A double-crosslinked hydrogel and its application as wound healing dressing

Abstract

The present invention discloses a double-crosslinked hydrogel, which is formed by interweaving two crosslinked networks: poly(N-isopropylacrylamide) formed by free radical polymerization of N-isopropylacrylamide and N,N'-methylenebisacrylamide, and a crosslinked network formed by nucleophilic addition reaction of hyaluronic acid and glutaraldehyde. The double-crosslinked hydrogel of the present invention combines the multiple requirements of accelerating wound closure, promoting wound healing, and having tissue adhesion ability and temperature sensitivity to meet the needs of wound healing.

Description

Technical Field

[0001] The present invention belongs to the technical field of wound healing dressings, and in particular relates to a double-crosslinked hydrogel and an application thereof as a wound healing dressing. Background Art

[0002] As the body's largest defensive organ and first line of defense, the skin is susceptible to external stimuli, including strong impacts, intense friction, and sharp cuts, which often lead to skin trauma. Severe skin trauma, if not treated promptly and effectively, can often lead to wound infection, delayed healing, scarring, and even tissue necrosis.

[0003] Various gauzes and bandages that are currently widely used in clinical practice have the function of protecting wounds, but they still have adverse effects on wound healing, such as foreign body reaction, adhesion of dressings to wounds, wound infection, etc. These materials can only provide physical protection for wounds and have limited benefits on wound closure and healing. Currently, many hydrogels with unique biochemical functions have been developed. These hydrogels focus on promoting the speed of wound healing, that is, by introducing drugs or bioactive factors to change the microenvironment of the wound, accelerate various wound healing reactions, and promote wound healing by exerting the biochemical functions of the material. However, a dressing that can accelerate wound closure, promote wound healing, has tissue adhesion ability, and is sensitive to temperature is still a technical problem to be solved. Summary of the Invention

[0004] On the one hand, the present invention provides a double-crosslinked hydrogel (PNI-HA), which is formed by interweaving two crosslinked networks, one of which is: poly (N-isopropylacrylamide) (PNIPAM) formed by a free radical polymerization reaction between N-isopropylacrylamide (NIPAM) and N,N'-methylenebisacrylamide (MBA), and the other is: a crosslinked network formed by a nucleophilic addition reaction between hyaluronic acid (HA) and glutaraldehyde (GTA).

[0005] In one aspect, the present invention provides a method for preparing a double-crosslinked hydrogel, comprising the following steps:

[0006] 1) NIPAM and MBA were added to the HA solution, followed by the initiator, stirring, and then the accelerator to obtain a semi-interpenetrating cross-linked network (SIPN);

[0007] 2) SIPN was directly immersed in an acetone solution containing GTA, and then immersed in distilled water to remove GTA and acetone to obtain PNI-HA.

[0008] Furthermore, the stirring process is carried out at room temperature for 30 minutes. The stirring process can also be carried out under nitrogen protection, cryogenic protection, or an ice bath. Condition screening revealed that room temperature does not affect the preparation of the material. The room temperature refers to 18-25°C.

[0009] Furthermore, the acetone is an 80% acetone solution, and the immersion time in the acetone solution is 24 hours.

[0010] Furthermore, the pH value of the acetone solution is lower than 4, preferably between 1 and 2.

[0011] Furthermore, the molecular weight of HA is selected to be between 500 kDa and 2000 kDa, preferably between 700 and 800 kDa.

[0012] Furthermore, the concentration of the HA solution does not exceed 1%.

[0013] Furthermore, the quality ratio of NIPAM to MBA is 1:0.06.

[0014] Furthermore, the initiator is selected from persulfate, azo-containing organic initiator and the like.

[0015] Furthermore, the accelerator is selected from tetramethylethylenediamine, triethylamine and the like.

[0016] In one aspect, the present invention provides a use of a double-crosslinked hydrogel as a wound healing dressing.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1. Based on a deep understanding of the biological principles of wound healing, the present invention has prepared a new type of double-cross-linked hydrogel that combines multiple requirements such as accelerated wound closure, promotion of wound healing, tissue adhesion, and temperature sensitivity to meet the needs of wound healing.

[0019] 2. Compared with existing wound dressings, PNI-HA firmly adheres to the skin through the action of Schiff bases, and synergizes with thermal response self-contraction ability to contract wounds through adhesion and mechanical action; at the same time, it has the biochemical effects of reducing inflammatory response, reducing collagen deposition, and promoting angiogenesis, thereby promoting wound healing.

[0020] 3. PNI-HA has low production environment requirements, simple preparation method, readily available raw materials, and can be prepared in large quantities.

[0021] 4. The preparation components used in the hydrogel described in the present invention are not only considered based on their respective functions. The coordination between the components is an important factor in achieving the performance of the hydrogel. PNIPAM has thermal responsive self-shrinkage. The addition of HA enhances the structural density of the hydrogel and improves the mechanical properties; at the same time, it enhances the softness of the hydrogel, helps the hydrogel fit the wound surface, and avoids secondary damage to the wound caused by the dressing; in addition, it improves the biocompatibility of the hydrogel and enhances the ability of the hydrogel to promote wound healing. The addition of GTA increases the carrying rate of HA; at the same time, it enhances the adhesion of the hydrogel, helps the hydrogel fit closely to the wound surface, avoids microbial invasion, and cooperates with the thermal responsive self-shrinkage ability of PNIPAM to promote wound closure.

[0022] 5. The thermoresponsive material NIPAM, composed of a hydrophilic amide group (-CONH-) and a hydrophobic isopropyl group (-CH(CH3)2), can be cross-linked with MBA to form PNIPAM. The lower critical solution temperature (LCST) of PNIPAM in aqueous solution is approximately 33°C. When the temperature rises above the LCST (for example, 37°C body temperature), PNIPAM changes from a hydrophilic state to a hydrophobic state and forms intramolecular hydrogen bonds between polymer chains, thereby exhibiting a significant sol-gel transition and self-shrinkage properties.

[0023] 6. HA is a natural macromolecule present in the extracellular matrix of the skin. It has significant effects in maintaining a moist microenvironment, inhibiting inflammatory responses, and accelerating scar-free skin regeneration. However, traditional HA materials have low tissue adhesion to the skin. GTA-crosslinked HA hydrogel (HA-GTA) can enhance the material's tissue adhesion because the aldehyde groups exposed by glutaraldehyde undergo a Schiff base reaction with amino groups on the tissue surface.

[0024] 7. In an interpenetrating polymer network (IPN), PNIPAM imparts excellent thermally responsive self-shrinkage to the hydrogel, while HA-GTA enhances tissue adhesion and imparts biochemical functionality to the hydrogel. Compared to other currently available wound dressings, this material combines biomechanical activity with biochemical functionality to simultaneously achieve both rapid wound closure and enhanced wound healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The accompanying drawings, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0026] Figure 1 : Electron micrographs of PNI, PNI-0.5HA, and PNI-1.0HA.

[0027] Figure 2: Thermal responsive self-shrinkage of PNI-HA, including (A) thermal responsive self-shrinkage images of PNI, PNI-0.5HA and PNI-1.0HA, (B) change in shrinkage rate of hydrogel when moving from 25°C to 37°C, (C) change in shrinkage rate of hydrogel at 37°C with time, (D) SR change of PNI-1.0HA after three "shrinkage-expansion" cycles at different temperatures (25°C and 37°C).

[0028] Figure 3 : Thermal responsive adhesion of PNI-HA, including (A) heat map of adhesion strength of hydrogels with uniform final size at different temperatures (25°C, 29°C, 33°C, 37°C), and (B) change of adhesion strength of hydrogels with uniform initial size at 25°C with temperature.

[0029] Figure 4 : PNI-HA mediated wound closure, including: (A) images of PNI and PNI-1.0HA mediated wound closure at 0, 2, and 4 h, (B) stacked comparison of wound areas in image (A), and (CD) quantitative analysis of wound areas at 2 h and 4 h.

[0030] Figure 5 : PNI-HA mediated wound healing, including: (A) images of 21-day wound healing mediated by PNI and PNI-1.0HA, (BC) superimposed wound area and quantitative wound closure rate to form image (A), (DE) H&E staining (green arrows indicate inflammatory cells), (F) quantification of inflammatory cells in H&E staining (160×240μm), (G1) Masson staining, CD31 (red arrows indicate CD31-positive blood vessels) and integrin β1 immunohistochemical staining. DETAILED DESCRIPTION

[0031] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments and comparative examples.

[0032] Example 1: Preparation of PNI

[0033] NIPAM (50 mg / mL) and MBA (3 mg / mL) were dissolved in distilled water. Ammonium persulfate (APS) (5 mg / mL) was then added to the NIPAM / MBA solution as an initiator and stirred at room temperature for 30 minutes. Subsequently, N,N,N',N'-tetramethylethylenediamine (TEMED) (1.5 mg / mL) was added as a promoter to accelerate the formation of the network. After thorough rinsing with distilled water, the PNI hydrogel was obtained. Electron microscopy images are shown in Figure 2. Figure 1 .

[0034] Example 2: Preparation of PNI-HA

[0035] Different amounts (5 mg / mL, 10 mg / mL) of HA (Mw = 700-800 kDa) were dissolved in distilled water to prepare two HA solutions. Then NIPAM (50 mg / mL) and MBA (3 mg / mL) were dissolved in the HA solution, and ammonium persulfate (APS) (5 mg / mL) was added as an initiator and stirred at room temperature for 30 minutes. Tetramethylethylenediamine (TEMED) (1.5 mg / mL) was added as a promoter to accelerate the formation of the PNI network and obtain a semi-interpenetrating cross-linked network (SIPN). In order to further promote the cross-linking of HA in the hydrogel, the SIPN hydrogel was directly immersed in an 80% acetone solution containing 100 mM GTA and 0.01 M HCl for 24 hours. After soaking in distilled water for 4 hours to remove glutaraldehyde and acetone, PNI-0.5HA (0.5% HA hydrogel) and PNI-1.0HA (1% HA hydrogel) were obtained. See the electron microscope image. Figure 1 .

[0036] Example 3: Thermal responsive self-shrinkage of PNI-HA

[0037] Methods: PNI-HA was placed in distilled water at different temperatures (25°C, 29°C, 33°C, and 37°C), and images of the samples were recorded. To investigate the relationship between the thermal response shrinkage of the hydrogel and time, the change in area shrinkage of the hydrogel after it was placed from 25°C to 37°C was calculated. Simultaneously, to calculate the change in swelling ratio (SR) caused by thermal response shrinkage, the sample weight was also weighed. To analyze the repeatability of thermal response shrinkage, the SR change of PNI-1.0HA after incubation in distilled water at 25°C or 37°C for 3 hours and undergoing three "shrinkage-swelling" cycles was measured.

[0038] Results: PNI, PNI-0.5HA and PNI-1.0HA had typical thermal response self-shrinkage phenomenon ( Figure 2 A). The hydrogel was moved from 25°C to 37°C to measure the autogenous shrinkage rate ( Figure 2 B). The hydrogel can be observed to shrink significantly in the first 240 min, reaching a maximum at 360 min, and then remaining relatively stable in the subsequent time. The swelling equilibrium of PNI and PNI-HA at different temperatures was determined ( Figure 2 C). As the temperature increased, the volume of all hydrogels decreased significantly. At the same time, the SR decreased significantly. Changes in the swelling equilibrium of the hydrogels were also observed during repeated cycles at 25-37°C. Figure 2 D). The results show that the swelling equilibrium did not change significantly after three cycles, indicating that the thermal responsive self-shrinkage of PNI-HA has good repeatability.

[0039] Example 4: Thermally Responsive Adhesion of PNI-HA

[0040] The tissue adhesion of PNI-1A at different temperatures (25°C, 29°C, 33°C, and 37°C) was tested using a modified tensile test method on a benchtop tensile testing machine. Briefly, two pieces of pigskin were attached to either side of a hydrogel with uniform initial dimensions (12 mm diameter and 1 mm thickness) at 25°C, or to either side of a hydrogel with uniform final dimensions (5 mm diameter and 1 mm thickness) at different temperatures (25°C, 29°C, 33°C, and 37°C). The pigskins were then pulled in opposite directions perpendicular to the hydrogel surface, and the maximum tensile force and associated adhesion strength were recorded before complete separation.

[0041] Results: The tissue adhesion of PNI-HA was quantitatively determined using a modified tensile test method. It was observed that all hydrogels exhibited temperature-sensitive adhesion strength. Figure 3 As shown in A, at the same size, the introduction of HA and the increase of temperature significantly enhanced the adhesion ability of the hydrogel. Using hydrogels with the same initial size at 25°C, the effect of temperature on the adhesion strength of the hydrogel was observed. Figure 3 As shown in Figure B, increasing temperature also significantly enhanced the hydrogel's tissue adhesion. PNI-HA not only adheres to wounds like other clinically used glues, but its higher adhesion strength helps accelerate wound closure through thermally responsive self-contraction, thereby promoting wound healing.

[0042] Example 5: PNI-HA mediated wound closure

[0043] Methods: 1. Male Kunming mice aged 5-6 weeks and weighing 30-40 g were selected to create a full-thickness skin defect model under sterile conditions.

[0044] 2. The wounds in the control group were exposed to the air without treatment, while the wounds and surrounding skin in the experimental group were covered with PNI-HA.

[0045] 3. To analyze the role of PNI-HA in wound closure mediated by self-contraction under thermal stimulation, images of wound closure were recorded at 0, 2, and 4 hours. The wound area was then marked and the stacking position of the mouse wounds was compared over time. The wound closure rate (WCR) (%) was calculated using formula (1):

[0046]

[0047] Where Area0 is the initial area of ​​the wound (0h), Area n is the wound area at nh.

[0048] Results: As Figure 4As shown in A, PNI-HA significantly promotes the closure of rat skin wounds through thermal response self-contraction. In order to observe the closure changes more intuitively, we stacked the wounds at different times ( Figure 4 B). The results once again confirmed that the thermal response of PNI-1.0HA has the most significant effect in accelerating wound closure. The degree of wound area reduction at 2h and 4h compared with 0h was further quantitatively compared ( Figure 4 C / D), PNI-1.0HA reduced wound area by approximately 26.6% at 2 hours and by approximately 45.9% at 4 hours, significantly higher than the PNI group (11.0% reduction at 2 hours and 21.1% reduction at 4 hours) and the control group (4.6% reduction at 2 hours and 10.5% reduction at 4 hours). Overall, PNI-1.0HA exhibited good thermal responsiveness and self-shrinkage, accelerating wound closure.

[0049] Example 6: PNI-HA mediated wound closure PNI-HA mediated wound healing

[0050] Methods: 1. Male Kunming mice aged 5-6 weeks and weighing 30-40 g were selected to create a full-thickness skin defect model under sterile conditions.

[0051] 2. The wounds in the control group were left exposed to air without treatment, while the wounds and surrounding skin in the experimental group were covered with PNI-HA. Images of wound healing were recorded on days 0, 3, 7, 10, 14, and 21.

[0052] 3. On the 21st day, wound tissue specimens were collected from each group for histological evaluation (H&E, Masson staining, CD31 immunohistochemistry, and integrin β1 immunohistochemistry).

[0053] result: Figure 5 As shown in A, the wound area of ​​each group gradually decreased over 21 days, and the wound of PNI-1.0HA healed the best. The results of wound superposition once again confirmed the ability of PNI-1.0HA to promote wound healing ( Figure 5 B). Specifically, the wound area of ​​the PNI-1.0HA group on day 3 was reduced by approximately 43.7% compared to day 0, and the wound healing rate was significantly faster than that of the control group (19.9%) and the PNI group (28.3%). Similarly, the wound healing effect of the PNI-1.0HA group was the best in the first 10 days ( Figure 5 C) Observe the formation of granulation tissue and perform H&E staining, as shown in Figure 5 As shown in D / E. The results showed that both PNI and PNI-1.0HA groups had obvious residual hydrogel material (marked by green arrows), but the degree of inflammatory cell infiltration in PNI and PNI-1.0HA groups was significantly lower than that in the control group. Quantification of inflammatory cells ( Figure 5F) showed that the number of inflammatory cells in the PNI-1.0HA group was significantly less than that in the PNI group, and the number of inflammatory cells in the PNI-1.0HA group was less than that in the PNI group. Masson staining was used to further evaluate the collagen deposition in the wound site ( Figure 5 G). Compared with PNI-1.0HA, the dark blue staining area of ​​the control group and PNI group was larger (greater collagen deposition), indicating a higher scar tissue formation rate.

[0054] Angiogenesis was assessed by CD31 immunohistochemical staining ( Figure 5 H), red arrows indicate CD31-positive blood vessels. Compared with the control group, the number of CD31-positive cells in the PNI-1.0HA group increased significantly, suggesting that PNI-1.0HA promotes angiogenesis and promotes wound healing. Integrin β1 plays a key role in epithelial cell regeneration, such as Figure 5 As shown in I, compared with the control group, the blue color of the epithelial area in the PNI-1.0HA group was darker and the expression level of integrin β1 was higher.

[0055] In summary, PNI-HA exhibits thermoresponsiveness and tissue adhesion, leveraging the thermoresponsive self-shrinkage of hydrogels and the excellent biological functions of HA to significantly promote wound closure and wound healing. These results suggest that PNI-HA provides a promising strategy for wound dressings that accelerate wound closure and promote wound healing.

Claims

1. A double-crosslinked hydrogel, characterized in that: It is composed of two cross-linked networks interwoven with each other, one of which is poly(N-isopropylacrylamide) formed by free radical polymerization of N-isopropylacrylamide and N,N'-methylenebisacrylamide, and the other is a cross-linked network formed by nucleophilic addition reaction of hyaluronic acid and glutaraldehyde.

2. The method for preparing the double-crosslinked hydrogel according to claim 1, wherein the steps as follows: 1) Adding N-isopropylacrylamide and N,N'-methylenebisacrylamide to a hyaluronic acid solution, followed by adding an initiator, stirring, and then adding an accelerator to obtain a semi-interpenetrating cross-linked network; 2) The semi-interpenetrating cross-linked network is directly immersed in an acetone solution containing glutaraldehyde, and then immersed in distilled water to remove the glutaraldehyde and acetone to obtain a double-cross-linked hydrogel.

3. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The stirring process is at room temperature.

4. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The hyaluronic acid has a molecular weight between 500 kDa and 2000 kDa.

5. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The concentration of the hyaluronic acid solution does not exceed 1%.

6. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The pH value of the acetone solution is lower than 4.

7. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The mass ratio of the N-isopropylacrylamide to N,N'-methylenebisacrylamide is 1:0.

06.

8. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The initiator is selected from persulfate and azo-containing organic initiator.

9. The method for preparing the double-crosslinked hydrogel according to claim 2, wherein: The accelerator is selected from tetramethylethylenediamine and triethylamine.

10. Use of the double-crosslinked hydrogel according to claim 1 in preparing a wound healing dressing.

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