A photo-crosslinking antibacterial chitosan adhesive as well as a preparation method and application thereof

Abstract

This invention discloses a photocrosslinked antibacterial chitosan adhesive, its preparation method, and its application, belonging to the field of biomedical polymer materials. The method includes: dissolving chitosan in an aqueous acetic acid solution until completely dissolved to obtain a chitosan solution; dissolving lipoic acid in ethanol, adding the dissolved chitosan to the chitosan solution, and reacting to obtain lipoic acid-modified chitosan; grafting the lipoic acid-modified chitosan with gallic acid to obtain bifunctionalized chitosan; preparing a precursor solution of the bifunctionalized chitosan and irradiating it with ultraviolet light to obtain the photocrosslinked antibacterial chitosan adhesive. This invention uses chitosan as a substrate, utilizing the photo-induced ring-opening polymerization characteristics of the disulfide bonds of lipoic acid to achieve rapid photocuring without an initiator; enhancing interfacial adhesion through the pyrogallol structure of gallic acid and synergistically achieving broad-spectrum antibacterial activity with the cationic properties of chitosan; and simultaneously scavenging reactive oxygen species through the polyphenolic hydroxyl groups of gallic acid to regulate the inflammatory microenvironment, ultimately forming a multifunctional integrated hydrogel system.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials technology, and more specifically, relates to a photocrosslinked antibacterial chitosan adhesive, its preparation method, and its application. Background Technology

[0002] Surgical adhesives are crucial materials for achieving rapid hemostasis, wound closure, and infection prevention in trauma care and minimally invasive surgery. While existing commercially available fibrin glue and cyanoacrylate adhesives can partially meet the needs, they have significant shortcomings: weak adhesion strength in wet tissue (<10kPa), making them prone to detachment in bleeding and exudative environments; slow curing kinetics (>1 minute), failing to meet the requirements for rapid hemostasis; and the materials themselves lack antibacterial activity, making postoperative wounds susceptible to bacterial infection.

[0003] Photopolymerizable hydrogels offer novel approaches to deep wound treatment due to their minimally invasive delivery and spatiotemporally controllable curing properties. However, existing photopolymerization systems suffer from key bottlenecks: they rely on exogenous photoinitiators, which can easily induce cytotoxicity; and they struggle to simultaneously achieve a multifunctional integration of rapid photopolymerization, strong wet adhesion, and highly effective antibacterial properties. Furthermore, traditional injectable hydrogels suffer from insufficient permeability and interfacial adhesion in deep wounds, further limiting their clinical application.

[0004] Chitosan, as a natural polysaccharide, possesses excellent biocompatibility, biodegradability, and inherent antibacterial activity; however, it lacks photopolymerization activity and strong adhesion properties. The polyhydroxy structure of pyrogallol compounds (such as gallic acid) can enhance interfacial adhesion through hydrogen bonding and π-π stacking, and also exhibits antibacterial and antioxidant activities. The disulfide bonds of lipoic acid can undergo photo-triggered ring-opening polymerization without the need for exogenous initiators, thus avoiding cytotoxic risks. Current technologies have not yet synergistically designed these three components, failing to simultaneously address multiple issues related to the curing speed, adhesion strength, antibacterial properties, and biosafety of existing adhesives.

[0005] In summary, existing biomedical photocrosslinking adhesives have drawbacks such as low curing efficiency, poor wet adhesion performance, insufficient biosafety, limited functionality, and insufficient penetration into deep wounds, thus limiting their clinical application. Summary of the Invention

[0006] To address the shortcomings and improvement needs of existing technologies, this invention provides a photocrosslinked antibacterial chitosan adhesive, its preparation method, and its application. The aim is to solve the problems of low curing efficiency, poor wet adhesion, insufficient biocompatibility, limited functionality, and insufficient penetration into deep wounds in existing biomedical photocrosslinked adhesives, thus restricting their clinical application.

[0007] To achieve the above objectives, in a first aspect, the present invention provides a method for preparing a photocrosslinked antibacterial chitosan adhesive, comprising the following steps: Chitosan was dissolved in an aqueous acetic acid solution until completely dissolved to obtain a chitosan solution; Lipoic acid was dissolved in ethanol, and the solution was added to the chitosan solution. After the reaction, lipoic acid-modified chitosan (TCS) was obtained. The lipoic acid-modified chitosan TCS was grafted with gallic acid to obtain bifunctionalized chitosan GTCS. The bifunctionalized chitosan GTCS was formulated into a precursor solution and then irradiated with ultraviolet light to obtain a photocrosslinked antibacterial chitosan adhesive.

[0008] Further, the lipoic acid is dissolved in ethanol, and then added to the chitosan solution. After reaction, lipoic acid-modified chitosan TCS is obtained, specifically as follows: Lipoic acid was dissolved in ethanol, and the solution was added dropwise to the chitosan solution. An activating reagent was added, and the reaction was carried out under nitrogen protection for a first preset time. The reaction solution was dialyzed in acidic deionized water through a dialysis bag and then freeze-dried to obtain thioctic acid-modified chitosan (TCS).

[0009] Furthermore, the activating reagent is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the molecular weight cutoff of the dialysis bag is 3.5 kDa, and the pH of the acidic deionized water is 5.5.

[0010] Furthermore, the process of grafting the lipoic acid-modified chitosan TCS with gallic acid to obtain bifunctionalized chitosan GTCS specifically involves: After dissolving the lipoic acid-modified chitosan TCS in an aqueous acetic acid solution, gallic acid was added and stirred to dissolve. Then, an activating agent was added and the reaction was stirred for a second preset time. The reaction solution was first dialyzed in acidic ultrapure water, then dialyzed in neutral ultrapure water to remove acid residue, and then freeze-dried to obtain bifunctional chitosan GTCS.

[0011] Furthermore, the lipoic acid grafting rate is 14.88%, and the gallic acid grafting rate is 9.59%-40.69%.

[0012] Further, the step of preparing the bifunctionalized chitosan GTCS into a precursor solution and irradiating it with ultraviolet light to obtain a photocrosslinked antibacterial chitosan adhesive is specifically as follows: The bifunctionalized chitosan GTCS was dissolved in deionized water to prepare a precursor solution; it was then irradiated with 365nm ultraviolet light at an intensity of 30mW / cm² to obtain a photocrosslinked antibacterial chitosan adhesive.

[0013] In a second aspect, the present invention provides a photocrosslinked antibacterial chitosan adhesive, wherein the photocrosslinked antibacterial chitosan adhesive is obtained by the preparation method of the photocrosslinked antibacterial chitosan adhesive described in the first aspect.

[0014] Furthermore, the photocrosslinked antibacterial chitosan adhesive is a porous injectable hydrogel.

[0015] Thirdly, the present invention provides the application of the photocrosslinked antibacterial chitosan adhesive described in the second aspect in the preparation of medical wound repair materials.

[0016] In summary, this invention uses chitosan (CS) as a substrate and introduces lipoic acid (TA) and gallic acid (GA) sequentially through a two-step grafting reaction to construct a bifunctional chitosan (GTCS) precursor. It utilizes the photo-induced ring-opening polymerization characteristics of lipoic acid's disulfide bonds to achieve rapid photocuring without an initiator. The pyrogallol structure of gallic acid enhances interfacial adhesion and, in conjunction with the cationic properties of chitosan, achieves broad-spectrum antibacterial activity. Simultaneously, the polyphenolic hydroxyl groups of gallic acid scavenge reactive oxygen species (ROS), regulating the inflammatory microenvironment, ultimately forming a multifunctional integrated hydrogel system of "polymerization-adhesion-antibacterial-anti-inflammatory," achieving the following beneficial effects: (1) Ultra-fast initiator-free curing: UV photopolymerization is completed within 11s, which is much faster than existing photopolymer materials (>1 minute), and no exogenous photoinitiator is required, which completely eliminates the risk of cytotoxicity and solves the contradiction between safety and curing efficiency of traditional photopolymer biomaterials; (2) Super strong wet adhesion performance: The shear adhesion strength of GTCS2 hydrogel to pigskin is 31.40±5.09kPa, which is more than 3 times that of commercial adhesives (<10kPa), and it maintains stable adhesion in aqueous environment, solving the problem of easy detachment of adhesion on wet wounds. (3) Multifunctional synergistic integration: Simultaneously realizes emergency hemostasis, broad-spectrum antibacterial, ROS clearance and inflammation regulation functions, shortens hemostasis time by 70%, kills Escherichia coli and Staphylococcus aureus at a rate of >98%, clears ROS at a rate of >85%, and does not require additional antibacterial agents or anti-inflammatory drugs, simplifying product composition; (4) Excellent biocompatibility: hemolysis rate <5%, survival rate of L929 fibroblasts and HUVEC endothelial cells >80%, no obvious toxicity to major organs (heart, liver, spleen, lungs and kidneys) in vivo, and can be completely degraded within 10 days, meeting the safety requirements of biomedical materials; (5) Wide range of applications: It is injectable and self-healing, with good permeability and interface adhesion. It is suitable for both surface wounds and deep tissue damage for emergency hemostasis and repair of infected wounds. It is especially suitable for minimally invasive surgery and emergency scenarios. Attached Figure Description

[0017] Figure 1 ¹H NMR spectra of LA hydrogel, CS hydrogel, and GTCS1, GTCS2, and GTCS3 hydrogels prepared in the embodiments of the present invention.

[0018] Figure 2 FTIR infrared spectra of CS hydrogel, TCS hydrogel, and GTCS1, GTCS2, and GTCS3 hydrogels prepared in the embodiments of the present invention.

[0019] Figure 3 Rheological time-scanning curves of TCS hydrogel and GTCS1, GTCS2, and GTCS3 hydrogels prepared in the embodiments of the present invention.

[0020] Figure 4 This is a scanning electron microscope image of the GTCS2 hydrogel prepared in Example 1 of the present invention.

[0021] Figure 5 The hemostatic effect diagrams of gelatin sponge, TCS hydrogel, and GTCS2 hydrogel provided in Embodiment 2 of the present invention are shown.

[0022] Figure 6 This is a comparison chart of hemostasis time and bleeding volume of gelatin sponge, TCS hydrogel, and GTCS2 hydrogel provided in Example 2 of the present invention.

[0023] Figure 7 The images show the wound closure effect of the 3M Tegaderm™ dressing, TCS hydrogel, and GTCS2 hydrogel provided in Embodiment 3 of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0026] It should be noted that, in the specific implementation of this invention, the raw material composition is as follows: Chitosan (CS): viscosity 1000 mPa•s, degree of deacetylation 95.2%; Alpha-lipoic acid (TA), gallic acid (GA); Activating agents: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC•HCl), N-hydroxysuccinimide (NHS); Solvents: 1.5% (v / v) aqueous acetic acid, ethanol (analytical grade); All other reagents were of analytical grade.

[0027] This invention provides a method for preparing a photocrosslinked antibacterial chitosan adhesive, specifically comprising: Step 1: Preparation of lipoic acid modified chitosan (TCS) (1) Take 1.2g of chitosan and add it to 60mL of 1.5% (v / v) acetic acid aqueous solution. Stir magnetically at 25℃ for 12h until completely dissolved to obtain chitosan solution; (2) Take another 0.2g of lipoic acid and dissolve it in 40mL of ethanol. Slowly add the solution to the above chitosan solution, and then add 0.8g of EDC•HCl and 0.2g of NHS in sequence. (3) Under nitrogen protection, react at 25°C for 4 hours; (4) The reaction solution was dialyzed in deionized water at pH 5.5 for 72 hours through a dialysis bag with a molecular weight cutoff of 3.5 kDa (the dialysate was changed every 8 hours). (5) After freeze drying, lipoic acid modified chitosan (TCS) was obtained, with a photosensitive disulfide bond grafting rate of 14.88%.

[0028] Step 2: Preparation of bifunctionalized chitosan (GTCS) (1) Dissolve 1.00g TCS in 50mL of 1.0% (v / v) acetic acid aqueous solution, and add 0.125g, 0.25g, and 0.5g gallic acid respectively (to prepare GTCS1, GTCS2, and GTCS3), and stir until completely dissolved; (2) Add 1.2g EDC•HCl and 0.7g NHS, and stir vigorously at room temperature for 2h; (3) The reaction solution was first dialyzed in ultrapure water with pH 5.5 for 72 hours (the dialysate was changed every 8 hours), and then dialyzed twice in neutral ultrapure water to remove acid residue; (4) After freeze-drying, a series of bifunctional chitosans (GTCS1, GTCS2, GTCS3) were obtained, with gallic acid grafting rates of 9.59%, 24.74%, and 40.69%, respectively.

[0029] Step 3: Preparation of photocrosslinked antibacterial chitosan adhesive GTCS (preferably GTCS2) was dissolved in deionized water to prepare a 2w / w% precursor solution. The solution was then irradiated with 365nm ultraviolet light (30mW / cm²), and polymerization was completed within 11s without the need for an exogenous photoinitiator, forming a stable photocrosslinked antibacterial chitosan adhesive.

[0030] The core innovation of this invention lies in the synergistic design of "dual-functional modification": the disulfide bonds of lipoic acid undergo ring-opening polymerization under UV light to form a stable polysaccharide thioether network, achieving rapid curing without initiators; the pyrogallol structure of gallic acid forms strong interactions with functional groups such as amino and hydroxyl groups on the tissue surface through multiple hydrogen bonds, π-π stacking, and Michael addition reactions, significantly improving wet adhesion; simultaneously, the polyphenolic hydroxyl groups of gallic acid synergistically work with the cationic properties of chitosan to achieve broad-spectrum antibacterial effects by disrupting bacterial cell membranes and inhibiting bacterial metabolism, and the polyphenolic hydroxyl groups can also act as electron donors to scavenge ROS and regulate the expression of inflammatory factors. The synergistic effect of the above functional modules precisely solves the core pain points of existing adhesives such as "slow curing, weak adhesion, single function, and insufficient safety," achieving full-chain optimization of "polymerization-adhesion-antibacterial-anti-inflammatory-repair."

[0031] The dual cross-linking network formed by disulfide ring-opening polymerization and polyphenol-thiol reaction endows the hydrogel with excellent self-healing properties (complete healing of the fracture surface within 30 minutes) and mechanical stability, adapting to the dynamic environment of the wound. The adjustable gallic acid grafting rate (9.59%-40.69%) allows for flexible control of the hydrogel's adhesion strength, antibacterial activity, and curing speed, meeting the treatment needs of different wounds. No complex equipment is required; the precursor solution can be stored at room temperature and rapidly cured by UV light irradiation, making it simple to operate and conducive to clinical application. Related experimental characterization results are available in the [reference needed]. Figures 1 to 3 .

[0032] Example 1: Preparation and characterization of GTCS2 hydrogel with optimal performance (1) Prepare GTCS2 (gallic acid grafting rate 24.74%) according to steps 1-3 above, and prepare a 2w / w% precursor solution; (2) Ultraviolet light polymerization: 365nm UV light (30mW / cm²) was irradiated for 11s to obtain GTCS2 hydrogel; (3) Characterization results: Chemical structure: such as Figure 1 and Figure 2 As shown, ¹H NMR verifies the successful grafting of TA and GA, and FTIR detects characteristic functional groups; Rheological properties: such as Figure 3 As shown, the storage modulus (G') is higher than the loss modulus (G''), exhibiting excellent gel strength, with a G' recovery rate of 95% under alternating strain (1%-1000%) cycles; Microstructure: such as Figure 4 As shown, scanning electron microscopy reveals a porous network structure that facilitates cell infiltration and substance exchange.

[0033] Example 2: Application of GTCS2 hydrogel in emergency hemostasis (1) Animal model: 8-week-old SD rats (220-250g) with liver perforation injury (5mm long × 2mm deep); (2) Treatment plan: Inject 0.5 mL of GTCS2 precursor solution into the damaged area and cure by UV light irradiation for 60 s; (3) Control group: commercial gelatin sponge, TCS hydrogel; (4) Results: such as Figure 5 and Figure 6 As shown, the GTCS2 group had a hemostasis time of 32±3.6 s and a blood loss of 0.81±0.16 g, which were significantly better than the gelatin sponge group (158.33±13.05 s, 2.62±0.42 g) and the TCS group (65±8.19 s, 1.42±0.26 g).

[0034] Example 3: Application of GTCS2 hydrogel in healing infected wounds (1) Animal model: A 15 mm diameter full-thickness wound on the back of SD rats was inoculated with a mixed bacterial suspension of Escherichia coli and Staphylococcus aureus (1×10⁻⁶ each). 7 CFU), to construct an infected wound model; (2) Treatment plan: Inject 1 mL of GTCS2 precursor solution into the wound and cure by UV light irradiation for 60 s; (3) Control group: Commercial 3M Tegaderm™ dressing, TCS hydrogel; (4) Results: such as Figure 7 As shown, the wound closure rate of the GTCS2 group reached 93.23% after 14 days, which was significantly higher than that of the 3M group (86.05%) and the TCS group (88.40%); the wound bacterial clearance rate was >99%, the collagen deposition amount reached 63.40±2.48%, and the expression of inflammatory factors (TNF-α, IL-6) was significantly downregulated.

[0035] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing a photocrosslinked antibacterial chitosan adhesive, characterized in that, Includes the following steps: Chitosan was dissolved in an aqueous acetic acid solution until completely dissolved to obtain a chitosan solution; Lipoic acid was dissolved in ethanol, and the solution was added to the chitosan solution. After the reaction, lipoic acid-modified chitosan (TCS) was obtained. The lipoic acid-modified chitosan TCS was grafted with gallic acid to obtain bifunctionalized chitosan GTCS. The bifunctionalized chitosan GTCS was formulated into a precursor solution and then irradiated with ultraviolet light to obtain a photocrosslinked antibacterial chitosan adhesive.

2. The method for preparing the photocrosslinked antibacterial chitosan adhesive according to claim 1, characterized in that, The lipoic acid is dissolved in ethanol, and then added to the chitosan solution. After reaction, lipoic acid-modified chitosan (TCS) is obtained. Specifically: Lipoic acid was dissolved in ethanol, and the solution was added dropwise to the chitosan solution. An activating reagent was added, and the reaction was carried out under nitrogen protection for a first preset time. The reaction solution was dialyzed in acidic deionized water through a dialysis bag and then freeze-dried to obtain thioctic acid-modified chitosan (TCS).

3. The method for preparing the photocrosslinked antibacterial chitosan adhesive according to claim 2, characterized in that, The activating reagents are 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, the molecular weight cutoff of the dialysis bag is 3.5 kDa, and the pH of the acidic deionized water is 5.

5.

4. The preparation method of the photocrosslinked antibacterial chitosan adhesive according to claim 1, characterized in that, The process of grafting gallic acid onto lipoic acid-modified chitosan TCS to obtain bifunctionalized chitosan GTCS is as follows: After dissolving the lipoic acid-modified chitosan TCS in an aqueous acetic acid solution, gallic acid was added and stirred to dissolve. Then, an activating agent was added and the reaction was stirred for a second preset time. The reaction solution was first dialyzed in acidic ultrapure water, then dialyzed in neutral ultrapure water to remove acid residue, and then freeze-dried to obtain bifunctional chitosan GTCS.

5. The method for preparing the photocrosslinked antibacterial chitosan adhesive according to claim 4, characterized in that, The grafting rate of lipoic acid was 14.88%, and the grafting rate of gallic acid was 9.59%-40.69%.

6. The method for preparing the photocrosslinked antibacterial chitosan adhesive according to claim 1, characterized in that, The process of preparing a precursor solution from the bifunctionalized chitosan GTCS and then irradiating it with ultraviolet light to obtain a photocrosslinked antibacterial chitosan adhesive is as follows: The bifunctionalized chitosan GTCS was dissolved in deionized water to prepare a precursor solution; it was then irradiated with 365nm ultraviolet light at an intensity of 30mW / cm² to obtain a photocrosslinked antibacterial chitosan adhesive.

7. A photocrosslinked antibacterial chitosan adhesive, characterized in that, The photocrosslinked antibacterial chitosan adhesive is obtained by the preparation method of the photocrosslinked antibacterial chitosan adhesive according to any one of claims 1 to 6.

8. The photocrosslinked antibacterial chitosan adhesive according to claim 7, characterized in that, The photocrosslinked antibacterial chitosan adhesive is a porous injectable hydrogel.

9. The application of the photocrosslinked antibacterial chitosan adhesive according to claim 7 or 8 in the preparation of medical wound repair materials.

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