A self-shrinking hydrogel and a preparation method and application thereof

Abstract

The application provides a self-shrinking hydrogel as well as a preparation method and application thereof, wherein the modified chitosan derivative has a structure of formula I. After the chitosan is modified in the application, the solubility of the chitosan is effectively improved, so that the chitosan can be uniformly dispersed in PBS or ultrapure water, the application prospect is widened, and the modified chitosan derivative is more suitable for accelerating wound repair in biochemical functions. The self-shrinking hydrogel containing the modified chitosan derivative is sensitive to temperature in the application, can produce deformation and shrinkage with the increase of temperature, can generate centripetal force on the edge tissue of a wound when applied to the wound, and is beneficial to wound healing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biomedical materials, and particularly relates to a self-shrinking hydrogel as well as a preparation method and application thereof. BACKGROUND

[0002] Various wounds, including acute postoperative wounds, trauma, burns and chronic wounds caused by diabetes or circulatory disorders, have always been the focus of clinical attention, and have a significant impact on patient quality of life and social resource allocation. Wound healing is a complex physiological process involving stages such as hemostasis, inflammation, proliferation and tissue remodeling. The functions of many dressings are also designed and developed around these stages, such as enhancing absorption of tissue exudate, effectively resisting bacterial infection, eliminating inflammatory factors, etc. A large number of studies have shown that mechanical stress stimulation is essential for the growth and development of body tissues. The development of existing wound dressings focuses more on biochemical aspects, but few pay attention to providing positive stress stimulation to wound tissues through dressings to accelerate healing. SUMMARY

[0003] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the first aspect of the present application proposes a modified chitosan derivative with temperature-sensitive properties and photo-crosslinking properties.

[0004] The second aspect of the present application proposes a preparation method of the modified chitosan derivative.

[0005] The third aspect of the present application proposes a self-shrinking hydrogel comprising the modified chitosan derivative.

[0006] The fourth aspect of the present application proposes a preparation method of the self-shrinking hydrogel.

[0007] The fifth aspect of the present application proposes an application of the modified chitosan derivative and / or the self-shrinking hydrogel.

[0008] According to the first aspect of the present application, a modified chitosan derivative of formula I is proposed:

[0009]

[0010] wherein DD is the degree of deacetylation, and the degree of deacetylation is 30% to 99%, preferably 50% to 95%.

[0011] In some embodiments of the present application, the degree of substitution of -NH2 in formula I is 1.0 to 2.0, preferably 1.1 to 1.9; preferably, the degree of substitution of -NH2 in formula I is 0.1 to 0.5, preferably 0.1 to 0.3; further preferably, the degree of substitution of -NH2 in formula I is 0.1 to 0.3. ​​the degree of substitution is 0.1-0.5, preferably 0.1-0.3.

[0012] In some preferred embodiments of the present application, the molecular weight of the modified chitosan derivative is 100000-300000.

[0013] According to a second aspect of the present application, a method for preparing the modified chitosan derivative of formula I is provided, comprising the following steps:

[0014] S1: after alkalization of the chitosan, reacting with 1,2-epoxybutane to obtain hydroxybutyl chitosan;

[0015] S2: after dissolution of the hydroxybutyl chitosan, adding unsaturated acid anhydride to react to obtain double bond hydroxybutyl chitosan;

[0016] S3: after dissolution of the double bond hydroxybutyl chitosan, reacting with arginine to obtain the modified chitosan derivative of formula I.

[0017] In some embodiments of the present application, in S1, the mass-volume ratio of the chitosan to the 1,2-epoxybutane is 1:(10-30) g / mL.

[0018] In some preferred embodiments of the present application, in S2, the mass ratio of the hydroxybutyl chitosan to the unsaturated acid anhydride is 2:(3-10).

[0019] In some more preferred embodiments of the present application, in S3, the mass ratio of the double bond hydroxybutyl chitosan to the arginine is 2:(5-15).

[0020] In some more preferred embodiments of the present application, the unsaturated acid anhydride comprises at least one of methyl acrylate, acrylate or maleic anhydride.

[0021] In some more preferred embodiments of the present application, in S3, the reaction further comprises using N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDC) as activators.

[0022] In some more preferred embodiments of the present application, in S2, the reaction time is 12h-24h.

[0023] In some more preferred embodiments of the present application, in S3, the reaction time is 15h-48h.

[0024] In some more preferable embodiments of the present application, the preparation method further comprises a step of purifying the product of each step; preferably, S1 further comprises adjusting the pH of the reaction product to neutral, filtering, alcohol precipitation, and drying; further preferably, S2 further comprises adjusting the pH of the reaction product to neutral, dialysis for 3-5 days, and lyophilization; and more preferably, S3 further comprises dialysis for 3-5 days and lyophilization.

[0025] According to a third aspect of the present application, a self-shrinking hydrogel comprising the modified chitosan derivative is provided.

[0026] In some embodiments of the present application, the mass concentration of the modified chitosan derivative is 3-10%.

[0027] According to a fourth aspect of the present application, a preparation method of a self-shrinking hydrogel is provided, comprising the following steps: adding a water-soluble photo-crosslinking agent to a solution of the modified chitosan derivative, and preparing the self-shrinking hydrogel after photo-crosslinking.

[0028] According to a fifth aspect of the present application, the modified chitosan derivative and / or the self-shrinking hydrogel are used in the preparation of a wound repair material.

[0029] The present application has the following beneficial effects:

[0030] 1. In the present application, the solubility of chitosan is effectively improved after modification, so that the modified chitosan derivative can be uniformly dispersed in PBS or ultrapure water, thereby widening the application prospect, and the modified chitosan derivative is more suitable for accelerating wound repair in terms of biochemical function.

[0031] 2. The self-shrinking hydrogel containing the modified chitosan derivative in the present application has temperature sensitivity and ultraviolet sensitivity, and is in sol form before photo-crosslinking, can be seamlessly and closely fitted to various irregular wounds, and the strength and adhesion of the system are further enhanced after photo-crosslinking, so that the wound is effectively closed, and under the body temperature condition, the hydrogel can be promoted to shrink and deform after contacting the body, thereby producing a centripetal traction effect on the edge tissue of the wound, and actively shrinking the wound to promote healing from a physical level. The introduction of arginine groups enhances the adhesion of the system to the tissue, and arginine as an endogenous component of the body can accelerate blood vessel regeneration and promote healing from a biochemical level.

[0032] 3. The self-shrinking hydrogel containing the modified chitosan derivative in the present application remains sensitive to temperature, and can produce deformation and shrinkage with the increase of temperature, and can produce a centripetal force on the edge tissue of the wound when applied to the wound, which is beneficial to wound healing. BRIEF DESCRIPTION OF DRAWINGS

[0033] The present application will be further described below in conjunction with the drawings and examples, wherein:

[0034] Figure 1 The physical picture of the modified chitosan derivative material of the present application embodiment 1-embodiment 3.

[0035] Figure 2 The solubility of the unmodified chitosan and the modified chitosan derivative prepared in embodiment 1-embodiment 3 of the present application.

[0036] Figure 3 The storage modulus and loss modulus of the modified chitosan derivative of the present application embodiment 1-embodiment 3 with temperature change.

[0037] Figure 4 The tensile strength picture of different modified chitosan in the present application embodiment 3.

[0038] Figure 5 The influence of the modified chitosan derivative of the present application embodiment 3 on the expression amount of HIF-1α of wound tissue.

[0039] Figure 6 The area shrinkage rate of different self-shrinking hydrogels prepared in the present application embodiment 4.

[0040] Figure 7 The physical picture of different self-shrinking hydrogels prepared in the present application embodiment 4 at different temperatures. DETAILED DESCRIPTION

[0041] The concept and the technical effect of the present application will be described clearly and completely in combination with the embodiments, so as to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the present application.

[0042] Embodiment 1

[0043] A modified chitosan derivative is prepared in this embodiment, and the specific process is as follows:

[0044] S1: Synthesis of hydroxybutyl chitosan (HBC): 4 g of refined chitosan with 50% degree of deacetylation was weighed into a 250 mL beaker, and 30 mL of 50% sodium hydroxide solution was added dropwise under stirring at 25°C and 200 rpm / min for 24 h. Then the alkali solution was squeezed out and washed with distilled water twice to obtain alkali chitosan. The alkali chitosan was transferred to a round-bottom flask, and 80 mL of a mixed solution of isopropyl alcohol and water (v / v 1:1) was added dropwise under stirring at 25°C and 200 rpm / min for 24 h. Then 80 mL of 1,2-epoxybutane was added dropwise, and the reaction was heated to 55-60°C and continued for 12 h. The reaction was terminated, 1% hydrochloric acid was added to adjust the pH to neutral, and the solution was completely dissolved to be transparent. The solution was transferred to a 3000 molecular weight dialysis bag and dialyzed for 3-4 days. The dialysate was transferred to 4°C overnight. Impurities were filtered out, frozen at -20°C, and freeze-dried.

[0045] S2: Synthesis of double bond hydroxybutyl chitosan (HBC_m): 2 g of HBC was dissolved in 200 mL of ultrapure water, and 6.4 g of methacrylic anhydride was added dropwise after being fully dissolved at room temperature. The reaction temperature was controlled at 60°C, and the stirring speed was about 1000 rpm / min. The reaction was continued for 12-24 h. The pH was adjusted to neutral with sodium bicarbonate. Dialysis was performed in a 3000 molecular weight dialysis bag for 3-4 days. Freeze-drying was performed to obtain the modified chitosan derivative.

[0046] S3: Synthesis of modified chitosan derivative (HBC_m_Arg): 2 g of HBC_m was dissolved in 200 mL of ultrapure water at room temperature, and 1.42 g of NHS and 1.92 g of EDC were added and stirred for 10 min. Then 12 g of arginine was added and stirred for 24 h to complete the reaction. After dialysis in ultrapure water for 3-4 days, the modified chitosan derivative was obtained after freeze-drying.

[0047] Example 2

[0048] In this example, a modified chitosan derivative was prepared, and the specific process was as follows:

[0049] S1: Synthesis of hydroxybutyl chitosan (HBC): 4 g of refined 90% deacetylated chitosan was weighed into a 250 mL beaker, and 30 mL of 50% sodium hydroxide solution was added dropwise under stirring at 25°C and 200 rpm / min for 24 h. The alkali solution was then squeezed out, and the chitosan was washed twice with distilled water to obtain alkali chitosan. The alkali chitosan was transferred to a round-bottom flask, and 80 mL of a mixed solution of isopropyl alcohol and water (v / v 1:1) was added dropwise under stirring at 25°C and 200 rpm / min for 24 h. Then, 80 mL of 1,2-epoxybutane was added, and the reaction was heated to 55-60°C and continued for 12 h. The reaction was terminated, and 1% hydrochloric acid was added to adjust the pH to neutral. The solution was completely dissolved and transparent. The solution was transferred to a 3000 molecular weight dialysis bag, and dialysis was performed for 3-4 days. The dialysate was transferred to 4°C overnight. Impurities were filtered out, and the solution was frozen at -20°C and lyophilized.

[0050] S2: Synthesis of double bond hydroxybutyl chitosan (HBC_m): 2 g of HBC was dissolved in 200 mL of ultrapure water, and 6.4 g of methacrylic anhydride was added dropwise at room temperature. The reaction was controlled at 60°C, and the stirring speed was about 1000 rpm / min. The reaction was continued for 12-24 h. The pH was adjusted to neutral with sodium bicarbonate. Dialysis was performed in a 3000 molecular weight dialysis bag for 3-4 days. The modified chitosan derivative was obtained after lyophilization.

[0051] S3: Synthesis of modified chitosan derivative (HBC_m_Arg): 2 g of HBC_m was dissolved in 200 mL of ultrapure water at room temperature. 1.42 g of NHS and 1.92 g of EDC were added and stirred for 10 min. Then, 12 g of arginine was added, and the reaction was stirred for 24 h. The reaction was dialyzed in ultrapure water for 3-4 days, and the modified chitosan derivative was obtained after lyophilization.

[0052] Example 3

[0053] In this example, a modified chitosan derivative was prepared, and the specific process was as follows:

[0054] S1: Synthesis of hydroxybutyl chitosan (HBC): 4 g of refined 95% deacetylation degree chitosan was weighed into a 250 mL beaker, and 30 mL of 50% sodium hydroxide solution was added dropwise under stirring at 25°C and 200 rpm / min for 24 h, followed by extrusion of the alkali solution and washing with distilled water twice to obtain alkali chitosan; the alkali chitosan was transferred to a round-bottom flask, and 80 mL of a mixed solution of isopropyl alcohol and water (v / v 1:1) was added dropwise, and fully dispersed under stirring at 25°C and 200 rpm / min for 24 h; 80 mL of 1,2-epoxybutane was added dropwise, and heated to 55-60°C for 12 h; the reaction was terminated, 1% HCl was added to adjust the pH to neutral, and the solution was completely dissolved to be transparent; it was transferred to a 3000 molecular weight dialysis bag, dialyzed for 3-4 days, and the dialysate was transferred to 4°C overnight; impurities were filtered out, frozen at -20°C, and freeze-dried;

[0055] S2: Synthesis of double bond hydroxybutyl chitosan (HBC_m): 2 g of HBC was dissolved in 200 mL of ultrapure water, and 6.4 g of methacrylic anhydride was added dropwise after fully dissolved at room temperature; the reaction temperature was controlled at 60°C, and strong stirring was performed at a rotation speed of about 1000 rpm / min, and the reaction was continued for 12-24 h; sodium bicarbonate was used to adjust the pH to neutral; dialysis was performed in a 3000 molecular weight dialysis bag for 3-4 days; and freeze-drying was performed.

[0056] S3: Synthesis of modified chitosan derivative (HBC_m_Arg): 2 g of HBC_m was dissolved in 200 mL of ultrapure water at room temperature; 1.42 g of NHS and 1.92 g of EDC were added and stirred for 10 min; 12 g of arginine was added, and the reaction was fully stirred for 24 h; dialysis was performed in ultrapure water for 3-4 days; and the modified chitosan derivative was obtained after freeze-drying.

[0057] The modified chitosan derivative materials prepared in Examples 1-3 are shown in the physical map of Figure 1

[0058] Example 4

[0059] In this example, a self-shrinking hydrogel was prepared, and the specific process was as follows:

[0060] The chitosan derivative (HBC_m_Arg) in Examples 1-3 was weighed appropriately, and ultrapure water was used to prepare a 5% concentration (magnetic stirring was performed at a rotation speed of about 300 rpm / min in a 4°C refrigerator); a water-soluble photocrosslinking agent LAP was added, and a LAP concentration of 1%-2% was prepared; it was injected into a silica gel mold, and transferred to 365 nm photocrosslinking for 10-20 min to obtain the self-shrinking hydrogel corresponding to Examples 1-3.

[0061] Test Example 1

[0062] ​This experiment tested the solubility of unmodified chitosan and the modified chitosan derivatives prepared in Examples 1 to 3. The specific process is as follows:

[0063] Appropriate amounts of chitosan with deacetylation degrees of 50%, 90%, and 95%, and the modified chitosan derivatives prepared in Examples 1-3 were weighed out and mixed with ultrapure water to a concentration of 5%. The mixture was stirred at 4°C for 24 hours; after standing for 15 minutes, the clarity of the solution and the presence or absence of precipitation were observed. The results are as follows: Figure 2 As shown.

[0064] The results showed that unmodified chitosan was insoluble in PBS or ultrapure water, regardless of low temperature, room temperature, or heating. After standing, obvious precipitation occurred, and the solubility was approximately zero. In contrast, the modified chitosan derivatives prepared in Examples 1 to 3 had a solubility of up to 5% in PBS or ultrapure water below 10°C. After standing, they remained uniformly dispersed without precipitation.

[0065] Experimental Example 2

[0066] This experiment tested the rheological behavior of the modified chitosan derivatives prepared in Examples 1-3. The specific procedure was as follows: the modified chitosan derivatives prepared in Examples 1-3 were dissolved at a concentration of 5% by mass at 4°C, then loaded into a rheometer. Temperature scanning was performed at 4°C to 50°C, and changes in storage modulus and loss modulus were recorded. The results are as follows: Figure 3 As shown.

[0067] from Figure 3 It can be seen that in Examples 1 to 3, as the degree of deacetylation of chitosan increases, the phase transition temperature of the modified chitosan derivative at a concentration of 5% gradually decreases. With the increase of the degree of deacetylation, the temperature sensitivity of the product is enhanced, and the shrinkage and deformation efficiency is higher and the deformation range is greater when the gel is placed at 37°C.

[0068] Experimental Example 3

[0069] This experimental example tests the adhesion of products obtained in different steps of Example 3. The specific process is as follows:

[0070] Prepare 5% HBC, HBC_m, and HBC_m_Arg hydrogels respectively; coat them onto gelatin-coated glass slides and crosslink them using 365nm light for 30 seconds; then stretch the opposite glass slides at a rate of 5mm / min using a universal testing machine; record the stress and strain results. The results are as follows: Figure 4 As shown.

[0071] from Figure 4It can be seen that the tensile strength of HBC prepared with chitosan of 95% deacetylation was 15.9 kPa for gelatin-glass slide adhesion, while that of HBC_m was 25.5 kPa and that of HBC_m_Arg was 167.7 kPa. The tensile strength of HBC_m_Arg hydrogel was significantly higher than that of other experimental groups, indicating that it should exhibit better adhesion to biological tissues and show stronger traction on tissues when deformed.

[0072] Test Example 4

[0073] This experimental example tests the biofunctionality of the self-shrinking hydrogel corresponding to Example 3. The specific process is as follows:

[0074] The HBC and HBC_m_Arg obtained in Example 3 were used to prepare a hydrogel; this hydrogel was applied to the entire skin defect of rats and left for 10 days; skin tissue from the healed site of the rats was used to determine the expression level of HIF-1α protein (using β-actin as an internal control). The results are as follows: Figure 5 As shown.

[0075] from Figure 5 It can be seen that HBC_m_Arg promotes HIF-1α expression more effectively than HBC, and should therefore promote angiogenesis more effectively (Example 5).

[0076] This experimental example tests the area shrinkage rate of different self-shrinking hydrogels prepared in Example 4. The specific process is as follows:

[0077] The self-shrinking hydrogels corresponding to Examples 1 to 3 in Example 4 were placed on transparent glass slides; the outline of the hydrogel was outlined with a marker at 20°C and photographed; the hydrogels were incubated at 37°C for 5 minutes and photographed again; the initial and final outlines of the hydrogels were traced using ImageJ software and the corresponding areas were calculated. The results are as follows: Figure 6 , Figure 7 As shown.

[0078] from Figure 6 , Figure 7 It can be seen that the HBC_m_Arg hydrogels prepared with chitosan of 50%, 90%, and 95% deacetylation degrees exhibit greater deformation percentages with increasing deacetylation degree (average area shrinkage of 9.85% for the 50% deacetylation group, 16.05% for the 90% group, and 22.43% for the 95% group), indicating higher deformation efficiency. The temperature-sensitive deformation properties of the HBC_m_Arg hydrogel can be controlled.

[0079] The above has described the embodiments of the present application in detail, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge of those skilled in the art without departing from the spirit of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Formula I modified chitosan derivatives: ； in, DD represents the degree of deacetylation, which is 50% to 95%; in Formula I, The degree of substitution is 1.1~1.9; The degree of substitution is 0.1~0.3; The degree of substitution is 0.1~0.3; the molecular weight of the modified chitosan derivative is 100,000~300,000; the preparation method of the modified chitosan derivative of Formula I includes the following steps: S1: Deacetylated chitosan is reacted with 1,2-epoxybutane to obtain hydroxybutyl chitosan; S2: Hydroxybutyl chitosan is dissolved and then reacted with unsaturated acid anhydride to obtain double-bonded hydroxybutyl chitosan. S3: The double-bonded hydroxybutyl chitosan is dissolved and reacted with arginine to obtain the modified chitosan derivative of Formula I; in S2, the mass ratio of the hydroxybutyl chitosan to the unsaturated acid anhydride is 2:(3~10).

2. The modified chitosan derivative according to claim 1, characterized in that: In S3, the mass ratio of the double-bonded hydroxybutyl chitosan to the arginine is 2:(5~15).

3. A self-shrinking hydrogel, characterized in that: Including the modified chitosan derivative as described in claim 1.

4. A method for preparing a self-shrinking hydrogel, characterized in that: Includes the following steps: A water-soluble photocrosslinking agent was added to a modified chitosan derivative solution, and the self-shrinking hydrogel as described in claim 3 was obtained after photocrosslinking.

5. The application of a modified chitosan derivative as described in claim 1 and / or a self-shrinking hydrogel as described in claim 3 in the preparation of wound repair materials.

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