A high-adhesion chitosan-based hydrogel and its preparation method and application

Abstract

The present invention discloses a high-adhesion chitosan-based hydrogel, a preparation method and an application thereof. The main raw materials used in the present invention are natural polysaccharide chitosan and TMG as a biological buffer. The raw material sources are abundant and have excellent biocompatibility and biodegradability. Chitosan is modified with TMG and a hydrogel is formed by hydrogen bond cross-linking to obtain a hydrogel based on a natural polysaccharide base. The obtained hydrogel forms hydrogen bonds with the skin surface, thereby giving the hydrogel excellent adhesion properties, and can be used for wound dressings and acute hemostatic materials. In addition to the skin surface, the hydrogel obtained by the present invention also shows excellent adhesion properties on the surfaces of materials such as glass and metal, and has potential application prospects in adhesives.

Description

Technical Field

[0001] The present invention relates to the field of polymer materials, and in particular to a high-adhesion chitosan-based hydrogel and a preparation method and application thereof. Background Art

[0002] Skin wounds caused by acute trauma, surgery and chronic diseases remain a huge challenge facing mankind. Designing effective wound dressings has become an urgent problem that needs to be solved in modern medical systems. Compared with other wound adhesives, hydrogel adhesives have the following advantages: (1) similar to native extracellular matrix, which can better maintain tissue function and promote cell migration; (2) excellent cell, drug and bioactive carriers; (3) high water content, which can maintain a moist wound environment. Therefore, hydrogel adhesives are widely used in different types of wound healing. Hydrogel is a three-dimensional network structure composed of high molecular polymers. Hydrogels constructed with natural polysaccharides and their derivatives have unique biological activity, good biocompatibility, low immune response, easy physical and chemical modification, abundant sources, and low price. They are widely used in hemostasis and wound healing. Natural polysaccharides commonly used to prepare adhesive hydrogels include chitosan, hyaluronic acid, cellulose, alginate, dextran, chondroitin sulfate, pullulan, etc.

[0003] Chitosan is an alkaline polysaccharide obtained by deacetylation of chitin in an alkaline environment. It is abundant in nature and easily available. Free amino groups are easily available in chitosan, carrying a positive charge that can react with many negatively charged substances. Chitosan has the characteristics of biodegradability, biocompatibility, adhesion, antibacterial properties and low toxicity. In recent years, it has been widely studied and applied in drug delivery, antibacterial coatings, drug delivery systems, wound dressings and cartilage regeneration. At the same time, the research on chitosan-based adhesive hydrogels still faces huge challenges. For example, the low solubility of chitosan and the high viscosity of the solution result in a brittle and weak chitosan physical network. Therefore, it is still a difficult task to modify chitosan with suitable substances to improve its mechanical properties and adhesion properties. Summary of the Invention

[0004] The present invention aims to overcome the shortcomings and deficiencies of the prior art and provide a method for preparing a highly adhesive chitosan-based hydrogel. The method primarily involves an amidation reaction between TMG and chitosan, followed by cross-linking via multiple hydrogen bonds to form a TMG-modified highly adhesive chitosan-based hydrogel.

[0005] Another object of the present invention is to provide a highly adhesive chitosan-based hydrogel prepared by the above preparation method, wherein the hydrogel has good biocompatibility and good adhesion performance.

[0006] Another object of the present invention is to provide applications of the high-adhesion chitosan-based hydrogel.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for preparing a high-adhesion chitosan-based hydrogel comprises the following steps:

[0009] (1) Preparation of chitosan acetate solution

[0010] Dissolve chitosan in acetic acid solution and stir until the chitosan is fully dissolved, add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide), and continue stirring to obtain a chitosan acetic acid solution;

[0011] (2) Preparation of TMG-modified chitosan

[0012] The chitosan acetate solution obtained in step (1) was added with TMG (tris(hydroxymethyl)methylglycine) and stirred for reaction. After the reaction was completed, the reaction product was placed in a dialysis bag, dialyzed with water, and freeze-dried to obtain TMG-modified chitosan;

[0013] (3) Preparation of high-adhesion chitosan-based hydrogel

[0014] The TMG-modified chitosan obtained in step (2) is dissolved in water, slowly stirred and then allowed to stand to obtain a high-adhesion chitosan-based hydrogel.

[0015] The volume fraction (v / v, ml / ml) of the acetic acid solution in step (1) is 0.5% to 2%, preferably 1%.

[0016] The average molecular weight of the chitosan in step (1) is 3 to 10 Kda, preferably 5 Kda.

[0017] The ratio of the mass of the chitosan in step (1) to the volume of the acetic acid solution is 1:200-30, preferably 1:100.

[0018] The molar ratio of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride to chitosan in step (1) is 1-2:1-2; preferably 1:1.

[0019] The molar ratio of N-hydroxysuccinimide to chitosan in step (1) is 1-2:1-2; preferably 1.2:1.

[0020] The continued stirring in step (1) is stirring for 30 minutes.

[0021] The reaction temperature in step (2) is room temperature.

[0022] The reaction time of step (2) is 24 to 72 hours, preferably 48 hours.

[0023] The molar ratio of TMG to chitosan in step (2) is 1 to 5:1, preferably 3:1.

[0024] The molecular weight cut-off of the dialysis bag used in step (2) is 3000 to 4000, preferably 3500.

[0025] The dialysis time in step (2) is 3 to 5 days, preferably 3 days.

[0026] The mass ratio of the TMG-modified chitosan to the volume ratio of water (mass concentration, w / v) in step (3) is 1:5 to 30, preferably 1:10.

[0027] The stirring speed in step (3) is 200 r / min.

[0028] The stirring time in step (3) is 1 h.

[0029] The standing time described in step (3) is 24h.

[0030] The high-adhesion chitosan-based hydrogel prepared by the above preparation method.

[0031] Application of the above-mentioned high-adhesion chitosan-based hydrogel as a medical tissue adhesive.

[0032] Application of the high-adhesion chitosan-based hydrogel in the preparation of adhesives.

[0033] The present invention has the following advantages and effects compared to the prior art:

[0034] (1) The main raw materials used in the present invention are natural polysaccharide chitosan and TMG as a biological buffer, which are abundant in source and have excellent biocompatibility and biodegradability.

[0035] (2) In the present invention, TMG and chitosan undergo amidation reaction without the need for an external cross-linking agent. The reaction conditions are mild and the operation is simple, which has application prospects in biomedicine.

[0036] (3) In the present invention, TMG is used to modify chitosan and form a hydrogel through hydrogen bond cross-linking to obtain a hydrogel with natural polysaccharide as the raw material.

[0037] (4) The hydrogel obtained in the present invention forms hydrogen bonds with the skin surface, thereby imparting excellent adhesion properties to the hydrogel, making it suitable for use in wound dressings and acute hemostatic materials. In addition to skin surfaces, the hydrogel obtained in the present invention also exhibits excellent adhesion properties on surfaces such as glass and metal, showing potential application prospects in adhesives. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 Schematic diagram of the structure of the product in Example 1 and H NMR spectrum.

[0039] Figure 2 is a Fourier transform infrared (FTIR) spectrum of the product in Example 1.

[0040] Figure 3 is a scanning electron microscope image of the gel sample in Example 3.

[0041] Figure 4 This is the rheological data diagram of the product in Example 4 during strain sweep.

[0042] Figure 5 This is the rheological data diagram of the product in Example 4 during frequency scanning.

[0043] Figure 6 This is a curve showing the change in swelling degree of the gel sample in Example 5 over time.

[0044] Figure 7 This is a curve showing the change in water loss degree of the gel sample in Example 6 over time.

[0045] Figure 8 is the adhesion strength of the gel sample in Example 7 on the pigskin surface.

[0046] Figure 9 is the adhesion strength of the gel sample in Example 8 on the tinplate sheet and glass.

[0047] Figure 10 Schematic diagram of the device for the pressure burst experiment in Example 9.

[0048] Figure 11 This is the pressure burst data diagram of the gel sample in Example 9.

[0049] Figure 12 is the cell viability of the gel sample in Example 10.

[0050] Figure 13 is the hemolysis rate of the gel sample in Example 11.

[0051] Figure 14 is the bleeding volume of the gel sample in Example 12. DETAILED DESCRIPTION

[0052] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0053] If specific experimental conditions are not specified in the following embodiments, conventional experimental conditions or those recommended by the reagent company will generally be used. Materials and reagents used were commercially available unless otherwise specified.

[0054] Example 1

[0055] (1) Take 2 ml of acetic acid and add it dropwise to 200 ml of deionized water to form a 1% acetic acid solution.

[0056] (2) Weigh 2.0 g of chitosan and add it to the solution obtained in step (1), stirring continuously until the chitosan is completely dissolved.

[0057] (3) Weigh 2.2 g of EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 1.5 g of NHS (N-hydroxysuccinimide), add them to the acetic acid solution containing chitosan obtained in step (2), and react for 30 min.

[0058] (4) Weigh 6 g of TMG (tris(hydroxymethyl)methylglycine) and add it to the solution obtained in step (3), and react at room temperature for 48 hours.

[0059] (5) After the reaction, the reaction product was placed in a cellulose dialysis bag with a molecular weight cutoff of 3500, dialyzed with deionized water for 4 days, and freeze-dried to obtain TMG-modified chitosan (CTMG).

[0060] The prepared chitosan was detected by nuclear magnetic resonance. Figure 1 The H NMR spectrum of the product is shown in Figure 2. Compared with CS, CTMG has a new proton peak at 3.85, which is the characteristic peak of the methylene group on the TMG structural unit, indicating that CTMG was successfully synthesized. The Fourier transform infrared (FTIR) spectrum of the product is shown in Figure 2. Figure 2 As shown, 1655.04, 1595.86, 1422.02cm -1 The peaks near the chitosan are amide functional groups (CONH). Compared with the CS spectrum, the peak intensity of the CTMG spectrum in this range has increased significantly, indicating that an amidation reaction has occurred between CS and TMG. At the same time, the peaks at 3360, 47 cm -1 There is a clear absorption peak at , and the absorption peak of hydroxyl groups asymmetrically broadens and shifts toward lower wavenumbers. This is due to the fact that TMG monomers polymerize to form long-chain macromolecules, which increases the hydrogen bond density. The experimental results show that TMG is successfully modified onto the chitosan structure.

[0061] Example 2

[0062] According to Table 1, different masses of the product of Example 1 were weighed and dissolved in a certain volume of deionized water to form hydrogels of different concentrations, namely, high-adhesion chitosan-based hydrogels.

[0063] Table 1

[0064]

[0065] Example 3

[0066] The three hydrogels with different concentrations obtained in Example 2 were freeze-dried and vacuum-coated with gold. The cross-sectional morphology of the hydrogels was observed using a Quanta 400FEG field emission scanning electron microscope produced by FEI / OXFORD / HKL. Figure 3 This is a scanning morphology image of the obtained product, from which it can be seen that the obtained product has a three-dimensional network structure.

[0067] Example 4

[0068] The three hydrogels prepared in Example 2 were used to measure the hydrogels using Kinexus Pro + The rheological properties of the gel were tested using a rotational rheometer.

[0069] The fixture consisted of 25mm-diameter parallel plates, the experimental temperature was 25°C, and the gap between the parallel plates was 1mm. First, a dynamic strain sweep was performed on the hydrogel to determine its linear viscoelastic range. The sweep frequency was fixed at 1 rad / s and the sweep range was 0.01-100%. Then, a dynamic frequency sweep was performed on the sample, with the strain fixed at 1% and the frequency sweep range from 0.01-10 Hz.

[0070] The experimental results are as follows Figures 4-5 As shown, Figure 4 The dynamic strain scanning curve of the hydrogel shows that with the increase of CTMG concentration, the maximum linear viscoelastic region strain value of the sample increases, indicating that the increase in concentration increases the cross-linking points of the system and the network structure formed is more compact. The dynamic frequency scanning curve of the hydrogel is shown in FIG. Figure 5 As shown in the figure, the G' of the 15CTMG hydrogel sample is always greater than G", showing the properties of a solid, indicating that the hydrogel has formed a relatively complete network structure at this time.

[0071] Example 5

[0072] The 15CTMG hydrogel sample prepared in Example 2 was freeze-dried and dried in an oven at 40°C to constant weight. The mass after drying, W0, was measured. The dried hydrogel sample was placed in a Petri dish filled with deionized water. The gel was removed from the water at regular intervals and the surface moisture was absorbed with filter paper. The mass of the gel after water absorption, W1, was weighed and the swelling ratio (SR) was calculated as follows:

[0073]

[0074] The swelling properties of the hydrogel were obtained. Figure 6 As shown in the figure, the swelling rate of the hydrogel increased sharply in the first 30 minutes, and then tended to slow down. After reaching the swelling equilibrium, the swelling rate was as high as 1075%, indicating that the hydrogel has good water absorption and swelling ability and is suitable for preparing wound dressings.

[0075] Example 6

[0076] Take the hydrogel that has reached swelling equilibrium in Example 5, dry the surface water, measure the gel mass W2, place the gel in a 27°C oven, take out the gel at regular intervals, and weigh the gel mass W3. Calculate the water retention ratio (WR) of the gel:

[0077]

[0078] The water retention properties of the hydrogel were obtained. Figure 7 As shown in the figure, the hydrogel loses water quickly in the first 3 hours, and then tends to lose water slowly. After reaching equilibrium, the water retention rate is 16%, indicating that the CTMG hydrogel has certain water retention properties.

[0079] Example 7

[0080] The three gels prepared in Example 2 were subjected to lap shear tests using a microcomputer-controlled electronic universal testing machine from Shenzhen Kebi Testing Equipment Co., Ltd. to measure the adhesion of the hydrogels to pigskin. The pigskin was sized 25 mm x 30 mm and adhered tightly to a tinplate sheet using 502 glue. The hydrogel was evenly coated between the two pieces of pigskin, maintaining the hydrogel dimensions at 25 mm x 20 mm x 1 mm and a contact area S of 5 cm. 2 After standing for 12 hours, a uniaxial tensile test was performed at a stretching rate of 0.2 mm / min to obtain the maximum tensile stress F, from which the adhesion strength of the hydrogel sample on the pig skin was calculated:

[0081]

[0082] The adhesion strength of the gel sample on the pigskin surface was obtained, as Figure 8 As shown in the figure, compared with medical cyanoacrylate glue (Compat medical glue of Swedish Molnik Medical Co., Ltd.), the three hydrogels showed better adhesion properties on the pig skin surface, among which 10CTMG had the highest adhesion strength of 113.2KPa, indicating that CTMG hydrogel has excellent adhesion properties.

[0083] Example 8

[0084] The three hydrogel samples prepared in Example 2 were subjected to lap shear tests using a microcomputer-controlled electronic universal testing machine from Shenzhen Kebi Testing Equipment Co., Ltd. to measure the adhesion properties of the hydrogel samples on the surfaces of different substrates. The substrates were cut into 25mm×75mm sizes, degreased by alkaline washing, and rinsed three times with deionized water to remove surface stains. The hydrogel was evenly coated between the substrates, with the size of the hydrogel controlled to be 25mm×20mm×1mm and the contact area S to be 5cm. 2 , and let it stand for 12 hours. Then, a uniaxial tensile test was performed at a stretching rate of 0.2 mm / min to obtain the maximum tensile stress F, from which the adhesion strength of the hydrogel sample on the substrate material was calculated:

[0085]

[0086] Figure 9 The adhesion strength of the three hydrogel samples on tinplate and glass indicates that CTMG hydrogel also exhibits excellent adhesion properties on the surfaces of glass, metal and other materials, and has potential application value in adhesives.

[0087] Example 9

[0088] The pressure burst test is used to measure the pressure that the hydrogel can withstand. Figure 10 The figure shows the schematic diagram of the device. A 2 mm long incision was made on the surface of pigskin. The three hydrogel samples prepared in Example 2 were adhered to the incision. A pressure of 0.5 kPa was applied for 1 minute to ensure complete adhesion of the hydrogel to the pigskin surface. The device was completely sealed. Air was then slowly injected at a rate of 10 ml / min until the hydrogel burst. The test was repeated three times for each sample.

[0089] The maximum pressure that the three hydrogel samples can withstand is as follows Figure 11 As shown in the figure, the bursting pressure that the three hydrogel samples can withstand is greater than that of medical hemostatic sponge (Jiangxi Xiangen Medical Technology Development Co., Ltd.), and much higher than the normal arterial blood pressure of adults (0.012MPa~0.018MPa), indicating that the three hydrogel samples prepared have the conditions to become hemostatic materials.

[0090] Example 10

[0091] The hydrogel sample prepared in Example 2 was freeze-dried, ground into powder and sterilized by ultraviolet irradiation. Then, it was dispersed in cell culture medium to prepare solutions of 10, 20, 50, 100 and 200 μg / mL. Logarithmic phase human normal hepatocytes (LO2) were collected for CCK-8 cytotoxicity assay. 1×10 5 Logarithmic phase LO2 cells were added with the above-mentioned hydrogel solutions of different concentrations, and a blank control group was set up. After incubation in a 37°C incubator for 24 hours, CCK-8 dye was added and incubated for another 2 hours. The absorbance was measured at 450 nm using a microplate reader, and the cell survival rate was finally calculated based on the blank control. Five parallel experiments were performed for each group.

[0092] Figure 12 is the survival rate of normal human liver cells (LO2) at each hydrogel sample concentration, Figure 12 It can be seen that the cell activity co-cultured with all concentrations reached more than 80%, indicating that CTMG hydrogel has low cytotoxicity to LO2 cells and good biocompatibility.

[0093] Example 11

[0094] The three hydrogel samples prepared in Example 2 were taken, freeze-dried, and the blood compatibility of the CTMG samples was measured by in vitro hemolysis experiments. 5 mg of the three hydrogel freeze-dried samples were dissolved in 3 mL of PBS solution and incubated at 37 ° C for 24 hours to obtain sample extracts. Then 125 μL of the sample extract was thoroughly mixed with 875 μL of 2% fresh anticoagulated red blood cell solution, incubated in a 37 ° C incubator for 1 hour and centrifuged (3000 rpm, 10 minutes), 100 μL of the supernatant was transferred to a 96-well plate, and the absorbance of the supernatant was measured at 540 nm using a multifunctional microplate reader. PBS was used as the negative control group, TritonX-100 was used as the positive control group, and 5 parallel samples were obtained for each sample.

[0095] The hemolysis rate of the sample is Figure 13 As shown in the figure, the hemolysis rates of the three hydrogel samples were all lower than 5%, indicating that the three hydrogel samples had good blood compatibility and met the needs of clinical application.

[0096] Example 12

[0097] The hemostatic properties of CTMG gel and commercial hemostatic sponge were evaluated using the SD rat liver bleeding model. The three hydrogels prepared in Example 2 were taken and freeze-dried. SD rats (female, weighing 180g-220g, 6 weeks old) were anesthetized with 10% chloral hydrate solution and fixed on a soft surgical board with an inclination angle of 30°; the rat abdomen was cut open with a scalpel to expose the liver, and the body fluid near the rat liver was carefully wiped. A piece of filter paper with a mass of W0 was placed under the liver, and a scalpel was used to make a wound 10mm long and 5mm deep on the surface of the liver. 7CTMG, 10CTMG, and 15CTMG hydrogel freeze-dried samples were placed on the wound respectively, and the bleeding of the wound was observed every 30s. When the bleeding stopped, the weight of the filter paper after absorbing the blood was weighed as W1. The blank control group did not apply any material to the wound. The positive control group used a medical hemostatic sponge (Jiangxi Xiangen Medical Technology Development Co., Ltd.) as a dressing after puncturing the wound.

[0098] The amount of bleeding of CTMG gel and medical hemostatic sponge is as follows Figure 14 As shown in the figure, compared with medical hemostatic sponge, CTMG gel has a good hemostatic effect, indicating that the three hydrogel samples prepared have the conditions to become hemostatic materials.

[0099] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a high-adhesion chitosan-based hydrogel, characterized in that The steps include: (1) Preparation of chitosan acetate solution Dissolve chitosan in acetic acid solution and stir until the chitosan is fully dissolved. Add EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) and continue stirring to obtain chitosan acetic acid solution. (2) Preparation of TMG-modified chitosan Take the chitosan acetic acid solution obtained in step (1), add TMG (tris(hydroxymethyl)methylglycine) and stir to react. After the reaction is completed, place the reaction product in a dialysis bag, dialyze with water, and freeze-dry to obtain TMG-modified chitosan; (3) Preparation of high-adhesion chitosan-based hydrogel The TMG-modified chitosan obtained in step (2) is dissolved in water, slowly stirred and then allowed to stand to obtain a highly adhesive chitosan-based hydrogel.

2. The preparation method according to claim 1, wherein: The volume fraction of the acetic acid solution in step (1) is 0.5% to 2%; The ratio of the mass of the chitosan in step (1) to the volume of the acetic acid solution is 1:200-30.

3. The preparation method according to claim 1, wherein: The molar ratio of EDC to chitosan in step (1) is 1-2:1-2; The molar ratio of NHS to chitosan in step (1) is 1-2:1-2.

4. The preparation method according to claim 1, wherein: The molar ratio of TMG to chitosan in step (2) is 1 to 5:

1.

5. The preparation method according to claim 1, wherein: The reaction temperature in step (2) is room temperature; The reaction time of step (2) is 24 to 72 hours; The dialysis bag used in step (2) has a molecular weight cut-off of 3000 to 4000; The dialysis time in step (2) is 3 to 5 days.

6. The preparation method according to claim 1, wherein: The volume ratio of the mass of the TMG-modified chitosan to water in step (3) is 1:5-30.

7. The preparation method according to claim 1, wherein: The stirring speed in step (3) is 200 r / min; The stirring time in step (3) is 1 h; The standing time in step (3) is 24 h.

8. A high-adhesion chitosan-based hydrogel, characterized in that Prepared according to any one of claims 1 to 7.

9. Use of the high-adhesion chitosan-based hydrogel according to claim 8 in the preparation of an adhesive.

10. Use of the high-adhesion chitosan-based hydrogel according to claim 8 in the preparation of medical tissue adhesives.

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