obtain perfluorotributylamine nanoemulsion.

[0057] Furthermore, the mass ratio of lecithin to cholesterol is 5:1 to 10:1.

[0058] Furthermore, the mass ratio of lecithin to cholesterol is 6:1-7:1.

[0059] Furthermore, the mass ratio of cholesterol to DSPE-PEG2000 is 1:1 to 1:2.

[0060] Furthermore, the mass ratio of cholesterol to DSPE-PEG2000 is 12.37:13.09.

[0061] Furthermore, the mass concentration of the cholesterol dissolved in dichloromethane is 1 mg / ml to 10 mg / ml.

[0062] Furthermore, the cholesterol concentration after being dissolved in dichloromethane is 1.005 mg / ml.

[0063] Furthermore, dichloromethane is removed by rotary evaporation.

[0064] Furthermore, rotary evaporation was carried out at 25℃-35℃.

[0065] Furthermore, rotary evaporation was carried out at 30°C.

[0066] Furthermore, rotary evaporation was carried out at 100 rpm-200 rpm.

[0067] Furthermore, rotary evaporation was performed at 156 rpm.

[0068] Furthermore, the pH value of the PBS solution is 7.4.

[0069] Furthermore, two ultrasound sessions were administered in an ice bath.

[0070] Furthermore, each ice bath ultrasound session lasts two minutes.

[0071] Furthermore, there was a 1-minute interval between the two ice bath ultrasound sessions.

[0072] Furthermore, the volume concentration of the perfluorotributylamine is 4.5%-15%.

[0073] Furthermore, the volume concentration of the perfluorotributylamine is 4.5%.

[0074] Furthermore, the method also includes adding oxygen to the perfluorotributylamine nanoemulsion to obtain an oxygen-loaded perfluorotributylamine nanoemulsion.

[0075] The fourth aspect of the present invention provides for any of the following applications:

[0076] (1) The use of the hydrogel system of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention in the preparation of a medicament for treating diseases caused by insulin deficiency or insufficiency;

[0077] (2) Application of angiogenesis factor and perfluorotributylamine in the preparation of drugs for treating diseases caused by insulin deficiency or insufficiency;

[0078] (3) Application of carboxymethyl chitosan and oxidized hyaluronic acid in the preparation of drugs for treating diseases caused by insulin deficiency or insufficiency;

[0079] (4) The use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in promoting insulin secretion;

[0080] (5) The use of the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention in inhibiting pancreatic islet cell apoptosis;

[0081] (6) The use of the hydrogel system of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention in promoting angiogenesis.

[0082] In this invention, the diseases caused by insulin deficiency or insufficiency include, but are not limited to, hyperglycemia, type 1 diabetes, type 2 diabetes, type 1 or type 2 diabetes-related complications, and other rare types of diabetes.

[0083] In this invention, type 1 diabetes is mainly caused by the abnormal activation of the patient's own immune system, which leads to the destruction of β cells in Langerhans pancreatic islets by the autoimmune system, ultimately resulting in insulin deficiency.

[0084] Furthermore, the disease caused by insulin deficiency or insufficiency is selected from type 1 diabetes.

[0085] In this invention, pancreatic islet transplantation can be used to treat diseases caused by insulin deficiency or insufficiency.

[0086] Furthermore, islet transplantation can be performed using the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0087] In this invention, "islets of Langerhans" can be broadly understood to include any cell or cell aggregate that secretes insulin.

[0088] Furthermore, islets can be taken from individuals, such as donors, including allogeneic donors, autologous donors, or xenogeneic donors.

[0089] Furthermore, islets can be derived from stem cells (including ESCs and iPSCs) or islet progenitor cells.

[0090] In this invention, "promoting insulin secretion" means allowing pancreatic islet cells to enhance their ability to secrete insulin. For example, insulin secretion may include an increase of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more.

[0091] In this invention, "inhibiting pancreatic islet cell apoptosis" refers to the ability of pancreatic islet cells to reduce their apoptosis. For example, the apoptosis rate or percentage of pancreatic islet cells may include a reduction of at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% or more.

[0092] The fifth aspect of the present invention provides any of the following methods:

[0093] (1) A method for promoting angiogenesis in vitro, the method comprising applying the hydrogel system of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention;

[0094] (2) A method for promoting insulin secretion in vitro, the method comprising applying the hydrogel system of the first aspect of the present invention or the pharmaceutical composition of the second aspect of the present invention;

[0095] (3) A method for inhibiting pancreatic islet cell apoptosis in vitro, the method comprising applying the hydrogel system described in the first aspect of the present invention or the pharmaceutical composition described in the second aspect of the present invention.

[0096] In this invention, "extracorporeal" refers to any scenario outside the body.

[0097] In this invention, "in vitro angiogenesis promotion" can include, but is not limited to, applying the hydrogel system described in the first aspect of this invention or the pharmaceutical composition described in the second aspect of this invention to human umbilical vein endothelial cells in a laboratory to promote the differentiation of human umbilical vein endothelial cells and the formation of vascular networks.

[0098] The advantages and beneficial effects of this invention are as follows:

[0099] This invention provides a hydrogel system, its preparation method, and its applications. The hydrogel system provided by this invention comprises oxidized hyaluronic acid, chitosan, perfluorotributylamine, and angiogenic factors. This hydrogel system can provide oxygen to pancreatic islet cells, promote in situ islet vascular reconstruction, inhibit islet cell apoptosis, and promote insulin secretion, providing a new strategy for islet transplantation and even the treatment of diseases caused by insulin deficiency or insufficiency. Attached Figure Description

[0100] Figure 1 Fourier transform infrared absorption spectra of hyaluronic acid and oxidized hyaluronic acid.

[0101] Figure 2 This is a graph showing the oxidation degree test results of oxidized hyaluronic acid material.

[0102] Figure 3 This is a transmission electron microscope image of perfluorotributylamine nanoemulsion.

[0103] Figure 4 This is a DLS light scattering pattern of the perfluorotributylamine nanoemulsion.

[0104] Figure 5 The image shows the microstructure and pore size of the composite hydrogel.

[0105] Figure 6 This is a photograph of the composite hydrogel in its gelation process.

[0106] Figure 7 The figure shows the results of the composite hydrogel degradation experiment.

[0107] Figure 8 This is a graph showing the results of alternating scans by a rotating rheometer.

[0108] Figure 9 This is a diagram illustrating the self-healing process of the composite hydrogel.

[0109] Figure 10 The figure shows the experimental results of the rheological properties of the hydrogel.

[0110] Figure 11 This is a diagram showing the results of a hydrogel cytotoxicity experiment.

[0111] Figure 12 This is a diagram showing the results of a hydrogel blood compatibility test.

[0112] Figure 13 This is a diagram showing the results of an in vitro angiogenesis experiment.

[0113] Figure 14 This is a graph showing the results of an in vitro insulin secretion experiment.

[0114] Figure 15 This is a diagram showing the results of an in vitro pancreatic islet hypoxia experiment.

[0115] Figure 16 The figure shows the results of an experiment on pancreatic islet cell apoptosis under hypoxic conditions.

[0116] Figure 17 The figures show the results of in vivo insulin secretion experiments. Figure A shows the results of the mouse blood glucose experiment, Figure B shows the results of the glucose tolerance experiment, Figure C shows the data analysis of Figure A, and Figure D shows the results of insulin secretion recovery.

[0117] Figure 18 This is a schematic diagram illustrating the preparation process and functional verification of the composite hydrogel. Detailed Implementation

[0118] The present invention will be further described below with reference to embodiments. The following description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make equivalent modifications to the disclosed technical content to create equivalent embodiments. Any simple modifications or equivalent changes made to the following embodiments based on the technical essence of the present invention without departing from the scope of the invention are all within the protection scope of the present invention.

[0119] Example 1: Preparation of Vascular Growth Factor-Oxidized Hyaluronic Acid (VEGF-OHA) Solution

[0120] OHA Synthesis: Dissolve 1.0-1.2 g of HA in 100-120 mL of deionized water, and dissolve 0.5-0.7 g of NaIO4 in 20 mL of deionized water, then add the dissolved NaIO4 solution dropwise to the HA solution. Stir the reaction mixture in the dark at room temperature for 6 hours to ensure complete reaction. Then, neutralize the unreacted NaIO4 with 1.0 mL of ethylene glycol and allow the mixture to stir again for 2 hours. Dialyze the resulting product against double-distilled water for 3 days (MWCO 12,000), changing the dialysate three times daily to remove residual periodate and ethylene glycol. After lyophilization, a white, cotton-like substance, oxidized hyaluronic acid, was obtained and stored under vacuum.

[0121] Fourier transform infrared (FTIR) spectroscopy: After freeze-drying, the material was thoroughly ground with potassium bromide at a mass ratio of 1:100 and pressed into thin sheets. The freeze-dried hydrogel sample was then analyzed using an ATR attachment via FTIR spectroscopy in the wavelength range of 500–4000 cm⁻¹. -1 The range was scanned. The Fourier transform infrared absorption spectra of HA and OHA are shown below. Figure 1 As shown.

[0122] Oxidation degree measurement: 0.05 g of lyophilized OHA was dissolved in 20 ml of 0.25 mol / L hydroxylamine hydrochloride (0.3475 g adjusted to pH 4.5) containing 0.002 wt% methyl orange reagent. The mixture was stirred at room temperature for 24 h, and the aldehyde was converted to oxime. The released hydrochloric acid was then titrated with 1 mol / L sodium hydroxide solution until a red to yellow endpoint was reached. The pH change with the volume of sodium hydroxide solution added was recorded. The relevant reactions and calculation formulas are as follows:

[0123] HA-(CHO)n+nH2N-OH·HCl=HA-(CH=N-OH)n+nH2O+nHCl(1);

[0124] HCl + NaOH = NaCl + H₂O (2);

[0125] Oxidation degree (%) = 403ΔV × c × 10 -3 / 2w;

[0126] In the formula, V is the volume of sodium hydroxide solution consumed, in milliliters; c is the concentration of sodium hydroxide solution, in mol / L; w is the weight of OHA, in grams; and 403 is the molecular weight of the repeating unit, in g / mol.

[0127] Experimental results are as follows Figure 2 As shown, the oxidation degree of OHA is approximately 48%.

[0128] OHA was dissolved in PBS (pH 7.4) to prepare an OHA solution with a mass concentration of 2%-4%.

[0129] VEGF was dissolved in OHA solution to obtain VEGF-OHA solution, with a VEGF mass concentration of 100-200 ng / ml.

[0130] Example 2 Preparation of carboxymethyl chitosan-perfluorotributylamine (CMC-PFTBA) solution

[0131] Synthesis of oxygen-loaded PFTBA nanoemulsion: To prepare the PFTBA nanoemulsion, 60.586 mg of lecithin, 10.05 mg of cholesterol, and 10.635 mg of DSPE-PEG2000 were dissolved in 10 mL of dichloromethane. After removing the dichloromethane by rotary evaporation at 30 °C and 156 rpm, the resulting mixture was diluted in 5.0 mL of PBS solution (pH 7.4) and subjected to two sonication treatments in an ice bath (300 W, 3 min each time), with a 1 min interval between the two treatments. Subsequently, similarly, 4.775 mL of the sonicated solution was mixed with 0.225 mL of perfluorotributylamine, and the ice bath sonication treatment was repeated twice to produce PFTBA nanoemulsions with a volume concentration of 4.5%–15% (equivalent to a mass concentration of 7%–20%). The resulting emulsion was then placed in a hyperbaric oxygen chamber for 30 min to achieve complete oxygen saturation. The solution was finally passed through a 0.22 μm sterile filter to obtain a sterile oxygen-carrying PFTBA nanoemulsion (PFTBA-O2 emulsion).

[0132] The morphology of PFTBA-O2 was examined using transmission electron microscopy (TEM, Talos L120CG2, Thermo Scientific). The experimental results are as follows: Figure 3 As shown. The hydrated particle size was determined using a Malvern Zetasizer NanoS-90 dynamic light scattering (DLS) instrument, and the experimental results are as follows. Figure 4 As shown.

[0133] Carboxymethyl chitosan (CMC) was dissolved in PFTBA nanoemulsion to obtain a CMC-PFTBA solution, with a CMC-PFTBA solution having a CMC-PFTBA solution concentration of 1%-3%.

[0134] First, add VEGF-OHA (VO) solution to the islet cell pellet to resuspend the islet cells. Then, add CMC-PFTBA (CP) solution at a volume ratio of 1:2 to mix into a gel to complete the pre-transplantation preparation.

[0135] Example 3: Optimization of VO to CP volume ratio and mass concentration

[0136] The microstructure and pore size of the composite hydrogel were observed using scanning electron microscopy. The experimental results are as follows: Figure 5 As shown.

[0137] The gelation time of the hydrogel was evaluated using the inverted sample vial method: the precursor solution was introduced into the sample vial and gelled at 25°C. The flowability of the mixture was assessed, and the gelation time was recorded as the moment when the sample stopped flowing when the sample vial was inverted. Figure 6 This is a photograph of the composite hydrogel in its gelation process.

[0138] Different experimental groups were set up, and experiments were conducted according to the volume ratios of VO:CP = 1:2, 2:1, 3:2, 1:1, and 2:3. The results showed that the volume ratios of 2:1, 3:2, 1:1, and 2:3 were not good for gelation or the gelation time was too long.

[0139] The mass concentration screening and optimization experiment was conducted using a VO:CP volume ratio of 1:2, where the mass concentration of OHA in VO was set to 2%, 3%, and 4%, respectively; and the mass concentration of CMC in CP was set to 1%, 2%, and 3%, respectively. The experimental results are shown in Table 1.

[0140] Table 1. Screening and optimization experiments for VO and CP mass concentrations

[0141]

[0142] Example 4: Optimization of PFTBA volumetric concentration screening

[0143] Degradation experiments, rheological characterization using a rheometer, and cytotoxicity experiments were conducted to screen materials with different volume concentrations of PFTBA, assessing their degradation performance, self-healing properties, injectability, cytotoxicity, and in vitro angiogenesis. Experiments were performed at a VO:CP volume ratio of 1:2, with both OHA and CMC concentrations at 2%. (When both OHA and CMC concentrations are 2%, vocp0 represents no PFTBA, vocp3 represents a final PFTBA volume concentration of 3%, and similarly, vocp5 represents a final PFTBA volume concentration of 5%).

[0144] (1) Degradation experiment

[0145] The degradation of VOCs, VOCP3, and VOCP5 (n=3) was assessed by measuring weight loss in PBS over 21 days. The initial weight (W0) of the lyophilized samples was measured at specified intervals (1, 4, 7, 14, and 21 days) after immersion in PBS at 37°C. At each time point, the samples were extracted from PBS, lyophilized, and subsequently weighed again (Wd). The degradation rate was determined using the following method: Degradation rate (%) = The experimental results are shown in Table 2, and the weight decrease of the hydrogel is shown in the graph. Figure 7 As shown, the percentage weight reduction of hydrogels with added PFTBA was less than that of hydrogels without added PFTBA.

[0146] Table 2 Performance test results

[0147]

[0148] (2) Self-healing properties of rheometer hydrogel

[0149] (At low strain, the storage modulus is greater than the loss modulus, maintaining the gel state; at high strain, the storage modulus is less than the loss modulus, indicating that the gel breaks down into a fluid. After a 60s self-healing time, the strain changes from high to low and testing continues. If the storage modulus is greater than the loss modulus, it indicates that the gel state has been restored and the storage modulus (which can be considered as hardness) remains stable without decreasing, indicating good self-healing performance.) The rheological properties of the hydrogel were evaluated using an Anton Paar MCR-302e rotational rheometer. Cylindrical hydrogels with a diameter of 12mm and a height of 3mm were prepared and placed between 25mm parallel plates with a gap of 1.00mm. The perimeter was sealed with silicone oil to inhibit moisture evaporation. The Anton Paar MCR-302e rotational rheometer was used to alternately scan VOC and VOCP for three periods at low strain (1%) and high strain (500%) to study their self-healing properties. The experimental results are shown in Table 2. Figure 8 .

[0150] Self-healing experiment of hydrogels: We initially fabricated two circular VOCP gels using an aqueous dye to evaluate their self-healing behavior. After gelation, each VOCP gel was divided in half, with one half of the red hydrogel combined with one half of the transparent hydrogel to form a new structure. After a 30-minute self-healing period at room temperature, tensile stress was manually applied by reconnecting the samples with tweezers to observe the adhesion area. The experimental procedure is as follows. Figure 9 As shown (from left to right: initial, self-healing 30 min, stretching).

[0151] (3) Injectability

[0152] (The viscosity decreases with increasing shear rate, representing shear thinning behavior and indicating good injectability): The rheological properties of the hydrogel were evaluated using an Anton Paar MCR-302e rotational rheometer. Cylindrical hydrogels with a diameter of 12 mm and a height of 3 mm were prepared and placed between 25 mm parallel plates with a gap of 1.00 mm. The perimeter was sealed with silicone oil to suppress moisture evaporation. Furthermore, the shear viscosity was measured from 0.1 to 100 (1 / s) shear rates. The experimental results are shown in Table 2. Figure 10 Hydrogels supplemented with 3% PFTBA have the best injection performance.

[0153] (4) Cell proliferation and cytotoxicity assays

[0154] (Viability was assessed according to ISO 10993 standards; <70% viability indicates cytotoxicity.): Material extracts were prepared according to ISO 10993-12 guidelines by incubating sterile samples in serum-free DMEM (Thermo) or RPMI-1640 (Life Technology) at 37°C for 24 and 48 hours. These extracts included OHA-CMC hydrogels (OC), PFTBA-free (VOC) VEGF@OHA-CMC hydrogels, and VEGF@OHA-CMC hydrogels supplemented with 3% or 5% PFTBA (3% VOCP, 5% VOCP). The extracts were sterilized by filtration (0.22 μm) before use. INS-1 and HeLa cells were seeded in 96-well plates and cultured in DMEM or RPMI-1640 medium supplemented with 10% FBS (Sigma) and 1% penicillin-streptomycin (Gibco) for 12–24 hours to allow cell adhesion, then the medium was replaced with 100% extract solution. After incubation at 37°C for 24–48 hours, cell proliferation and cytotoxicity were assessed using CCK-8 assay (450 nm). Morphological changes were examined under a phase-contrast microscope, and cell viability was assessed according to ISO 10993 standards; viability <70% indicated cytotoxicity. The experimental results are shown in Table 2. Figure 11 The results showed that the hydrogel provided by the present invention is non-toxic to cells.

[0155] (5) Blood compatibility test of hydrogels:

[0156] The hemolysis test was used to determine the blood compatibility of the hydrogel. Red blood cells were separated by centrifugation at 4000 rpm for 5 minutes, followed by washing three times with PBS, also centrifuged at 4000 rpm for 5 minutes each time. The red blood cells were resuspended in PBS at a concentration of 2% v / v. Subsequently, 10 mg of the lyophilized hydrogel was incubated with 1 mL of the red blood cell suspension at 37°C for 4 hours. The hydrogel was extracted, and the red blood cell suspension was centrifuged at 4000 rpm for 5 minutes. The absorbance of the supernatant was recorded at 540 nm. Double-distilled water was used as a positive control, and PBS as a negative control. The hemolysis ratio was determined using the following formula:

[0157] Hemolysis ratio (%) = (As−Ab) - (Ap−Ab) × 100%;

[0158] The absorbance of As refers to the absorbance of the supernatant incubated with the hydrogel, while Ab represents the absorbance of the negative control and Ap represents the absorbance of the positive control.

[0159] Experimental results are as follows Figure 12 As shown, the hydrogel provided by the present invention has good blood compatibility.

[0160] (6) In vitro angiogenesis experiment

[0161] Human umbilical vein endothelial cells (HUVECs) were cultured at a density of 2 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 cells per well in 48-well plates and resuspended in DMEM Glutamax (Thermo) supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were seeded on uncoated plastic surfaces (control) or on substrates pre-coated with 20 μL of LDEV-free matrix gel (Corning), and then VOC, VOCP3, or VOCP5 hydrogels were carefully placed into each well. Each condition was tested in triplicate.

[0162] After incubation at 37°C for 12 hours, renal tubule formation was assessed using a 10× magnification phase-contrast microscope (Nikon). For quantitative analysis, three central regions randomly selected for each filter were examined. Images were analyzed using the AngiogenesisAnalyzer plugin for ImageJ to quantify key parameters, including total tubular length, number of nodes, and branching points.

[0163] Experimental results are as follows Figure 13As shown, compared with commercially available Matrigel, our material performs better in vitro angiogenesis, with superior total tube length, number of nodes, and number of tubes. Among these, vocp3 has the best angiogenesis ability, so we used this concentration in our animal experiments.

[0164] Example 5: In vitro insulin secretion study

[0165] Insulin secretion stimulated by glucose was evaluated in mouse islets cultured under different conditions. The groups were: PFTBA-containing group without VEGF (CPO2-S), VEGF-containing group without PFTBA (VOC-E), and VEGF-containing group with PFTBA (VOCPO2-E).

[0166] In vitro mouse islet isolation: Pancreas was harvested from 8-week-old mice via a midline abdominal incision. Cold collagenase P solution (Roche) was injected into the pancreas through a hollow bile duct to ensure uniform enzyme perfusion. The pancreas was then excised and incubated at 37°C for 16 minutes to promote enzyme digestion. The digested tissue was passed through a mesh screen to remove large undigested fragments, and the filtrate was subjected to a discontinuous dextran gradient (Histopaque, Merck Millipore) for islet purification. After gradient centrifugation, the solution was allowed to stand for 10–15 minutes for further separation of islets from acinar tissue. Purified islets were manually selected and counted under a microscope. All mouse data were derived from at least three independent islet isolates.

[0167] Freshly isolated mouse islets were washed three times with PBS, and 20 islets were allocated to each well. For suspension culture, the islets were maintained in RPMI-1640 medium supplemented with 10% FBS and 3% penicillin-streptomycin, or stored in the same medium containing 15 μL of CMC-3% PFTBAO2 (CPO2-S). For embedded cultures, the islets were embedded in 15 μL of hydrogel formulations, including VEGF@OHA-CMC (VOC-E) or VEGF@OHA-CMC-3% PFTBAO2 (VOCPO2-E), and immersed in the medium. After incubation for 48 or 72 hours, the culture medium was removed, and the islets were washed three times with cold PBS. They were then pre-incubated for 1 hour at 37°C in 200 μL of 2.8 mM glucose-Krebs buffer (118.5 mM NaCl, 2.54 mM CaCl2, 1.19 mM KH2PO4, 1.19 mM MgSO4, 10 mM HEPES, 2% BSA, pH 7.4). After centrifugation, the supernatant was discarded, and the supernatant was replaced with 200 μL of fresh 2.8 mM glucose-Krebs buffer. The islets were then incubated for another 1 hour at 37°C to collect basal insulin secretion samples. The islets were then incubated sequentially in 200 μL of 16.8 mM glucose-Krebs buffer for 1 hour to assess glucose-stimulated insulin secretion (GSIS), followed by a final incubation for 1 hour in 30 mM KCl-Krebs buffer for further stimulation. The islets were then washed twice with cold PBS and subsequently lysed in RIPA buffer containing protease inhibitors. The lysates were sonicated for 10 seconds to quantify total insulin content and protein. Insulin levels were quantified using a mouse insulin ELISA kit (Mercodia).

[0168] Experimental results are as follows Figure 14 As shown, the VOCPO2-E group was significantly higher than other groups under high glucose stimulation; in the 72h group, when the glucose concentration was 16.8mM, there were no significant differences between the control group and the CP group, and between the control group and the VOC group. These experimental results indicate that PFTBA exerts a synergistic effect with VEGF and OHA.

[0169] Example 6: In vitro assay of pancreatic islet hypoxia activity

[0170] Freshly isolated mouse islets (from 8-12 week old mice) were cultured in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin-streptomycin at 37°C in a humidified incubator under 5% CO2 and 95% air. The islets were divided into normoxic and hypoxic groups, the latter further subdivided into four categories: unencapsulated islets, islets encapsulated in 15 μL VOC, VOCP, or VOCPO2 hydrogels, and cultured under hypoxic conditions for 6 hours in a hypoxic workstation (0% O2, 5% CO2, 37°C).

[0171] Following the instructions, the Image-iT™ Hypoxia Reagent (Invitrogen) was used to assess the degree of hypoxia within the islets. Fluorescence imaging was performed using a Nikon fluorescence microscope and a Lecia confocal microscope (excitation / emission: 488 nm). Two independent observers analyzed hypoxia levels in a blinded manner, and fluorescence intensity was quantified using ImageJ. Three independent experiments were analyzed, each involving 6-60 islets from 6 mice. Results are as follows... Figure 15 As shown in the figure, mouse islets under different oxygen conditions were tested for hypoxia. Hypoxia caused them to emit green light, and the brighter the light, the more hypoxic the condition. The islets encapsulated in our oxygen-carrying hydrogel and the islets cultured under full oxygen conditions did not emit light, which proves the oxygen supply capacity of the material.

[0172] Following the manufacturer's instructions, the Calcein-AM / PI live / dead cell staining kit (Elabscience) was used to assess cell viability within pancreatic islets. Calcein-AM is hydrolyzed by intracellular esterases in live cells, emitting green fluorescence (Ex / Em = 494 / 517 nm), while propidium iodide (PI) selectively enters damaged membrane cells and binds to double-stranded DNA, producing red fluorescence (Ex / Em = 535 / 617 nm). Islets were incubated with Calcein-AM and PI at 37°C for 15 min, washed with PBS, and imaged using Nikon fluorescence microscopy and Leica confocal microscopy. The ratio of Calcein-AM to PI signal intensity for each islet was quantified. Three independent experiments were analyzed, each involving 60 islets from 6 mice. Results are shown below. Figure 16 As shown, our oxygen-enriched hydrogel material encapsulates the islets with the fewest apoptotic events when cultured under full oxygen conditions.

[0173] Example 7: Study on In vivo insulin secretion

[0174] Islet transplantation was performed using 8-week-old male C57BL / 6 mice. Selective ablation of endogenous β-cells was achieved via a single intraperitoneal injection of streptozotocin (STZ, 150 mg / kg; Sigma-Aldrich). For intramuscular islet transplantation, 270 isolated islets (approximately 1-2 million cells) were resuspended in 50 μL LOCPO2 hydrogel and implanted into the leg muscle using a 21-gauge needle. Blood glucose levels were monitored every 2-3 days while the mice were fed. An intraperitoneal glucose tolerance test (IPGTT) was performed on day 6 post-transplantation after overnight fasting. Mice were injected intraperitoneally with D-glucose (2 g / kg), and blood glucose levels were measured at baseline (fasting, 0 minutes) and at 15, 30, 60, and 120 minutes after glucose administration. For glucose-stimulated insulin secretion (GSIS), blood was collected in heparin-coated tubes in a fasting state and 30 minutes after glucose administration. Plasma was obtained by centrifugation (2,000g, 15 min, 4℃), and insulin concentration was quantified using a mouse insulin ELISA kit (Mercodia). Mice were housed under standard conditions in a Guangzhou laboratory. All animal experiments were conducted according to protocols approved by the Guangzhou Laboratory Animal Care and Use Committee.

[0175] Experimental results are as follows Figure 17 As shown, Figure A represents that the material provided in this application can control blood glucose in mice; Figure B represents the glucose tolerance test, which also shows that blood glucose can be reduced under high glucose stimulation, indicating that blood glucose control has been restored; Figure C represents the data analysis of Figure A; and Figure D represents the glucose-responsive insulin secretion stimulation, proving that insulin secretion was restored after pancreatic islet cells encapsulated by hydrogel material were transplanted into mice. Figure 18 This is a schematic diagram illustrating the preparation process and functional verification of the composite hydrogel.

[0176] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A hydrogel system, characterized in that, The hydrogel system includes oxidized hyaluronic acid, chitosan, perfluorotributylamine and angiogenic factor, wherein the chitosan is carboxymethyl chitosan and the perfluorotributylamine is oxygen-loaded perfluorotributylamine. The oxygen-loaded perfluorotributylamine is prepared by the following method: lecithin, cholesterol, and DSPE-PEG2000 are dissolved in dichloromethane, the dichloromethane is removed, the mixture is diluted with PBS solution, and sonicated in an ice bath. The mixture is then mixed with perfluorotributylamine and sonicated to obtain a perfluorotributylamine nanoemulsion. Oxygen is added to the perfluorotributylamine nanoemulsion to obtain an oxygen-loaded perfluorotributylamine nanoemulsion. The mass ratio of chitosan to perfluorotributylamine is 1:20-3:7; The mass ratio of chitosan to oxidized hyaluronic acid is 1:2-3:1; The chitosan has a mass concentration of 0.67%-2%; The mass concentration of the angiogenesis factor was 33.3-66.7 ng / ml.

2. The hydrogel system according to claim 1, characterized in that, The mass ratio of chitosan to perfluorotributylamine is 2:

7.

3. The hydrogel system according to claim 1, characterized in that, The mass ratio of chitosan to oxidized hyaluronic acid is 2:

1.

4. The hydrogel system according to claim 1, characterized in that, The chitosan has a mass concentration of 1.33%.

5. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the hydrogel system according to any one of claims 1-4, and pancreatic islet cells.

6. The pharmaceutical composition according to claim 5, characterized in that, The pharmaceutical composition also includes a pharmaceutically acceptable carrier and / or excipients.

7. A method for preparing the hydrogel system according to any one of claims 1-4, characterized in that, The method includes mixing a blood vessel growth factor-oxidized hyaluronic acid solution with a chitosan-perfluorotributylamine solution; The volume ratio of the vascular endothelial growth factor-oxidized hyaluronic acid solution to the chitosan-perfluorotributylamine solution is 3:5-3:

10. The mass concentration of oxidized hyaluronic acid in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 2%-4%; The mass concentration of vascular endothelial growth factor in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 100-200 ng / ml; The mass concentration of perfluorotributylamine in the chitosan-perfluorotributylamine solution is 7%-20%; The chitosan-perfluorotributylamine solution has a chitosan concentration of 1%-3%.

8. The method according to claim 7, characterized in that, The volume ratio of the vascular endothelial growth factor-oxidized hyaluronic acid solution to the chitosan-perfluorotributylamine solution is 1:

2.

9. The method according to claim 7, characterized in that, The mass concentration of oxidized hyaluronic acid in the vascular endothelial growth factor-oxidized hyaluronic acid solution is 2%.

10. The method according to claim 7, characterized in that, The chitosan-perfluorotributylamine solution has a perfluorotributylamine mass concentration of 7%.

11. The method according to claim 7, characterized in that, The chitosan-perfluorotributylamine solution has a chitosan mass concentration of 2%.

12. The method according to any one of claims 7-11, characterized in that, The method also includes a method for preparing a vascular endothelial growth factor-oxidized hyaluronic acid solution, wherein the preparation method includes dissolving vascular endothelial growth factor in an oxidized hyaluronic acid solution.

13. The method according to any one of claims 7-11, characterized in that, The method also includes a method for preparing an oxidized hyaluronic acid solution, the preparation method comprising dissolving oxidized hyaluronic acid in a PBS solution.

14. The method according to claim 13, characterized in that, The pH of the PBS solution is 7.

4.

15. The method according to any one of claims 7-11, characterized in that, The method also includes a method for synthesizing oxidized hyaluronic acid, which includes dissolving hyaluronic acid in deionized water, adding NaIO4 solution, adding ethylene glycol, and dialyzing.

16. The method according to any one of claims 7-11, characterized in that, The method also includes a method for preparing a chitosan-perfluorotributylamine solution, wherein the preparation method includes dissolving chitosan in a perfluorotributylamine nanoemulsion.

17. The method according to any one of claims 7-11, characterized in that, The method also includes a method for synthesizing oxygen-loaded perfluorotributylamine nanoemulsion, the synthesis method comprising the following steps: dissolving lecithin, cholesterol and DSPE-PEG2000 in dichloromethane, removing the dichloromethane, diluting the mixture with PBS solution, sonicating in an ice bath, mixing the mixture with perfluorotributylamine, sonicating to obtain perfluorotributylamine nanoemulsion, and adding oxygen to the perfluorotributylamine nanoemulsion to obtain oxygen-loaded perfluorotributylamine nanoemulsion.

18. Use of the pharmaceutical composition according to any one of claims 5-6 in the preparation of a medicament for treating diseases caused by insulin deficiency or insufficiency.

19. The application according to claim 18, characterized in that, The diseases caused by insulin deficiency or insufficiency include hyperglycemia, type 1 diabetes, type 1 or type 2 diabetes-related complications, and other rare types of diabetes.

20. The application according to claim 19, characterized in that, The disease caused by insulin deficiency or insufficiency is selected from type 1 diabetes.

21. Use of the pharmaceutical composition according to any one of claims 5-6 in the preparation of a medicament for promoting insulin secretion.

22. The use of the hydrogel system according to any one of claims 1-4 or the pharmaceutical composition according to any one of claims 5-6 in the preparation of a medicament for inhibiting pancreatic islet cell apoptosis.

Images