Photocured hydrogel scaffold and its preparation method and application

Abstract

The present application relates to a kind of photo-cured hydrogel scaffolds, the photo-cured hydrogel scaffold includes collagen, o-nitrobenzyl alcohol polyethylene glycol and nanoparticle;The collagen includes phenyl boronic acid grafting methacryl collagen;The nanoparticle includes polydopamine-coated adsorbed with epigallocatechin gallate zeolite imidazolate framework-8 nanoparticle.This photo-cured hydrogel scaffold can be applied to complex infectious oral mucosa injury repair.

Description

Technical Field

[0001] This invention relates to the field of hydrogel scaffold technology, and in particular to a photocurable hydrogel scaffold, its preparation method, and its application. Background Technology

[0002] Oral ulcers are a prevalent oral disease worldwide, characterized by persistent erosion or destruction of epithelial tissue, affecting more than 25% of the world's population. Without effective intervention, progressive deterioration of the epithelial layer at the ulcer site can lead to tissue depression and even necrosis, severely impairing chewing, swallowing, and speech functions, and indirectly affecting the digestive process. More importantly, the loss of mucosal barrier function makes the wound susceptible to bacterial invasion, further creating a vicious cycle that hinders healing. The ulcer healing process involves continuous but overlapping phases, including hemostasis, inflammation, proliferation, and structural remodeling of the injured tissue. Activation of inflammatory cytokines typically occurs during the hemostasis phase, with the inflammatory response peaking within 24–48 hours and lasting for several days. Furthermore, chronic inflammation and impaired angiogenesis disrupt granulation tissue remodeling, leading to prolonged wound healing time. Inhibiting the secretion of pro-inflammatory cytokines and enhancing endothelial cell proliferation can accelerate wound healing.

[0003] Current clinical treatment options include vitamin preparations, commercially available patches containing flavonoids / chitosan, and growth factor gels. However, the dynamically moist oral environment limits the duration of these preparations' retention on the moist wound, preventing the active ingredients from sustaining their effect and limiting treatment efficacy. Furthermore, growth factor gels and cytokine dressings are expensive and carry the risk of exogenous additives. Therefore, developing biomaterials with long-lasting wet adhesion capabilities and providing anti-inflammatory and antibacterial effects is crucial. Thus, developing a composite scaffold system with highly efficient adhesion, multiple functions including antibacterial, anti-inflammatory, and antioxidant properties, and responsive modulation to external stimuli is a core requirement for improving the efficiency of oral ulcer treatment.

[0004] To address the shortcomings of existing technologies, this invention aims to solve the problems of short residence time and insufficient antibacterial and anti-inflammatory properties in oral ulcer repair materials, which lead to poor treatment outcomes. While existing materials can provide some repair, most lack prolonged adhesion in a moist state and are ineffective in promoting the healing of oral mucosal injuries. Furthermore, some materials suffer from difficulties in forming and brittleness during processing, failing to meet clinical needs. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a photocurable hydrogel scaffold comprising collagen (Col), o-nitrobenzyl glycol-modified polyethylene glycol (PEGNB), and nanoparticles; the collagen comprising phenylboronic acid-grafted methacryloyl collagen (ColMA-PBA); and the nanoparticles comprising polydopamine (PDA)-coated zeolite imidazole salt backbone-8 (ZIF8) nanoparticles adsorbed with epigallocatechin gallate (EGCG).

[0006] In one embodiment, the raw materials for preparing the phenylboronic acid-grafted methacrylamide collagen include collagen, 3-aminophenylboronic acid, and methacrylic anhydride; the collagen includes type I collagen (Col1).

[0007] A second aspect of the present invention also provides a method for preparing a photocurable hydrogel scaffold, comprising the following steps:

[0008] Preparation of phenylboronic acid-grafted methacrylamide collagen: Collagen was dissolved, and 3-aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were dissolved, mixed, reacted, dialyzed, and lyophilized to obtain phenylboronic acid-grafted collagen (Col-PBA); phenylboronic acid-grafted collagen was dissolved, methacrylic anhydride was added, reacted, dialyzed, and lyophilized to obtain phenylboronic acid-grafted methacrylamide collagen;

[0009] Preparation of o-nitrobenzyl alcoholized polyethylene glycol: polyethylene glycol is dissolved, o-nitrobenzyl alcohol (NB) is added, 1-hydroxybenzotriazole (HOBt) is added, the pH is adjusted, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) is added. The mixture is stirred, dialyzed, and freeze-dried to obtain o-nitrobenzyl alcoholized polyethylene glycol.

[0010] Preparation of polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate (ZIF8-PDA-EGCG): Zinc nitrate and 2-methylimidazole were dissolved and reacted to obtain a suspension. After standing, centrifugation and washing, zeolite imidazole salt framework-8 nanoparticles were obtained and freeze-dried. Epigallocatechin gallate was dissolved and sonicated. Zeolite imidazole salt framework-8 nanoparticles were added and sonicated, shaken, and centrifuged to obtain zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate (ZIF8-E). The nanoparticles obtained in the previous step were dissolved, dopamine hydrochloride was added, stirred, centrifuged and washed to obtain polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate (ZP-E).

[0011] Preparation of photocurable hydrogel scaffold: Phenyboric acid-grafted methacrylamide collagen and o-nitrobenzyl glycol were dissolved separately. Polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate were added to the phenylboric acid-grafted methacrylamide collagen and mixed evenly. Then o-nitrobenzyl glycol was added and mixed evenly. The mixture was then irradiated to obtain the photocurable hydrogel scaffold (CP@ZP-E).

[0012] In one embodiment, the preparation of the phenylboronic acid-grafted methacrylamide collagen includes the following steps: dissolving collagen in water and stirring until completely dissolved; dissolving 3-aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in a mixed solvent of water and DMSO and adding the solution to the collagen; reacting at room temperature; dialyzing with a dialysis bag; and lyophilizing to obtain phenylboronic acid-grafted collagen; dissolving the phenylboronic acid-grafted collagen in water; adding methacrylic anhydride; reacting at room temperature; dialyzing with a dialysis bag; and lyophilizing to obtain phenylboronic acid-grafted methacrylamide collagen.

[0013] In one embodiment, the stirring until completely dissolved is performed at 40-50°C.

[0014] In one embodiment, the mixed solvent of water and DMSO is in a volume ratio of (4~6):1 for water to DMSO.

[0015] In one embodiment, the room temperature reaction time is 2 to 4 days.

[0016] In one embodiment, the molecular weight cutoff of the dialysis bag in the phenylboronic acid-grafted collagen obtained by dialysis and freeze-drying is 3400-3600 Da.

[0017] In one embodiment, the temperature at which the phenylboronic acid-grafted collagen is dissolved is 40-50°C.

[0018] In one embodiment, the reaction time at room temperature after the addition of methacrylic anhydride is 3-5 hours.

[0019] In one embodiment, the molecular weight cutoff of the dialysis bag in the phenylboronic acid-grafted methacrylamide collagen obtained by dialysis and freeze-drying is 7500~8500 Da.

[0020] In one embodiment, the preparation of o-nitrobenzyl alcohol-modified polyethylene glycol includes the following steps: dissolving polyethylene glycol in water, adding o-nitrobenzyl alcohol and then adding 1-hydroxybenzotriazole, adjusting the pH to 4-5, adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stirring, dialyzing with a dialysis bag, and freeze-drying to obtain o-nitrobenzyl alcohol-modified polyethylene glycol.

[0021] In one embodiment, the mass of the 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is 150~250 mg.

[0022] In one embodiment, the stirring time is 45-50 hours.

[0023] In one embodiment, the dialysis with a dialysis bag after stirring includes dialysis with dilute hydrochloric acid containing sodium chloride, followed by dialysis with deionized water.

[0024] In one embodiment, the pH of the dilute hydrochloric acid is 3 to 4.

[0025] In one embodiment, the dialysis time with dilute hydrochloric acid containing sodium chloride is 1 to 3 days.

[0026] In one embodiment, the dialysis time with deionized water is 1 to 3 days.

[0027] In one embodiment, the molecular weight cutoff of the dialysis bag used for dialysis after stirring is 3400~3600 Da.

[0028] In one embodiment, the preparation of the polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate includes the following steps: zinc nitrate and 2-methylimidazole are dissolved in organic solvents respectively, and the two are stirred and mixed at room temperature to obtain a suspension. The suspension is allowed to stand, centrifuged, and washed to obtain zeolite imidazole salt framework-8 nanoparticles, which are then freeze-dried; epigallocatechin gallate is dissolved in an organic solvent, sonicated, and then the zeolite imidazole salt framework-8 nanoparticles are added. The mixture is sonicated, dispersed, and shaken to obtain a mixture. The mixture is centrifuged to obtain zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate, which are then freeze-dried; the nanoparticles obtained in the previous step are added to a buffer solution, and then dopamine hydrochloride is added and stirred. After centrifugation and washing, the mixture is freeze-dried to obtain polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate.

[0029] In one embodiment, the organic solvent includes anhydrous methanol.

[0030] In one embodiment, the suspension obtained by stirring the two at room temperature is obtained by magnetic stirring for 10-25 minutes.

[0031] In one embodiment, the centrifugation time for washing after centrifugation is 3 to 8 minutes.

[0032] In one embodiment, the ultrasound duration is 3 to 8 minutes.

[0033] In one embodiment, the ultrasonic dispersion time for ultrasonic dispersion and oscillation is 0.5 to 1.5 minutes, and the oscillation time is 23 to 25 hours.

[0034] In one embodiment, the centrifugation time for the mixture is 3 to 8 minutes.

[0035] In one embodiment, the freeze-drying time is 45-50 hours.

[0036] In one embodiment, the buffer solution comprises a tris(hydroxymethyl)aminomethane hydrochloride solution.

[0037] In one embodiment, the pH of the buffer solution is 8 to 9.

[0038] In one embodiment, the stirring temperature for adding dopamine hydrochloride is 35-40°C, and the stirring time is 10-15 hours.

[0039] In one embodiment, in the preparation of phenylboronic acid grafted methacrylamide collagen, the collagen : 3-aminophenylboronic acid mass ratio is (1~3): 1;

[0040] The phenylboronic acid-grafted collagen: the methacrylic anhydride is 1.0 g: (0.4~0.8) mL;

[0041] In the preparation of o-nitrobenzyl alcohol-modified polyethylene glycol, the mass ratio of polyethylene glycol to o-nitrobenzyl alcohol is 6: (0.1~0.4).

[0042] In the preparation of the polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate, the mass ratio of zinc nitrate to 2-methylimidazole is (1~2):(1~2).

[0043] The epigallocatechin gallate: the zeolite imidazole salt skeleton-8 nanoparticles are (1~2) mL: (1~2) mg.

[0044] According to the mass ratio, the zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate ester are 1: (0.3~0.5).

[0045] In one embodiment, the light irradiation time in the preparation step of the photocurable hydrogel scaffold is 10~30s.

[0046] In one embodiment, the illumination is blue light.

[0047] A third aspect of the present invention also provides the application of the above-described photocurable hydrogel scaffold or the photocurable hydrogel scaffold obtained by the above preparation method in the preparation of products for the treatment of oral ulcers.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] 1. Photothermal antibacterial and EGCG release work synergistically to meet the needs of the complex microenvironment of oral mucosal damage;

[0050] 2. It can be photothermally controlled to achieve a triple function of "antibacterial + anti-inflammatory + antioxidant";

[0051] 3. PDA modification of ZIF8 improves dispersibility and photothermal activity, and reduces the influence of PDA on the photocuring of NB groups;

[0052] 4. In-situ photopolymerization platform, precise bracket molding and controllable structure;

[0053] 5. The oral environment is moist, bacteria-rich, and subject to mechanical stresses such as chewing; CP@ZP-E hydrogel membrane can simultaneously meet the following requirements: ① wet adhesion (firmly adheres to moist, dynamic oral mucosal wounds); ② rapid light curing (precisely fits irregular oral wounds); ③ mechanical support (resistance to oral mechanical forces); ④ antibacterial and anti-inflammatory (coping with complex bacterial flora); ⑤ promotes repair (promotes the regeneration of oral mucosa, a special type of epithelium); an integrated solution for complex clinical needs.

[0054] 6. It has broad application prospects and is suitable for the repair of complex infectious oral mucosal injuries. Attached Figure Description

[0055] Figure 1 Gel formation images of Col1MA-PBA (10%, 15%, and 20%) and PEGNB;

[0056] Figure 2 SEM images of the gelation of Col1MA-PBA (10%, 15% and 20%) and PEGNB;

[0057] Figure 3 The release of EGCG in Example 5 is shown under NIR and non-NIR irradiation conditions;

[0058] Figure 4 The dynamic wet adhesion effect of the bracket in Example 5;

[0059] Figure 5 Photothermal heating curves for nanoparticle concentrations of 0 and 400 μg / mL;

[0060] Figure 6 The levels of TNF-α and IL-6 in mice after 7 days of wound treatment in each group. Detailed Implementation

[0061] To facilitate understanding of the present invention, a more complete description will be given below with reference to the accompanying drawings. Preferred embodiments of the invention are shown in the drawings. However, the invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the invention.

[0062] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0063] This invention designs a photocurable hydrogel membrane with photothermal regulation and pH-responsive release, exhibiting antibacterial, anti-inflammatory, antioxidant, and repair-promoting functions, for the repair of oral mucosal injuries. The hydrogel membrane uses methacrylamide collagen-phenylboronic acid (Col1MA-PBA) as the matrix, and incorporates an o-nitrobenzyl glycol-modified polyethylene glycol (PEGNB) photosensitive crosslinking network. In-situ rapid photocuring of the hydrogel (CP) is achieved using 405nm light irradiation. We formed a highly structurally controllable composite scaffold CP@ZP-E by mixing solutions of Col1MA-PBA, PEGNB, and ZP-E in a specific ratio and photocuring under 405nm light irradiation. In the scaffold, Col1MA-PBA utilizes the dynamic non-covalent bonding between phenylboronic acid (PBA) groups and wound tissue proteins to provide excellent wet adhesion. The in-situ rapid photocuring of the hydrogel using the PEGNB photosensitive crosslinking network and 405nm light irradiation allows for precise matching of the wound contour and provides good mechanical support. ZP-E NPs achieve programmed temperature-controlled photothermal stimulation under 405nm light irradiation, rapidly killing bacteria at high temperatures and continuously stimulating tissue growth at low temperatures. Simultaneously, EGCG is gradually released within the nanoparticles, enhancing the immunomodulatory and antibacterial effects of photothermal therapy and promoting angiogenesis and cell regeneration. This composite scaffold, through a synergistic mechanism of photothermal response and active substance release, achieves simultaneous infection control, inflammation resistance, oxidative stress reduction, and wound repair, demonstrating excellent in vitro antibacterial, anti-inflammatory, antioxidant, and tissue repair potential, providing a new approach for the treatment of oral mucosal damage.

[0064] Example

[0065] This invention provides a method for preparing the above-mentioned photocurable hydrogel scaffold with antibacterial, anti-inflammatory, antioxidant, and tissue-repair-promoting properties, comprising the following steps:

[0066] 1. Synthesis of phenylboronic acid-grafted methacrylylated type I collagen (Col1MA-PBA)

[0067] 2.0 g of Col1 was dissolved in 50 mL of water and stirred at 45 °C until completely dissolved. 1.0 g of 3-aminophenylboronic acid, 1.0 g of EDC, and 0.5 g of NHS were dissolved in 50 mL of water + 10 mL of DMSO solution, and then the mixture was added to the Col1 solution. The reaction was carried out at room temperature for 3 days, dialyzed through a 3500 Da dialysis bag, and lyophilized to obtain Col1-PBA. 1.0 g of Col1-PBA was weighed and dissolved in 50 mL of aqueous solution. After complete dissolution at 45 °C, 0.6 mL of methacrylic anhydride was added, and the reaction was carried out at room temperature for 4 hours. The reaction was then dialyzed through an 8000 Da dialysis bag and lyophilized.

[0068] In this embodiment, the mass ratio of collagen to 3-aminophenylboronic acid is 2:1;

[0069] Benzylboronic acid grafted collagen: methacrylic anhydride 1g: 0.6mL.

[0070] 2. Preparation of o-nitrobenzyl alcohol-modified polyethylene glycol (PEGNB)

[0071] Polyethylene glycol (6g) was dissolved in 50mL of deionized water at room temperature. NB (0.224g) was added, followed by HOBt (153mg). The pH of the solution was adjusted to 4.5, and finally EDC (200mg) was added. The mixture was stirred for 48h, placed in a dialysis bag (MW=3500), and dialyzed for 2 days in dilute hydrochloric acid (pH=3.5) containing 0.1M NaCl. Then, it was dialyzed for 2 days in deionized water. The solution was freeze-dried to obtain PEGNB.

[0072] In this embodiment, the mass ratio of polyethylene glycol to o-nitrobenzyl alcohol is 3:0.112.

[0073] 3. Preparation of ZIF8-PDA-EGCG (ZP-E)

[0074] 2.2071 g of zinc nitrate and 2.2071 g of 2-methylimidazole were dissolved in 10 mL of anhydrous methanol. At room temperature, the zinc nitrate solution was rapidly added to the 2-methylimidazole solution with vigorous stirring, forming a white emulsion suspension within 5 min. The suspension was further stirred with a magnetic stirrer at 850 rpm for 15 min. After standing at 4 °C for 6 h, the suspension was centrifuged at 9000 rpm for 5 min and washed multiple times with anhydrous methanol to remove unreacted zinc nitrate and 2-methylimidazole residues and accelerate water evaporation, yielding zeolite imidazole salt framework-8 nanoparticles (ZIF-8). Finally, the synthesized ZIF-8 was freeze-dried for 48 h to obtain the ZIF-8 sample.

[0075] A specific amount of EGCG was dispersed in 100 mL of anhydrous methanol and sonicated for 5 min. Next, 100 mg of ZIF-8 was added to the solution and sonicated for 1 min to ensure complete dispersion in the EGCG methanol solution. The entire apparatus was placed in a rapid shaker at 150 rpm and shaken for 24 h. The suspension was then centrifuged at 9000 rpm for 5 min to achieve solid-liquid separation, yielding ZIF8 (ZIF8-E) adsorbed with epigallocatechin gallate. 1 mL of the supernatant was collected and diluted for EGCG residue content measurement.

[0076] Finally, ZIF8-E was freeze-dried for 48 h to produce ZIF8-E samples; 35 mg of freeze-dried ZIF8-E microspheres were added to 10 mM tris(hydroxymethyl)aminomethane hydrochloride (pH=8.5) buffer (10 mL), followed by 15 mg of dopamine hydrochloride, and gently stirred at 37 °C for 12 h. After centrifugation and rinsing three times with deionized water, polydopamine-coated ZIF8-E microspheres (ZP-E) were obtained by freeze-drying.

[0077] In this embodiment, the mass ratio of zinc nitrate to 2-methylimidazole is 1:1;

[0078] Epigallocatechin gallate: zeolite imidazole salt skeleton-8 nanoparticles at a ratio of 1 mL: 1 mg;

[0079] The mass ratio of zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate to dopamine hydrochloride is 7:3.

[0080] 4. Preparation of photocurable hydrogel scaffold (CP@ZP-E)

[0081] Col1MA-PBA was dissolved in pure water, and then a certain mass of PEGNB was dissolved in pure water. The solution was sonicated to completely dissolve the PEGNB. ZP-E was then added to the Col1MA-PBA solution to prepare a homogeneous mixed solution. The ZP-E and Col1MA mixed solution was then mixed with an equal volume of PEGNB and stirred until homogeneous. The solution was then irradiated with 405nm blue light for 20s to obtain the CP@ZP-E hydrogel scaffold.

[0082] Examples and comparative examples of the photothermal-regulated antibacterial, anti-inflammatory, and antioxidant multifunctional hydrogel composite scaffold of the present invention contain the following components: 10%-20% Col1MA-PBA, 10% o-nitrobenzyl alcohol-modified polyethylene glycol (PEGNB), and 200-600 μg / mL ZP-E.

[0083] The formulation of the hydrogel composite scaffold with multiple functions of photothermal regulation, antibacterial, anti-inflammatory, and antioxidant properties in this invention is shown in Table 1:

[0084] Table 1. Formulation Mass Fraction Table

[0085]

[0086] Examples 1-9:

[0087] Col1MA-PBA was dissolved in pure water to form solutions with a mass-volume fraction of 10%, 15%, and 20%. Then, a certain mass of PEGNB was dissolved in pure water and sonicated to completely dissolve it, forming a solution with a mass-volume fraction of 10%. Different concentrations of ZP-E were then added to the Col1MA-PBA solutions to prepare homogeneous mixed solutions. The mixed solutions of ZP-E and Col1MA with different concentrations were mixed with an equal volume of PEGNB and stirred until homogeneous. Finally, the solution was irradiated with 405nm blue light for 20s to obtain the CP@ZP-E hydrogel scaffold.

[0088] Comparative Example 1:

[0089] Col1MA-PBA was dissolved in pure water to form a 10% (w / v) solution. Then, a certain mass of PEGNB was dissolved in pure water and sonicated to completely dissolve it, forming a 10% (w / v) solution. The Col1MA-PBA solution was mixed with an equal volume of PEGNB and stirred until homogeneous. Subsequently, it was irradiated with 405nm blue light for 20s to finally obtain a 10% Col1MA-PBA, 10% PEGNB hydrogel scaffold (CP).

[0090] Comparative Example 2:

[0091] Col1MA-PBA was dissolved in pure water to form a 15% (w / v) solution. Then, a certain mass of PEGNB was dissolved in pure water and sonicated to completely dissolve it, forming a 10% (w / v) solution. The Col1MA-PBA solution was mixed with an equal volume of PEGNB and stirred until homogeneous. Then, it was irradiated with 405nm blue light for 20s to finally obtain a 15% Col1MA-PBA and 10% PEGNB hydrogel scaffold.

[0092] Comparative Example 3:

[0093] Col1MA-PBA was dissolved in pure water to form a 20% (w / v) solution. Then, a certain mass of PEGNB was dissolved in pure water and sonicated to completely dissolve it, forming a 10% (w / v) solution. The Col1MA-PBA solution was mixed with an equal volume of PEGNB and stirred until homogeneous. Subsequently, it was irradiated with 405nm blue light for 20s to finally obtain a 20% Col1MA-PBA and 10% PEGNB hydrogel scaffold.

[0094] Observe the gelation process of the prepared hydrogel. Figure 1 The results show that 10%, 15%, and 20% concentrations of Col1MA-PBA can all form hydrogels with PEGNB under 405nm light irradiation. The 10% Col1MA-PBA and PEGNB combination exhibits a slow gelation rate and forms a hydrogel with low strength, making it prone to deformation. The 15% Col1MA-PBA and PEGNB combination demonstrates a moderate gelation rate and a certain degree of cross-linking strength, resulting in a hydrogel that is less prone to deformation. The 20% Col1MA-PBA and PEGNB combination can also form a hydrogel rapidly, but its high precursor concentration results in poor flowability, making it difficult to conform to the wound morphology and form a hydrogel at the injury site.

[0095] Figure 2 The results show that the hydrogels formed by 10% Col1MA-PBA and PEGNB exhibit large network pores, sparse cross-linking points, and a loose structure. The hydrogels formed by 15% Col1MA-PBA and PEGNB have moderate network pore sizes, providing both sufficient mechanical strength and allowing for cell migration and nutrient diffusion. In contrast, the hydrogels formed by 20% Col1MA-PBA and PEGNB are very dense, with very small pores and an uneven structure, which is detrimental to cell migration and nutrient diffusion.

[0096] Figure 3 The image illustrates the release of EGCG under infrared light irradiation. It shows that under NIR light, PDA absorbs heat and increases temperature, which facilitates the disintegration of the hydrogel structure, thereby accelerating the release of EGCG. This achieves controlled photothermal antibacterial action and synergistic antibacterial effects from rapid EGCG release. The rapidly released EGCG also contributes to anti-inflammatory and antioxidant effects at the site of injury, thus mitigating the slow wound healing caused by inflammatory damage and oxidative stress, and accelerating the repair of the damaged area.

[0097] Observe the dynamic wet adhesion effect of the hydrogel scaffold. Figure 4 The results show that a dynamically moist oral environment can cause many medications to remain on wounds that are not moist for extended periods, preventing the active ingredients from continuously reaching the wound and resulting in limited therapeutic effects. Figure 4The results show that the CP@ZP-E scaffold has excellent wet adhesion properties, and can adhere to moist major organs such as the heart, liver, spleen, lungs and kidneys. This is conducive to the hydrogel staying at the site of oral mucosal injury for a longer time, and is more conducive to the hydrogel scaffold promoting cell proliferation and the functional components exerting their effects for a longer period of time, thus promoting the repair of the damaged site.

[0098] from Figure 5 The results show that photothermal antibacterial agents eliminate bacteria by directly destroying key structures such as the bacterial cell membrane, proteins, and DNA at high temperatures. Hydrogels, with their photothermal effect, can rapidly and efficiently kill bacteria, thus significantly shortening treatment time. Figure 5 The results showed that when the concentration of nanoparticles reached 400 ug / mL, the temperature could be rapidly raised to nearly 50°C within 10 minutes. This allowed for localized temperature control, achieving sterilization without damaging surrounding tissues. In contrast, the solution without nanoparticles showed almost no significant temperature increase after 10 minutes of near-infrared light irradiation. This indicates that the hydrogel loaded with ZP-E nanoparticles can promote wound healing by using photothermal sterilization at the site of oral mucosal injury.

[0099] To detect the anti-inflammatory effect of ZP-E nanoparticle-loaded hydrogels, the levels of TNF-α and IL-6 were measured in wound tissues treated with Control, CP, and CP@ZP-E. Figure 6 The results showed that both the control group and the hydrogel CP group without ZP-E nanoparticles still had severe inflammatory responses. However, the levels of TNF-α and IL-6 in the wound tissue of the hydrogel CP@ZP-E (Example 5) with ZP-E nanoparticles were significantly reduced. This indicates that ZP-E nanoparticles can reduce excessive inflammatory responses by regulating the expression of pro-inflammatory cytokines, thereby effectively promoting the repair of oral mucosal damage.

[0100] Implementation effect evaluation

[0101] 1. Water absorption performance analysis

[0102] Test method:

[0103] To evaluate the water absorption performance of the CP@ZP-E composite scaffold described in this invention, the equilibrium water absorption rate was used as an indicator to test the mass change of scaffolds from different embodiments and comparative examples after water absorption in deionized water. The hydrogels of each formulation were cured under ultraviolet light (405nm) to form scaffolds, and then vacuum dried to constant weight, with the dry weight recorded as W0. The scaffolds were then immersed in PBS at pH 7.4 for 24 hours, ensuring complete immersion. After removing the scaffolds, the surface moisture was gently blotted dry with filter paper, and the wet weight was measured as W. t The water absorption rate of the stent was calculated using the formula: W (%) = (W t -W0) / W0×100%.

[0104] Table 2 Water absorption rate of photocurable hydrogel scaffold

[0105]

[0106] Table 2 Results Explanation:

[0107] The content of Col1MA-PBA significantly affects the water absorption rate: Examples 1, 2, and 3, with lower Col1MA-PBA concentrations (10%), exhibited the highest water absorption rates (264.68%, 280.41%, and 268.12%, respectively). This is likely due to the lower Col1MA-PBA concentration resulting in lower cross-linking strength of the formed hydrogel, making it prone to swelling. However, the formed hydrogels are easily broken and have poor structural morphology, making them unsuitable for subsequent treatment. Examples 4, 5, and 6, with slightly lower water absorption rates (238.38%, 255.76%, and 248.15%, respectively), show slightly lower water absorption rates as the Col1MA-PBA concentration increases. This is likely due to increased cross-linking strength and a denser structure, leading to a decrease in water absorption. Furthermore, the water absorption rate initially increases and then decreases with increasing nanoparticle concentration, indicating that adding an appropriate concentration of nanoparticles helps enhance water absorption capacity. Examples 7, 8, and 9, which added a Col1MA-PBA concentration (20%), also demonstrated that increasing the Col1MA-PBA concentration reduced water absorption, with absorption rates of only 184.47%, 191.57%, and 188.03%, respectively. Therefore, Example 5, a hydrogel containing 15% Col1MA-PBA and PEGNB with 400 μg / mL ZP-E, represents the optimal formulation, balancing cross-linking strength, structural morphology, and water absorption / moisturizing properties. It is beneficial for promoting oral mucosal repair and is considered the best example.

[0108] Comparative examples: Among the comparative examples, Comparative Example 1 had the highest water absorption rate (244.42%), while as the Col1MA-PBA concentration increased to 20%, the water absorption rate decreased to 174.03%, which also proves that increasing the Col1MA-PBA concentration will gradually reduce the water absorption rate. Furthermore, the comparative examples do not contain functional materials that promote wound healing and are not suitable for practical applications.

[0109] 2. Rheological property characterization

[0110] Test method:

[0111] The storage modulus G' and loss modulus G” were dynamically tracked over time using a rotational rheometer.

[0112] Table 3 Rheological properties of photocurable hydrogel scaffolds

[0113]

[0114] Table 3 Results Explanation:

[0115] G' represents the material's elastic energy storage capacity, and G" represents the material's viscous loss capacity. A G' greater than G" indicates that the material has entered the elastic dominance region, meaning that the scaffold can be successfully cross-linked and formed. In Examples 1, 2, and 3, the G' value is higher than G", indicating the formation of a hydrogel. However, the difference between G' and G" is small, indicating that the hydrogel has low strength and is easily broken. Examples 4, 5, and 6 show significantly increased G' values ​​in their scaffolds, indicating a more stable hydrogel structure. The difference between G' and G” is greatest when the nanoparticle addition is 400 μg / mL, indicating a strong structure suitable for subsequent oral injury repair. Examples 7, 8, and 9 also show increased G' values ​​after gelation, but due to high initial viscosity, uneven cross-linking and uneven dispersion of nanoparticles may exist, hindering the controlled release and photothermal control of EGCG. Comparative Example 1, due to the low concentration of cross-linking monomers in the precursor solution, has a G' of only 750 Pa after curing, with a small difference between G' and G”, resulting in a loose scaffold structure and poor molding quality. Comparative Examples 2 and 3 show gradually improved rheological properties, but both lack therapeutic active substances, making them unsuitable for subsequent use. The results showed that adding 400 μg / mL nanoparticle material and a Col1MA-PBA concentration of 15% could maintain suitable structural properties, and the added nanoparticles could also be uniformly dispersed in the precursor solution to form a hydrogel with a uniform structure. The results also showed that while adding nanoparticles endowed the scaffold network with photothermal, antibacterial, anti-inflammatory and antioxidant functions, it did not affect the formation of the mechanical properties of the scaffold network.

[0116] 3. Characterization of antibacterial properties

[0117] Test method:

[0118] Staphylococcus aureus and Escherichia coli were inoculated into LB liquid medium and cultured at 37°C in a shaker until the logarithmic growth phase (OD600≈0.5). All scaffold samples were prepared to the same specifications (8 mm in diameter, 2 mm in thickness) and sterilized by irradiation of both sides with a UV lamp for 20 min each. Each group was placed in a container containing 1 mL of bacterial suspension (1×10⁻⁶). 6 Incubate in centrifuge tubes containing (CFU / mL) for 12 hours (all groups were irradiated with 808nm NIR laser: 1W / CB). 2 (5 min). After incubation, the bacterial suspension was serially diluted and spread onto LB plates. After 24 h of incubation, the colony forming units (CFU) were counted, and the inhibition rate was calculated.

[0119] C (%) = (N C -N S ) / N C ×100 formula (1)

[0120] Table 4 Antibacterial properties of photocurable hydrogel scaffolds

[0121]

[0122] Table 4 Results Explanation:

[0123] Examples containing ZP-E all exhibited significant broad-spectrum antibacterial properties under near-infrared irradiation, especially Example 5, which showed inhibition rates of 97.6% and 96.2% against Staphylococcus aureus and Escherichia coli, respectively. In contrast, comparative examples without ZP-E did not show photothermal antibacterial ability (e.g., Comparative Example 2), with inhibition rates of only 9.2% and 7.3% against Staphylococcus aureus and Escherichia coli, respectively, showing almost no antibacterial ability. This further verifies the photothermal driven antibacterial and EGCG release synergistic antibacterial mechanism of this material.

[0124] 4. Antioxidant capacity test

[0125] Test method:

[0126] (1) Prepare ABTS radical solution, incubate the prepared solution with each group of scaffolds, place the mixture at room temperature in the dark, and detect it using a spectrophotometer tuned to 734 nm wavelength. The solution without scaffolds is used as a control.

[0127] Antioxidant activity was assessed by calculating the scavenging rate of ABTS free radicals, using the following formula:

[0128] Clearance rate A (%) = (Control) 吸光度 -Sample 吸光度 ) / Control 吸光度 ×100 formula (2)

[0129] (2) Prepare DPPH radical solution, incubate the prepared solution with each group of scaffolds, place the mixture at room temperature in the dark for 30 min, and detect it using a spectrophotometer tuned to 517 nm wavelength. The solution without scaffolds is used as a control.

[0130] The antioxidant activity was calculated based on the DPPH free radical scavenging rate according to formula (2).

[0131] Table 5 Antioxidant properties of hydrogel scaffolds

[0132]

[0133] Table 5 Results Explanation:

[0134] ECCG is a type of catechin, a class of polyphenolic compounds abundant in tea, belonging to the flavanol family. The EGCG molecule contains multiple active phenolic hydroxyl groups, which endow it with strong antioxidant activity. Studies have shown that its ability to scavenge free radicals is far stronger than that of common antioxidants such as vitamin C and vitamin E. Excessive free radical oxidation occurs at sites of oral mucosal injury, damaging cells, proteins, and DNA. CP@ZP-E hydrogels with added EGCG exhibit strong free radical scavenging capabilities. In particular, Examples 5 and 6 show the highest antioxidant activity compared to other examples and comparative examples, which is beneficial for promoting the repair of oral mucosal damage. Based on these results, Example 5, with 400 μg / mL nanoparticles, is the optimal choice for treating infectious oral ulcers.

[0135] This invention constructs a photosensitive adhesive nanocomposite hydrogel dressing (CP@ZP-E). The hydrogel uses methacrylamide collagen-phenylboronic acid (Col1MA-PBA) as a matrix, utilizing the dynamic non-covalent bonding between the phenylboronic acid (PBA) groups and wound tissue proteins to provide excellent wet adhesion properties. Simultaneously, by introducing an o-nitrobenzyl alcohol-modified polyethylene glycol (PEGNB) photosensitive crosslinking network, in-situ rapid photocuring of the hydrogel is achieved using 405nm light, enabling precise matching of the wound contour and providing good mechanical support. The o-nitrobenzyl alcohol (NB) groups require 405nm ultraviolet-visible light for efficient and rapid photolytic crosslinking to achieve in-situ molding. However, polydopamine (PDA) has very strong absorption in this wavelength range (405nm). If simply mixed, PDA will prevent light from reaching the NB groups, leading to a sharp decrease in crosslinking efficiency, prolonged curing time, or even complete failure to cure, causing the hydrogel to lose its basic functions of rapid molding and precise wound matching. Therefore, we innovatively designed and synthesized ZP-E nanoparticles as a photothermal agent and drug carrier. These nanoparticles were carefully designed to have a significantly lower absorption at 405 nm than PDA, achieving spectral compatibility with the photocuring wavelength of PEGNB. Polydopamine-coated ZIF8-epigallocatechin gallate nanoparticles (ZIF8-PDA@EGCG NPs, ZP-E) exhibit a photothermal effect under light irradiation, directly exerting antibacterial activity while simultaneously triggering the accelerated release of the loaded active molecule epigallocatechin gallate (EGCG), thus achieving significantly enhanced antibacterial, anti-inflammatory, and antioxidant functions. The hydrogel prepared in situ via photocuring not only possesses wet adhesion but also utilizes photothermal and pH-responsive sustained-release functions to effectively inhibit infection and enhance the membrane's antibacterial properties. EGCG release synergistically inhibits inflammatory factors and promotes epithelial cell repair, while simultaneously solving the problems of difficult molding and insufficient wet adhesion inherent in traditional materials. The composite scaffold of this invention provides a novel solution for the treatment of oral ulcers and has promising prospects for clinical application.

[0136] In summary, the photocurable hydrogel scaffold prepared by this invention preferably contains 15% Col1MA-PBA and 10% PEGNB; it also preferably contains 200 μg / mL of ZP-E, and has the following advantages:

[0137] 1. Enhanced Synergistic Therapy: EGCG is known to have anti-inflammatory and antioxidant effects, while PDA has photothermal antibacterial effects. However, this invention has discovered that EGCG, released controllably under programmed photothermal stimulation, produces a remarkable synergistic effect with the low-temperature photothermal effect. Specifically: ① Enhanced antibacterial effect: EGCG further disrupts bacterial biofilms, complementing photothermal sterilization; ② Immune regulation: EGCG effectively inhibits excessive inflammatory responses that may be triggered by photothermal therapy (such as reducing the levels of pro-inflammatory factors like TNF-α and IL-6), polarizing macrophages from the pro-inflammatory M1 type to the pro-repair M2 type, creating a favorable immune microenvironment for tissue repair; ③ Synergistic antioxidant effect: Clears excess reactive oxygen species (ROS) that may be generated during the photothermal process, protecting surrounding healthy cells. This synergistic mechanism of "physical therapy (photothermal) + chemical therapy (drugs)" achieves simultaneous infection control, inflammation suppression, oxidative stress reduction, and tissue regeneration promotion, with repair effects far exceeding any single method or simple superposition.

[0138] 2. Programmable photothermal temperature control performance: By adjusting the intensity and time of 405nm light irradiation, precise control of local temperature can be achieved, realizing "heating on demand, precise sterilization, avoiding overheating damage to surrounding healthy tissues, and having higher safety and controllability."

[0139] 3. Enhanced material mechanical properties: Using Col1MA-PBA and PEGNB as matrix materials, it not only provides good biocompatibility and biodegradability, but also enables the precise construction of complex-shaped wound gels through in-situ photocuring, meeting the clinical needs of individualized wound repair.

[0140] 4. Capable of constructing a therapeutic microenvironment with sustained release function: The scaffold can slowly release EGCG in vivo, which, in addition to having a long-lasting antibacterial effect, can also regulate free radicals in the wound environment and reduce inflammation, promote angiogenesis and tissue regeneration, and provide long-term support for oral mucosal repair.

[0141] 5. Applicable to oral mucosal repair under complex pathological conditions such as infection and persistent inflammation: This invention is designed with a multifunctional composite scaffold for patients’ special wound environment (such as infection, inflammation, slow repair, etc.), which exhibits excellent antibacterial, anti-inflammatory and antioxidant properties, and is especially suitable for the treatment of chronic and difficult-to-heal oral mucosal defects.

[0142] 6. Simple manufacturing process with promotion and application value: The photocrosslinking technology of this invention is mature, the steps are controllable, the cost is moderate, and it has strong replicability and industrialization prospects, making it suitable for large-scale clinical promotion and application.

[0143] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0144] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A photocurable hydrogel scaffold, characterized in that, The photocurable hydrogel scaffold comprises collagen, o-nitrobenzyl glycol, and nanoparticles; The collagen includes phenylboronic acid-grafted methacrylamide collagen; The nanoparticles include polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate.

2. The photocurable hydrogel scaffold according to claim 1, characterized in that, The raw materials for preparing the phenylboronic acid-grafted methacrylamide collagen include collagen, 3-aminophenylboronic acid, and methacrylic anhydride; the collagen includes type I collagen.

3. The method for preparing the photocurable hydrogel scaffold according to any one of claims 1-2, characterized in that, Includes the following steps: Preparation of phenylboronic acid-grafted methacrylamide collagen: Collagen was dissolved, and 3-aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide were dissolved, mixed, reacted, dialyzed, and lyophilized to obtain phenylboronic acid-grafted collagen; phenylboronic acid-grafted collagen was dissolved, methacrylic anhydride was added, reacted, dialyzed, and lyophilized to obtain phenylboronic acid-grafted methacrylamide collagen; Preparation of o-nitrobenzyl alcohol-modified polyethylene glycol: polyethylene glycol is dissolved, o-nitrobenzyl alcohol is added, 1-hydroxybenzotriazole is added, the pH is adjusted, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride is added, the mixture is stirred, dialyzed, and freeze-dried to obtain o-nitrobenzyl alcohol-modified polyethylene glycol; Preparation of polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate: Zinc nitrate and 2-methylimidazole were dissolved and stirred to obtain a suspension. After standing, centrifugation and washing, zeolite imidazole salt framework-8 nanoparticles were obtained and freeze-dried. Epigallocatechin gallate was dissolved and sonicated. Zeolite imidazole salt framework-8 nanoparticles were added and sonicated, shaken, and centrifuged to obtain zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate. The nanoparticles obtained in the previous step were dissolved, dopamine hydrochloride was added, stirred, centrifuged and washed to obtain polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate. Preparation of photocurable hydrogel scaffold: Phenylated boric acid-grafted methacrylamide collagen and o-nitrobenzyl glycol were dissolved separately. Polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate were added to the phenylboronic acid-grafted methacrylamide collagen and mixed evenly. Then o-nitrobenzyl glycol was added and mixed evenly. The mixture was then irradiated with light to obtain the photocurable hydrogel scaffold.

4. The preparation method according to claim 3, characterized in that, The preparation of the phenylboronic acid-grafted methacrylamide collagen includes the following steps: Collagen was dissolved in water and stirred until completely dissolved. 3-Aminophenylboronic acid, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide were dissolved in a mixed solvent of water and DMSO and added to the collagen solution. The mixture was reacted at room temperature, dialyzed through a dialysis bag, and lyophilized to obtain phenylboronic acid-grafted collagen. Benzylboronic acid-grafted collagen was dissolved in water, methacrylic anhydride was added, and the mixture was reacted at room temperature, dialyzed through a dialysis bag, and lyophilized to obtain phenylboronic acid-grafted methacrylamide collagen.

5. The preparation method according to claim 3, characterized in that, The preparation of the o-nitrobenzyl glycol includes the following steps: Dissolve polyethylene glycol in water, add o-nitrobenzyl alcohol and then 1-hydroxybenzotriazole, adjust the pH to 4-5, then add 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, stir, dialyze through a dialysis bag, and freeze-dry to obtain o-nitrobenzyl alcohol-modified polyethylene glycol.

6. The preparation method according to claim 3, characterized in that, The preparation of the polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate includes the following steps: Zinc nitrate and 2-methylimidazole were dissolved separately in an organic solvent and stirred at room temperature to obtain a suspension. The suspension was allowed to stand, centrifuged, and washed to obtain zeolite imidazole salt framework-8 nanoparticles, which were then freeze-dried. Epigallocatechin gallate was dissolved in an organic solvent, sonicated, and then the zeolite imidazole salt framework-8 nanoparticles were added. The mixture was sonicated and shaken to obtain a mixture. The mixture was centrifuged to obtain zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate, which were then freeze-dried. The nanoparticles obtained in the previous step were added to a buffer solution, and then dopamine hydrochloride was added. After stirring, centrifuging, washing, and freeze-drying, polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate were obtained.

7. The preparation method according to any one of claims 3-6, characterized in that, In the preparation of phenylboronic acid grafted methacrylamide collagen, the mass ratio of collagen to 3-aminophenylboronic acid is (1~3):1; The phenylboronic acid-grafted collagen: the methacrylic anhydride is 1.0 g: (0.4~0.8) mL; In the preparation of o-nitrobenzyl alcohol-modified polyethylene glycol, the mass ratio of polyethylene glycol to o-nitrobenzyl alcohol is 6: (0.1~0.4). In the preparation of the polydopamine-coated zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate, the mass ratio of zinc nitrate to 2-methylimidazole is (1~2):(1~2). The epigallocatechin gallate: the zeolite imidazole salt skeleton-8 nanoparticles are (1~2) mL: (1~2) mg; According to the mass ratio, the zeolite imidazole salt framework-8 nanoparticles adsorbed with epigallocatechin gallate ester are 1: (0.3~0.5).

8. The preparation method according to claim 6, characterized in that, The organic solvent includes anhydrous methanol.

9. The preparation method according to claim 3, characterized in that, In the preparation step of the photocurable hydrogel scaffold, the light irradiation time is 10~30s.

10. The use of the photocurable hydrogel scaffold as described in any one of claims 1-2, or the photocurable hydrogel scaffold obtained by the preparation method described in any one of claims 3-9, in the preparation of products for the treatment of oral ulcers.

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