CN120000580B

CN120000580B - A hydrogel system, its preparation method and application

Info

Publication number: CN120000580B
Application number: CN202510241690.8A
Authority: CN
Inventors: 杜雯; 孙钊裕; 张雨薇
Original assignee: Guangzhou Medical University
Current assignee: Guangzhou Medical University
Priority date: 2025-03-03
Filing date: 2025-03-03
Publication date: 2025-11-21
Anticipated expiration: 2045-03-03
Also published as: CN120000580A

Abstract

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

Description

Hydrogel system and preparation method and application thereof

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a hydrogel system and a preparation method and application thereof.

Background

Diabetes severely affects the quality of life of the patient. Type 1 diabetes (T1D) is an emerging method for treating T1D in recent years due to the destruction or loss of islet function caused by autoimmune attack, absolute deficiency of insulin secretion, and islet transplantation.

However, both post-transplant immune attack and hypoxia damage the transplanted islets, ultimately leading to failure of islet transplantation. To solve the two problems, the establishment of an immune tolerant microenvironment and oxygen supply in the local part of the implant is a good strategy, good blood supply can provide necessary oxygen and nutrients, and vascular reconstruction after implantation is a key to ensure long-term survival and function of transplanted islet cells.

Hydrogels are gels having a three-dimensional network structure formed by physical or chemical crosslinking of polymers, and have been widely used in the field of bioengineering in recent years. The hydrogel not only can be used for packaging cells to simulate the microenvironment of the cells, but also has the function of slowly releasing therapeutic drugs. The islet graft is encapsulated with natural or synthetic materials, and a barrier with immune isolation function is formed on the surface of the graft, so that the immune rejection of a host can be relieved. In addition, the islet encapsulating material can also be used as a carrier of oxygen-carrying medicaments or medicaments for promoting blood vessel growth.

However, after the hydrogel is coated, the diffusion of oxygen is limited, so that islet cells are easy to hypoxia and necrosis. The existing hydrogel materials for islet transplantation comprise collagen hydrogel, alginate hydrogel, polyethylene glycol (PEG), polylactic acid-glycolic acid copolymer (PLGA) hydrogel and the like, which have the defects of relatively high degradation speed, possibility of immune reaction initiation, lack of bioactivity, lack of cell adhesion sites, frequent modification of bioactive molecules and the like, and do not solve the early hypoxia problem of islet cells and the requirements of in-situ islet vascular remodeling. The traditional oxygen-carrying materials such as hydrogen peroxide are decomposed to generate oxygen, free radicals can damage cells, hemoglobin has the problems of immunogenicity and stability, and the oxygen-carrying time is short.

Disclosure of Invention

In view of this, the present invention has been made in order to make up for the deficiencies of the prior art.

In a first aspect, the present invention provides a hydrogel system comprising oxidized hyaluronic acid, chitosan, perfluoro tributylamine, and a vascular growth factor.

In the invention, oxidized hyaluronic acid and chitosan are physically crosslinked into gel by utilizing Schiff base reaction and by utilizing amine aldehyde condensation reaction between natural polysaccharides.

Further, the chitosan includes, but is not limited to, carboxymethyl chitosan, hydroxyethyl chitosan, quaternized chitosan, thiolated chitosan, acylated chitosan, phosphorylated chitosan, glycosylated chitosan, pegylated chitosan.

Further, the chitosan is selected from carboxymethyl chitosan.

Further, the perfluoro tributylamine is oxygen-carrying perfluoro tributylamine.

Further, the mass ratio of the chitosan to the perfluoro tributylamine is 1:20-3:7.

Further, the mass ratio of the chitosan to the perfluorotributylamine is 2:7.

Further, the mass ratio of the chitosan to the oxidized hyaluronic acid is 1:2-3:1.

Further, the mass ratio of the chitosan to the oxidized hyaluronic acid is 2:1.

Further, the mass concentration of the chitosan is 0.67% -2%.

Further, the mass concentration of the chitosan was 1.33%.

Further, the mass concentration of the vascular growth factor is 33.3-66.7ng/ml.

In a second aspect, the invention provides a pharmaceutical composition comprising a hydrogel system according to the first aspect of the invention.

Further, the pharmaceutical composition further comprises islet cells.

Further, the pharmaceutical composition comprises pharmaceutically acceptable carriers and/or auxiliary materials thereof.

In the present invention, "pharmaceutically acceptable carrier and/or adjuvant" includes, but is not limited to, diluents, binders, surfactants, wetting agents, adsorption carriers, lubricants, disintegrants, emulsifiers, bioavailability enhancers, suspending agents, sweeteners, flavoring agents, colorants, excipients, preservatives, solubilizers, dispersing agents and/or wetting agents. Wherein the diluents include, but are not limited to, lactose, sodium chloride, dextrose, urea, starch, water, binders include, but are not limited to, starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methylcellulose, ethylcellulose, polyvinyl alcohol, polyethylene glycol, polyvinylpyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, and hydroxypropyl methylcellulose, surfactants include, but are not limited to, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, monoglyceride of stearic acid, cetyl alcohol, humectants include, but are not limited to, glycerin, starch, adsorbent carriers include, but are not limited to, starch, lactose, bentonite, silica gel, kaolin, soap clay, 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 stearyl fumarate, polyoxyethylene monostearate, monolauryl saccharate, sodium lauryl sulfate, magnesium lauryl sulfate.

In the present invention, the pharmaceutical composition is administered in the form of an injection administration type.

Further, the injectable administration form includes, but is not limited to, intravenous injection, intramuscular injection, subcutaneous injection, intradermal injection or intracavity injection.

Further, the injectable administration is selected from intravenous injection.

Further, the vein is a portal vein.

In the present invention, the pharmaceutical composition and at least one additional therapeutic agent or therapy may be administered sequentially, concurrently and/or alternately, wherein the at least one additional therapeutic agent or therapy includes, but is not limited to, insulin therapy, dietary management, exercise intervention, adjuvant drug therapy, emerging therapeutic therapy.

Further, the insulin therapy includes, but is not limited to, administration of ultra-short, medium, long acting insulin.

Further, the ultra-short acting insulin includes, but is not limited to, insulin aspart, insulin lispro, which is used for pre-meal injection to control postprandial blood glucose.

Further, the short acting insulin includes, but is not limited to, regular insulin, which is used for pre-meal injection or intravenous injection (e.g., in diabetic ketoacidosis).

Further, the medium-acting insulin includes, but is not limited to, neutral zinc protamine insulin, which is used for basal insulin supplementation.

Further, the long acting insulin includes, but is not limited to, insulin glargine, insulin detention, insulin deglutition, such insulin being used for basal insulin supplementation.

Further, the insulin administration modes include, but are not limited to, subcutaneous injection, insulin pump administration, inhalation administration.

Further, the adjuvant therapy includes, but is not limited to, administration of pramlintide, SGLT-2 inhibitors.

Further, the emerging therapeutic therapies include, but are not limited to, stem cell therapies, immunotherapy.

In the present invention, the pharmaceutical composition is administered in a therapeutically effective amount of dosage. The term "therapeutically effective amount" refers to the level or amount of an agent of interest that does not produce significant negative or adverse side effects (1) delay or prevent the onset of a disease or its complications resulting from an insulin deficiency or deficiency, (2) slow or prevent the progression, exacerbation or worsening of a disease or its complications resulting from an insulin deficiency or deficiency, (3) ameliorate the symptoms of a disease or its complications resulting from an insulin deficiency or deficiency, (4) reduce the severity or incidence of a disease or its complications resulting from an insulin deficiency or deficiency, and (5) cure a disease or its complications resulting from an insulin deficiency or deficiency.

In a third aspect the present invention provides a method of preparing a hydrogel system according to the first aspect of the invention, the method comprising mixing a solution of a vascular growth factor-oxidized hyaluronic acid with a solution of chitosan-perfluoro tributylamine.

Further, the volume ratio of the vascular growth factor-oxidized hyaluronic acid solution to the chitosan-perfluoro tributylamine solution is 3:5-3:10.

Further, the volume ratio of the vascular growth factor-oxidized hyaluronic acid solution to the chitosan-perfluoro tributylamine solution is 1:2.

Further, the mass concentration of the oxidized hyaluronic acid in the blood vessel growth factor-oxidized hyaluronic acid solution is 2% -4%.

Further, the mass concentration of oxidized hyaluronic acid in the blood vessel growth factor-oxidized hyaluronic acid solution is 2%.

Further, the mass concentration of the blood vessel growth factor in the blood vessel growth factor-oxidized hyaluronic acid solution is 100-200ng/ml.

Further, the mass concentration of the perfluoro tributylamine in the chitosan-perfluoro tributylamine solution is 7-20%.

Further, the mass concentration of the perfluorotributylamine in the chitosan-perfluorotributylamine solution is 7%.

Further, the mass concentration of the chitosan in the chitosan-perfluoro tributylamine solution is 1-3%.

Further, the mass concentration of chitosan in the chitosan-perfluoro tributylamine solution is 2%.

Further, the method also includes a method of preparing a vascular growth factor-oxidized hyaluronic acid solution, the method including dissolving the vascular growth factor in the oxidized hyaluronic acid solution.

Further, the method also includes a method of preparing an oxidized hyaluronic acid solution, the method including dissolving oxidized hyaluronic acid in a PBS solution.

Further, the pH of the PBS solution was 7.4.

Further, the method also comprises a synthesis method of oxidized hyaluronic acid, wherein the synthesis method comprises the steps of dissolving hyaluronic acid in deionized water, adding NaIO ₄ solution, adding ethylene glycol and dialyzing.

Further, the mass ratio of the hyaluronic acid to the NaIO ₄ is 1.43:1-2.4:1.

Further, the volume ratio of the deionized water to the NaIO ₄ solution is 5:1-6:1.

Further, naIO ₄ solution was added and stirred in the dark until the reaction was completed.

Further, lyophilization was performed after dialysis.

Further, the method also comprises a preparation method of the chitosan-perfluoro tributylamine solution, wherein the preparation method comprises the step of dissolving chitosan in the perfluoro tributylamine nanoemulsion.

Further, the method comprises the steps of dissolving lecithin, cholesterol and DSPE-PEG2000 in dichloromethane, removing the dichloromethane, diluting the mixture by using PBS solution, carrying out ice bath ultrasonic treatment, mixing the mixture with the perfluorotributylamine, and carrying out ultrasonic treatment to obtain the perfluorotributylamine nanoemulsion.

Further, the mass ratio of the lecithin to the cholesterol is 5:1-10:1.

Further, the mass ratio of the lecithin to the cholesterol is 6:1-7:1.

Further, the mass ratio of the cholesterol to the DSPE-PEG2000 is 1:1-1:2.

Further, the mass ratio of cholesterol to DSPE-PEG2000 was 12.37:13.09.

Further, the mass concentration of the cholesterol after dissolution in methylene chloride is 1mg/ml to 10mg/ml.

Further, the mass concentration of the cholesterol after dissolution in methylene chloride was 1.005mg/ml.

Further, methylene chloride was removed by rotary evaporation.

Further, rotary evaporation was performed at 25-35 ℃.

Further, rotary evaporation was performed at 30 ℃.

Further, the rotary evaporation was performed at 100rpm to 200 rpm.

Further, rotary evaporation was performed at 156 rpm.

Further, the pH of the PBS solution was 7.4.

Further, ice bath ultrasound was performed twice.

Further, ice bath ultrasound was performed for two minutes each time.

Further, the interval between two ice bath sonications was 1min.

Further, the volume concentration of the perfluoro tributylamine is 4.5-15%.

Further, the volume concentration of the perfluorotributylamine was 4.5%.

Further, the method also comprises the step of adding oxygen to the perfluoro tributylamine nanoemulsion to obtain the oxygen-carrying perfluoro tributylamine nanoemulsion.

A fourth aspect of the invention provides any one of the following applications:

(1) Use of a hydrogel system according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for the manufacture of a medicament for the treatment of a disease caused by insulin deficiency or insufficiency;

(2) The application of the vascular growth factor and the perfluoro tributylamine in preparing the medicine for treating the diseases caused by the deficiency or the insufficiency of the insulin;

(3) Application of carboxymethyl chitosan and oxidized hyaluronic acid in preparing medicine for treating diseases caused by insulin deficiency or deficiency;

(4) Use of the hydrogel system according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for promoting insulin secretion;

(5) Use of the hydrogel system according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention for inhibiting apoptosis of islet cells;

(6) Use of a hydrogel system according to the first aspect of the invention or a pharmaceutical composition according to the second aspect of the invention for promoting angiogenesis.

In the present invention, the diseases caused by the deficiency or deficiency of insulin include, but are not limited to, hyperglycemia, type 1 diabetes, type 2 diabetes, type 1 diabetes or complications associated with type 2 diabetes, other rare type diabetes.

In the present invention, type 1 diabetes is mainly caused by abnormal activation of the patient's autoimmune system, thereby causing destruction of beta cells in langerhans islets by the autoimmune system, ultimately leading to insulin deficiency.

Further, the disease caused by the deficiency or deficiency of insulin is selected from type 1 diabetes.

In the present invention, diseases caused by insulin deficiency or insufficiency can be treated by islet transplantation.

Further, islet transplantation can be performed using the hydrogel system according to the first aspect of the invention or the pharmaceutical composition according to the second aspect of the invention.

In the present invention, "islets" are to be understood broadly to include any cell or cell aggregate that secretes insulin.

Further, islets can be obtained from an individual, such as a donor, including an allogeneic donor, an autologous donor, or a xenogeneic donor.

Further, islets can be derived from stem cells (including ESCs and iPSCs) or islet progenitor cells.

In the present invention, "promoting insulin secretion" refers to allowing islet cells to promote 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.

In the present invention, "inhibiting islet cell apoptosis" refers to the ability of islet cells to reduce their apoptosis. For example, the proportion of apoptosis or rate of apoptosis of 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 more than 98%.

The fifth aspect of the invention provides a method as defined in any one of the following:

(1) A method of promoting angiogenesis in vitro, the method comprising administering the hydrogel system of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention;

(2) A method of promoting insulin secretion in vitro comprising administering the hydrogel system of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention;

(3) A method of inhibiting islet cell apoptosis in vitro comprising administering the hydrogel system of the first aspect of the invention or the pharmaceutical composition of the second aspect of the invention.

In the present invention, "in vitro" refers to any scene that is not in vivo.

In the present invention, "in vitro promoting angiogenesis" may be exemplified as including, but not limited to, administering the hydrogel system according to the first aspect of the present invention or the pharmaceutical composition according to the second aspect of the present invention to human umbilical vein endothelial cells in a laboratory to promote differentiation and formation of vascular networks of human umbilical vein endothelial cells.

The invention has the advantages and beneficial effects that:

Drawings

FIG. 1 is a chart showing Fourier infrared absorption spectra of hyaluronic acid and oxidized hyaluronic acid.

Fig. 2 is a graph showing the result of detecting the degree of oxidization of the oxidized hyaluronic acid material.

FIG. 3 is a transmission electron microscope image of a perfluorotributylamine nanoemulsion.

Fig. 4 is a DLS light scattering plot of perfluorotributylamine nanoemulsion.

FIG. 5 is a graph of the microtopography and pore size of the composite hydrogel.

FIG. 6 is a gel-forming physical diagram of the composite hydrogel.

FIG. 7 is a graph showing the results of the composite hydrogel degradation experiment.

Fig. 8 is a graph of the alternate scan results of the rotary rheometer.

FIG. 9 is a diagram showing the self-healing experimental process of the composite hydrogel.

FIG. 10 is a graph showing the results of the rheological property test of the hydrogel.

FIG. 11 is a graph showing the results of a hydrogel cytotoxicity test.

FIG. 12 is a graph showing the results of hydrogel hemocompatibility experiments.

FIG. 13 is a graph showing the results of in vitro vascularization experiments.

FIG. 14 is a graph showing the results of an in vitro insulin secretion assay.

FIG. 15 is a graph showing the results of an in vitro islet hypoxia experiment.

Fig. 16 is a graph showing experimental results of islet apoptosis under hypoxic conditions.

Fig. 17 is a graph showing the results of an in vivo insulin secretion test, wherein graph a shows the results of a blood glucose test in mice, graph B shows the results of a glucose tolerance test, graph C shows the data analysis graph of graph a, and graph D shows the results of insulin secretion recovery.

FIG. 18 is a schematic diagram showing the preparation flow and functional verification of the composite hydrogel.

Detailed Description

The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.

EXAMPLE 1 preparation of vascular growth factor-oxidized hyaluronic acid (VEGF-OHA) solution

OHA synthesis 1.0-1.2g HA was dissolved in 100-120mL deionized water, 0.5-0.7g NaIO ₄ was dissolved in 20mL deionized water, and then added dropwise to the HA solution. The reaction mixture was stirred at room temperature for 6 hours without light to ensure complete reaction. Subsequently, unreacted NaIO ₄ was neutralized with 1.0mL of ethylene glycol, and the mixture was allowed to react with stirring for another 2 hours. The resulting product was dialyzed against double distilled water (MWCO 12,000) for 3 days, with the dialysate changed three times per day to remove residual periodate and ethylene glycol. After lyophilization, a white cotton-like material, oxidized hyaluronic acid, was obtained and stored under vacuum.

Fourier infrared absorption spectrum, namely, freeze-drying the material, fully grinding the material and potassium bromide according to the mass ratio of 1:100, and pressing the material into slices. The freeze-dried hydrogel sample is scanned by means of an ATR accessory over a wavelength range of 500-4000 cm ^-1 by fourier infrared spectroscopy. The Fourier infrared absorption spectra of HA and OHA are shown in FIG. 1.

Measurement of the degree of Oxidation 0.05g of lyophilized OHA was dissolved in 20ml of 0.25mol/L hydroxylamine hydrochloride (0.3475 g of pH 4.5 adjusted) containing 0.002% by weight of methyl orange reagent, the mixture was stirred at room temperature for 24h, the aldehyde was converted to oxime, and then the released hydrochloric acid was titrated with 1mol/L sodium hydroxide solution until the red to yellow end point was reached. The change in pH with the volume of sodium hydroxide solution added was recorded. The correlation reaction and calculation formula are as follows:

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

HCl+NaOH=NaCl+H₂0(2);

Oxidation degree (%) =403 Δvxc10 ^-3/2 w;

Wherein 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.

As shown in FIG. 2, the oxidation degree of OHA was about 48%.

OHA is dissolved in PBS (PH 7.4) solution to prepare OHA solution with the mass concentration of 2-4 percent.

VEGF is dissolved in the OHA solution to obtain VEGF-OHA solution, and the mass concentration of VEGF is 100-200ng/ml.

EXAMPLE 2 preparation of carboxymethyl chitosan-perfluorotributylamine (CMC-PFTBA) solution

Synthesis of oxygen-carrying PFTBA nanoemulsion to prepare the PFTBA nanoemulsion 60.586mg of lecithin, 10.05mg of cholesterol and 10.635mgDSPE-PEG2000 were dissolved in 10mL of dichloromethane. After removal of the dichloromethane using rotary evaporation at 30℃and 156rpm, the resulting mixture was diluted in 5.0mLPBS solution (pH 7.4) and sonicated in an ice bath (300W for 3 minutes each) with a1 minute interval between treatments. Thereafter, 4.775mL of the sonicated solution was similarly mixed with 0.225mL of perfluorotributylamine and the ice bath sonicated twice to produce PFTBA nanoemulsion at a volume concentration of 4.5% -15% (converted to a mass concentration of 7% -20%). The resulting emulsion was then placed in a hyperbaric oxygen chamber for 30 minutes to reach full oxygen saturation. The solution was finally passed through a 0.22 μm sterile filter to give a sterile oxygen-loaded PFTBA nanoemulsion (PFTBA-O ₂ emulsion).

The morphology of PFTBA-O ₂ was examined using a transmission electron microscope (TEM, talosL120CG2, thermo scientific) and the experimental results are shown in FIG. 3. The hydrated particle size was determined using MalvernZetasizerNanoS-90 Dynamic Light Scattering (DLS) equipment and the experimental results are shown in FIG. 4.

And dissolving carboxymethyl chitosan (CMC) in the PFTBA nanoemulsion to obtain CMC-PFTBA solution, wherein the mass concentration of the carboxymethyl chitosan is 1% -3%.

VEGF-OHA (VO) solution is added into islet cell sediment to re-suspend islet cells, CMC-PFTBA (CP) solution is added according to the volume ratio of 1:2 to be mixed into glue, so as to complete preparation before transplantation.

Example 3 VO volume ratio to CP and Mass concentration screening optimization

The microscopic morphology and pore size of the composite hydrogel were observed using a scanning electron microscope, and the experimental results are shown in fig. 5.

Gel time of the hydrogels was assessed using a sample bottle inversion method by introducing a precursor solution into a sample bottle and gelling at 25 ℃. The flowability of the mixture was evaluated and the gelation time was recorded as the time when the sample stopped flowing when the sample bottle was inverted, and FIG. 6 is a gel-forming physical image of the composite hydrogel.

Different experimental groups are set, experiments are carried out according to the volume ratios of VO to CP=1:2, 2:1, 3:2, 1:1 and 2:3, and as a result, the volume ratios of 2:1, 3:2, 1:1 and 2:3 are not good in glue forming or the glue forming time is long.

Mass concentration screening optimization experiments were performed using VO: cp=1:2 volume ratio, wherein mass concentrations of OHA in VO were set to 2%, 3%, 4%, respectively, and mass concentrations of CMC in CP were set to 1%, 2%, 3%, respectively, and the experimental results are shown in table 1.

Table 1 VO and CP Mass concentration screening optimization experiment

Example 4 PFTBA volume concentration screening optimization

Through degradation experiments, rheometer rheological property characterization and cytotoxicity experiments are carried out to screen different degradation performances, self-healing performances, injectability, cytotoxicity and in-vitro angiogenesis experiments of volume concentration contained in PFTBA in the material, and experiments are carried out according to the volume ratio of VO: CP=1:2, wherein the mass concentration of OHA and CMC is 2%. (vocp represents no PFTBA, vocp represents a final volume concentration of PFTBA of 3% and vocp represents a final volume concentration of PFTBA of 5% in the case of 2% both of the OHA and CMC mass concentrations).

(1) Degradation experiment

Degradation of VOCs, VOCP3 and VOCP5 (n=3) was assessed by measuring weight loss in PBS over 21 days. Initial weight of lyophilized samples (W ₀) was measured by soaking in PBS at 37 ℃ for specified intervals (1, 4, 7, 14 and 21 days). At each time point, samples were extracted from PBS, lyophilized, and then re-weighed (Wd). Degradation rate was determined using the following method, degradation rate (%) =degradation rate (%). The results of the experiment are shown in Table 2 and the weight drop graph of the hydrogels are shown in FIG. 7, and the percent weight drop of the PFTBA-added hydrogels is smaller than that of the PFTBA-not-added hydrogels.

TABLE 2 results of Performance experiments

(2) Self-healing properties of rheometer hydrogels

(Storage modulus at low strain is larger than loss modulus, gel state is maintained, storage modulus at high strain is smaller than loss modulus, gel is broken to become fluid, after 60s of self-healing time, the gel is converted from high strain to low strain, the test is continued, storage modulus of the material is larger than loss modulus, gel state is recovered, storage modulus (which can be regarded as hardness) is not reduced and stable, self-healing performance is good) and the rheological property of the hydrogel is evaluated by using a An Dongpa MCR-302e rotary rheometer. A cylindrical hydrogel having a diameter of 12mm and a height of 3mm was prepared, placed between 25mm parallel plates having a gap of 1.00mm, and the periphery was sealed with silicone oil to suppress evaporation of water. Three cycles of small strain (1%) and large strain (500%) of VOCs and VOCPs were alternately scanned using An Dongpa MCR-302e rotary rheometer to investigate their self-healing properties. The experimental results are shown in Table 2 and FIG. 8.

Self-healing experiments on hydrogels we initially produced two circular VOCP gels using aqueous pigments to evaluate self-healing behavior. After gelation, each VOCP gel was split into two, and one semicircle of red hydrogel was combined with one semicircle of transparent hydrogel to form a new structure. After a 30 minute self-healing period at room temperature, tensile stress was applied manually by reattaching the samples with forceps to observe the adhesion area, the experimental procedure is shown in fig. 9 (initial, self-healing 30min, tensile plot, respectively, from left to right).

(3) Injectability of

(The viscosity decreases with increasing shear rate, representing shear thinning behavior, demonstrating good injectability) the rheological properties of hydrogels were evaluated using a An Dongpa MCR-302e rotary rheometer. A cylindrical hydrogel having a diameter of 12mm and a height of 3mm was prepared, placed between 25mm parallel plates having a gap of 1.00mm, and the periphery was sealed with silicone oil to suppress evaporation of water. In addition, the shear viscosity measurement range is 0.1 to 100 (1/s) shear rate. The results of the experiments are shown in Table 2 and FIG. 10, with the hydrogel supplemented with 3% PFTBA having the best injectability.

(4) Cell proliferation and cytotoxicity assays

(Viability was assessed according to ISO10993 standard, <70% viability indicated cytotoxicity.) Material extracts were prepared by incubating sterilized samples in serum-free DMEM (Thermo) or RPMI-1640 (Life technology) for 24 and 48 hours at 37 ℃, including OHA-CMC hydrogels (OC), VEGF@OHA-CMC hydrogels without PFTBA (VOC) and VEGF@OHA-CMC hydrogels supplemented with 3% or 5% PFTBA (3% VOCP, 5% VOCP) according to ISO10993-12 guidelines. The extract was 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 ℃ 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 viability was assessed according to ISO10993 standards, with viability of <70% indicating cytotoxicity. The experimental results are shown in Table 2, and the results in FIG. 11 show that the hydrogels provided by the present invention are nontoxic to cells.

(5) Blood compatibility experiments of hydrogels:

The hemolysis test was used to determine the hemocompatibility of the hydrogels. Erythrocytes were isolated by centrifugation at 4000rpm for 5 minutes, then washed 3 times with PBS, and centrifuged again at 4000rpm for 5 minutes each. Erythrocytes were resuspended in PBS at a concentration of 2% v/v. Subsequently, 10mg of the lyophilized hydrogel was incubated with 1mL of red blood cell suspension at 37 ℃ for 4 hours. The hydrogel was extracted and the red blood cell suspension was centrifuged at 4000rpm for 5 minutes. The absorbance of the centrifuged supernatant was recorded at 540 nm. Double distilled water served as positive control and PBS served as negative control. The ratio of hemolysis was determined using the following formula:

Hemolysis ratio (%) = (As-Ab) - (Ap-Ab) ×100%;

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.

The experimental result is shown in figure 12, and the hydrogel provided by the invention has better blood compatibility.

(6) In vitro angiogenesis experiments

Human Umbilical Vein Endothelial Cells (HUVECs) were seeded in 48-well plates at a density of 2x 10 ⁴ cells per well and resuspended in DMEMGlutamax (Thermo) supplemented with 10% fbs and 1% penicillin-streptomycin. Cells were seeded onto uncoated plastic surfaces (control) or substrates pre-coated with 20 μl of matrigel without LDEV (corning), and VOC, VOCP3 or VOCP5 hydrogels were then carefully placed into each well. Triplicate tests were performed for each condition.

After 12 hours incubation at 37 ℃, renal tubule formation was assessed using a phase contrast microscope (Nikon) at 10 x magnification. For quantitative analysis, three central areas randomly selected for each filter were examined. Images were analyzed using AngiogenesisAnalyzer plug-in for ImageJ, quantifying key parameters including total tubular length, node number, and branching points.

The experimental results are shown in fig. 13, and our materials perform better in vitro vascularization than the Matrigel (Matrigel) which has been commercialized, and are superior in both total length and node number and tube number, wherein we used this concentration in animal experiments because vocp3 vascularization was the best.

EXAMPLE 5 in vitro insulin secretion Studies

Glucose stimulated insulin secretion was assessed in islets of mice cultured under different conditions, with the group settings being the PFTBA-containing no VEGF group (CPO ₂ -S), the VEGF-containing no PFTBA group (VOC-E), the VEGF-containing and PFTBA groups (VOCPO ₂ -E), respectively.

In vitro islet isolation of mice pancreas was harvested from 8 week old mice after abdominal midline incision. Cold collagenase P solution (Roche) was injected into the pancreas through the hollow bile duct to achieve uniform enzyme perfusion of the tissue. The pancreas was then resected and incubated at 37 ℃ for 16 minutes to promote enzymatic 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, merckMillipore) for islet purification. After gradient centrifugation, the solution was allowed to stand for 10-15 minutes to further separate islets from acinar tissue. Purified islets were manually selected and counted under a microscope. All mouse data were from at least 3 independent islet isolates.

Freshly isolated mouse islets were washed 3 times with PBS and 20 islets were allocated per well. For suspension culture, islets were maintained in RPMI-1640 medium supplemented with 10% FBS and 3% penicillin-streptomycin, or kept in the same medium containing 15. Mu. LCMC-3% PFTBAO ₂（CPO₂ -S). For the embedding culture, islets were embedded in 15 μl of a hydrogel formulation, including VEGF@OHA-CMC (VOC-E) or VEGF@OHA-CMC-3% PFTBAO ₂（VOCPO₂ -E), and immersed in the culture medium. After 48 or 72 hours incubation, the medium was removed, islets were washed 3 times with cold PBS and then pre-incubated in 200 μl of 2.8mM glucose-krebs buffer （118.5mM NaCl、2.54mM CaCl₂、1.19mM KH₂PO₄、1.19mM MgSO₄、10mM HEPES、2%BSA,pH7.4） for 1 hour at 37 ℃. After centrifugation, the supernatant was discarded, replaced with 200 μl of fresh 2.8mM glucose-krebs buffer and incubated at 37 ℃ for an additional 1 hour to collect basal insulin secretion samples. Islets were incubated sequentially for 1 hour in 200 μl of 16.8mM glucose-Krebs buffer to assess glucose-stimulated insulin secretion (GSIS), then last incubated for 1 hour in 30mM KCl-Krebs buffer for further stimulation. Islets were then washed twice with cold PBS and subsequently lysed in RIPA buffer containing protease inhibitors. Lysates were sonicated for 10 seconds to quantify total insulin content and protein. Insulin levels were quantified using the mouse insulin ELISA kit (Mercodia).

The experimental results are shown in FIG. 14, wherein VOCPO ₂ -E group is significantly higher than other groups under high sugar stimulation, and when the glucose concentration is 16.8mM in 72h group, there is no significant difference between the control group and the CP group and between the control group and the VOC group. The above experimental results demonstrate that PFTBA exerts a synergistic effect with VEGF and OHA.

Example 6 in vitro islet hypoxia vitality assay

Freshly isolated mouse islets (from 8-12 week old mice) were cultured in RPMI-1640 medium supplemented with 10% fbs and 1% penicillin-streptomycin in a humidified incubator at 37 ℃ under 5% co ₂ and 95% air. Islets are divided into normoxic and hypoxic groups, the latter being subdivided into four cases, unencapsulated islets, islets encapsulated in 15ul voc, VOCP or VOCPO ₂ hydrogels. Hypoxia incubation was performed in a hypoxic workstation (0%O ₂、5%CO₂, 37 ℃) for 6 hours.

Image-iTTM hypoxia reagent (Invitrogen) was used to assess the extent of hypoxia within islets according to the instructions. Fluorescence imaging was performed using a Nikon fluorescence microscope and Lecia confocal (excitation/emission: 488 nm). Two independent observers analyzed hypoxia levels blindly and quantified fluorescence intensity using ImageJ. Three independent experiments were analyzed, each including-60 islets from 6 mice. The experimental results are shown in fig. 15, and the oxygen deficiency of the rat islets under different oxygen conditions is detected, the oxygen deficiency emits green light, the brighter the oxygen deficiency is, the islets wrapped by the oxygen-carrying hydrogel and the islets cultured under the total oxygen condition can be seen to be non-luminous, and the oxygen supply capability of the material is proved.

Cell viability within islets was assessed using calcein AM/PI viable dead cell staining kit (Elabscience) according to the instructions. calcein-AM is hydrolyzed by cytolactonase in living cells, emitting green fluorescence (Ex/em=494/517 nm), while Propidium Iodide (PI) selectively enters membrane-damaged cells and binds to double-stranded DNA, producing red fluorescence (Ex/em=535/617 nm). Islets were incubated with Calcein-AM and PI for 15 min at 37 ℃, washed with PBS, and imaged using Nikon fluorescence microscopy and Leica confocal microscopy. The ratio of each of the islet calcein-AM to PI signal intensities was quantified. Three independent experiments were analyzed, each comprising 60 islets from 6 mice. The experimental results are shown in fig. 16, and the number of islet apoptosis cultured under the condition of islet and total oxygen coated by the oxygen-supplying hydrogel material is minimum.

EXAMPLE 7 in vivo insulin secretion Studies

Islet transplantation was performed using 8 week old male C57BL/6 mice. Selective ablation of endogenous beta cells was targeted by a single intraperitoneal injection of streptozotocin (STZ, 150mg/kg; sigma-Aldrich). For intramuscular islet transplantation, 270 isolated islets (about 1-200 ten thousand cells) were resuspended in 50 μ LVOCPO ₂ hydrogel and transplanted into leg muscles using a 21 gauge needle. Blood glucose levels were monitored every 2-3 days in the fed state. After overnight fast, an intraperitoneal glucose tolerance test (IPGTT) was performed on day 6 post-implantation. Mice were intraperitoneally injected with D-glucose (2 g/kg) and blood glucose levels were measured at baseline (fasting, 0 minutes) and 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 (2,000 g,15min,4 ℃) and insulin concentration was quantified using the mouse insulin ELISA kit (Mercodia). Mice were kept in the Guangzhou laboratory under standard conditions. All animal experiments were performed according to the protocol approved by the committee for animal care and use, laboratory, guangzhou.

The experimental results are shown in fig. 17, wherein the graph A represents that the material provided by the application can control the blood sugar of a mouse, the graph B represents that the glucose tolerance experiment can reduce the blood sugar under the stimulation of high sugar to indicate that the blood sugar control is restored, the graph C represents that the data analysis of the graph A is performed, the graph D represents that the insulin secretion stimulation of glucose response proves that the insulin secretion is restored after the islet cells coated by the hydrogel material are transplanted into the body of the mouse. FIG. 18 is a schematic diagram showing the preparation flow and functional verification of the composite hydrogel.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. The hydrogel system is characterized by comprising oxidized hyaluronic acid, chitosan, perfluoro tributylamine and a blood vessel growth factor, wherein the chitosan is carboxymethyl chitosan, and the perfluoro tributylamine is oxygen-carrying perfluoro tributylamine;

Dissolving lecithin, cholesterol and DSPE-PEG2000 in methylene dichloride, removing the methylene dichloride, diluting the mixture by using PBS solution, carrying out ice bath ultrasound, mixing the mixture with the perfluorotributylamine, carrying out ultrasound to obtain perfluorotributylamine nanoemulsion, and adding oxygen into the perfluorotributylamine nanoemulsion to obtain the oxygen-carrying perfluorotributylamine nanoemulsion;

The mass ratio of the chitosan to the perfluoro tributylamine is 1:20-3:7;

the mass ratio of the chitosan to the oxidized hyaluronic acid is 1:2-3:1;

The mass concentration of the chitosan is 0.67% -2%;

The mass concentration of the vascular growth factor is 33.3-66.7ng/ml.

2. The hydrogel system of claim 1, wherein the mass ratio of chitosan to perfluorotributylamine is 2:7.

3. The hydrogel system of claim 1, wherein the mass ratio of chitosan to oxidized hyaluronic acid is 2:1.

4. The hydrogel system of claim 1, wherein the chitosan has a mass concentration of 1.33%.

5. A pharmaceutical composition comprising the hydrogel system of any one of claims 1-4, and islet cells.

6. The pharmaceutical composition of claim 5, further comprising a pharmaceutically acceptable carrier and/or adjuvant.

7. A method of making the hydrogel system of any one of claims 1-4, comprising mixing a solution of a vascular growth factor-oxidized hyaluronic acid with a solution of chitosan-perfluoro tributylamine;

The volume ratio of the vascular growth factor-oxidized hyaluronic acid solution to the chitosan-perfluoro tributylamine solution is 3:5-3:10;

The mass concentration of the oxidized hyaluronic acid in the blood vessel growth factor-oxidized hyaluronic acid solution is 2% -4%;

the mass concentration of the blood vessel growth factor in the blood vessel growth factor-oxidized hyaluronic acid solution is 100-200ng/ml;

the mass concentration of the perfluoro tributylamine in the chitosan-perfluoro tributylamine solution is 7-20%;

the mass concentration of the chitosan in the chitosan-perfluoro tributylamine solution is 1% -3%.

8. The method of claim 7, wherein the volume ratio of the vascular growth factor-oxidized hyaluronic acid solution to the chitosan-perfluorotributylamine solution is 1:2.

9. The method of claim 7, wherein the mass concentration of oxidized hyaluronic acid in the vascular growth factor-oxidized hyaluronic acid solution is 2%.

10. The method according to claim 7, wherein the mass concentration of perfluorotributylamine in the chitosan-perfluorotributylamine solution is 7%.

11. The method according to claim 7, wherein the mass concentration of chitosan in the chitosan-perfluoro tributylamine solution is 2%.

12. The method of any one of claims 7-11, further comprising a process for preparing a vascular growth factor-oxidized hyaluronic acid solution, the process comprising dissolving the vascular growth factor in the oxidized hyaluronic acid solution.

13. The method of any one of claims 7-11, further comprising a method of preparing an oxidized hyaluronic acid solution comprising dissolving oxidized hyaluronic acid in a PBS solution.

14. The method of claim 13, wherein the pH of the PBS solution is 7.4.

15. The method of any one of claims 7-11, further comprising a method of synthesizing oxidized hyaluronic acid, the method comprising dissolving hyaluronic acid in deionized water, adding NaIO ₄ solution, adding ethylene glycol, and dialyzing.

16. The method according to any one of claims 7-11, further comprising a process for preparing a chitosan-perfluorotributylamine solution comprising dissolving chitosan in a perfluorotributylamine nanoemulsion.

17. The method according to any one of claims 7 to 11, further comprising a method of synthesizing an oxygen-loaded perfluorotributylamine nanoemulsion, the method comprising the steps of dissolving lecithin, cholesterol and DSPE-PEG2000 in methylene chloride, removing methylene chloride, diluting the mixture with PBS solution, ice-bath ultrasound, mixing the mixture with perfluorotributylamine, ultrasound to obtain a perfluorotributylamine nanoemulsion, and oxidizing the perfluorotributylamine nanoemulsion to obtain an oxygen-loaded perfluorotributylamine nanoemulsion.

18. Use of a pharmaceutical composition according to any one of claims 5-6 for the manufacture of a medicament for the treatment of a disease caused by insulin deficiency or insufficiency.

19. The use according to claim 18, wherein the diseases caused by insulin deficiency or deficiency comprise hyperglycemia, type 1 diabetes or type 2 diabetes related complications, other rare type diabetes.

20. The use according to claim 19, wherein the disease caused by insulin deficiency or deficiency is selected from type 1 diabetes.

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

22. Use of the hydrogel system of any one of claims 1-4 or the pharmaceutical composition of any one of claims 5-6 for the preparation of a medicament for inhibiting islet cell apoptosis.