Multi- efficacy schiff base-light dual crosslinking drug-loaded hydrogel and preparation method and application thereof

Abstract

This invention discloses a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel. The hydrogel comprises Dendrobium officinale polysaccharide (ODop), gelatin-hydroxyphenylpropionic acid (Gtn-HPA), an amino-containing antibacterial drug, a photocrosslinking agent, and an oxidizing agent. Specifically, Dendrobium officinale polysaccharide (ODop) undergoes Schiff base crosslinking with the amino-containing antibacterial drug and gelatin-hydroxyphenylpropionic acid (Gtn-HPA). Gtn-HPA itself undergoes photocrosslinking to enhance mechanical strength. Under acidic pH conditions, the Schiff base crosslinking hydrolyzes, releasing the amino-containing antibacterial drug from the hydrogel. The hydrogel prepared by this invention exhibits excellent mechanical properties, controlled drug release capability, and superior bioactivity.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel, its preparation method, and its application. Background Technology

[0002] Wound healing comprises four stages: hemostasis, inflammation, proliferation, and remodeling. Chronic wounds often stagnate during the inflammatory phase, accompanied by significant tissue fluid exudation, pain, and wound infection. Hydrogels, due to their high water content, three-dimensional network structure, and tunable mechanical properties, are widely used in controlled drug release, wound dressings, and tissue engineering scaffolds. Hydrogels are materials composed of cross-linked hydrophilic materials, capable of maintaining wound moisture while ensuring wound breathability. They absorb large amounts of exudate, reduce adhesion, and thus prevent secondary damage to the wound, meeting the requirements of critical dressings and making them one of the most promising materials in wound care. Common hydrogel dressings can be categorized into natural polysaccharide hydrogels and synthetic polymer hydrogels. Natural polysaccharides exhibit good biocompatibility and typically do not produce harmful substances upon degradation. Common natural polysaccharide hydrogels include sodium alginate hydrogel, hyaluronic acid hydrogel, and chitosan hydrogel.

[0003] Dendrobium polysaccharide (Dop) is a natural active ingredient extracted from the orchid Dendrobium officinale, possessing anti-tumor, anti-inflammatory, antioxidant, and immunomodulatory effects (International Journal of Biological Macromolecules, 2024, 263: 130315). Dop can directly scavenge excess reactive oxygen species (ROS) in the wound microenvironment, reducing oxidative stress damage to fibroblasts and endothelial cells, and inhibiting apoptosis. Simultaneously, it can downregulate the expression of pro-inflammatory factors such as TNF-α and IL-6, promote macrophage polarization towards the M2 type, and accelerate the transition from the inflammatory phase to the proliferative phase. Chinese patent CN120983298A discloses a Dendrobium officinale polysaccharide hydrogel mask, its preparation method, and its application. The mask is composed of gelatin, sodium alginate, calcium chloride (a cross-linking agent), and Dendrobium officinale polysaccharide, exhibiting excellent antioxidant properties, skin repair properties, and skin moisturizing effects, while also possessing high safety. In addition, gelatin (Gtn) is a water-soluble biomolecule obtained by mild hydrolysis (acid, alkali or enzymatic hydrolysis) of collagen. It can promote fibroblast synthesis and collagen secretion, induce endothelial cell migration and lumen formation, accelerate wound angiogenesis and epithelialization, and shorten the wound healing cycle (Biomaterials, 2023, 299: 122161). Gelatin molecules retain a large number of carboxyl and amino groups, allowing for high flexibility in structural modification. Under weakly alkaline conditions, amino groups can undergo Schiff base crosslinking with aldehyde groups to form a crosslinked network. It can be chemically modified through esterification, grafting, and other methods. For example, a paper (Macromolecular bioscience, 2019, 19(6): 1900098) synthesized Gtn-MA by grafting methacryloyl onto gelatin. Gtn-MA can crosslink to form a hydrogel after being irradiated with blue light (400~450nm) under the action of photoinitiator ruthenium (Ru) and oxidant sodium persulfate (SPS).

[0004] However, despite the good biocompatibility of natural polymer hydrogels (chitosan, sodium alginate, gelatin, etc.), they generally suffer from drawbacks such as low mechanical strength, rapid swelling, and significant drug burst release. Hydrogels based on Dendrobium polysaccharides often employ physical cross-linking methods, resulting in poor mechanical properties and difficulty in controlling degradation rates. While chemically synthesized pH-responsive hydrogels (polyacrylic acid, polymethacrylic acid derivatives) exhibit sensitive responses, they suffer from the toxicity of degradation products and insufficient biocompatibility. Furthermore, single cross-linking methods are insufficient to meet the needs of complex wound environments. For example, hydrogels with a single cross-linking method and traditional drug-loaded hydrogels cannot achieve controlled drug release. The cross-linking rate and strength of hydrogels with a single Schiff base cross-linking method are pH-dependent, and they are prone to premature degradation in the in vivo microenvironment. They lack the ability to regulate external responses, making it difficult to precisely trigger drug release. Furthermore, existing pH-responsive hydrogels lack systematic research on active polysaccharides from traditional Chinese medicine. The photoinitiator of hydrogels with a single photocross-linking method may be cytotoxic. The cross-linked network is rigid and lacks self-repair, making it unable to cope with changes in wound tension and mechanical damage. Traditional drug-loaded hydrogels, such as silver ion / antibiotic hydrogels, have uncontrollable drug release, are prone to drug resistance, and may damage normal tissues with long-term use, and have limited ability to regulate inflammation. Summary of the Invention

[0005] The present invention aims to provide a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel with excellent mechanical properties, controlled drug release capability, and excellent bioactivity; another objective of the present invention is to provide a method for preparing the multifunctional Schiff base-photocrosslinked drug-loaded hydrogel; and yet another objective of the present invention is to provide an application of the multifunctional Schiff base-photocrosslinked drug-loaded hydrogel.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] This invention provides a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel, comprising Dendrobium officinale polysaccharide (ODop), gelatin-hydroxyphenylpropionic acid (Gtn-HPA), an amino-containing antibacterial drug, a photocrosslinking agent, and an oxidizing agent; wherein, Dendrobium officinale polysaccharide (ODop) undergoes Schiff base crosslinking with the amino-containing antibacterial drug and gelatin-hydroxyphenylpropionic acid (Gtn-HPA), and gelatin-hydroxyphenylpropionic acid (Gtn-HPA) itself undergoes photocrosslinking to enhance mechanical strength; under acidic pH conditions, the Schiff base crosslinking hydrolyzes, releasing the amino-containing antibacterial drug from the hydrogel.

[0008] Preferably, the amino-containing antibacterial drug is one of gentamicin sulfate (GEN) and neomycin sulfate; the concentration of the amino-containing antibacterial drug is 0~0.5%.

[0009] Preferably, the photocrosslinking agent is one of Ru(II) polypyridine complex and riboflavin.

[0010] Preferably, the oxidant is one of sodium persulfate (SPS) and ammonium persulfate.

[0011] Preferably, the concentration of the oxidized Dendrobium polysaccharide (ODop) is 4-8% (wt%).

[0012] Preferably, the mass ratio of the oxidized Dendrobium polysaccharide (ODop) to gelatin-hydroxyphenylpropionic acid (Gtn-HPA) is 1:2 to 2:1.

[0013] Preferably, the photocrosslinking is performed under blue light irradiation.

[0014] Preferably, the pH value is 5.5 to 7.4.

[0015] Preferably, the mass fraction of the photocrosslinking agent is 0.375% to 1.5%; and the mass fraction of the oxidant is 1.2% to 4.8%.

[0016] This invention also provides a method for preparing a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel, comprising the following steps:

[0017] (1) At room temperature, Dendrobium officinale polysaccharide (ODop), gelatin-hydroxyphenylpropionic acid (Gtn-HPA), amino-containing antibacterial drugs, oxidants, and photocrosslinking agents were prepared into stock solutions using PBS buffer and the pH was adjusted to weakly alkaline.

[0018] (2) After mixing gelatin-hydroxyphenylpropionic acid (Gtn-HPA) and amino-containing antibacterial drugs, add oxidant and photocrosslinking agent in sequence and mix evenly;

[0019] (3) Add Dendrobium oxidized polysaccharide (ODop) to the mixture prepared in step (2), mix evenly, and then place it under a blue light irradiator for 5~180s for photocrosslinking, and finally form a double crosslinked drug-loaded hydrogel.

[0020] Preferably, in step (1), the pH value is 7.0 to 8.0.

[0021] This invention also provides the application of a multifunctional Schiff base-photocrosslinked drug-loaded hydrogel in chronic wound medications.

[0022] The design principle of this invention is as follows: In the cross-linking system of this invention, the aldehyde groups of Dendrobium officinale polysaccharide form pH-responsive Schiff base dynamic cross-links with gelatin-hydroxyphenylpropionic acid and gentamicin sulfate, which release gentamicin in response to the microenvironment of the infected wound to achieve intelligent drug release and achieve antibacterial and bactericidal effects. The oxidative coupling of phenolic groups on gelatin-hydroxyphenylpropionic acid is initiated by ruthenium / sodium persulfate photoinitiation, improving the mechanical properties of the hydrogel and constructing a stable secondary network to enhance hydrogel stability. Dendrobium officinale polysaccharide scavenges excess reactive oxygen species and downregulates the expression of pro-inflammatory factors, thereby reducing wound inflammation. Simultaneously, gelatin-hydroxyphenylpropionic acid promotes wound healing by promoting fibroblast proliferation and collagen synthesis. The controlled release of drugs in the hydrogel avoids secondary damage caused by frequent dressing changes. The synergistic effect of Dendrobium officinale polysaccharide, gelatin-hydroxyphenylpropionic acid, and gentamicin sulfate solves the problems of inflammation and infection in chronic wounds, promoting wound healing. The bi-crosslinked ODop / Gtn-HPA@GEN hydrogel constructed in this invention achieves Schiff base crosslinking between the aldehyde groups of ODop and the amino groups on Gtn-HPA and GEN, and polymerization crosslinking of the phenolic hydroxyl groups on Gtn-HPA under blue light irradiation using a Ru / SPS photoinitiator. These three components form a stable bi-crosslinked network structure with both Schiff base and photocrosslinking. In the microenvironment of an infected wound, with a pH of 5.5–6.5, the Schiff base crosslinking structure formed between ODop, Gtn-HPA, and GEN hydrolyzes under these pH conditions. Gtn-HPA, due to its larger molecular chain and self-formed photocrosslinking, maintains the overall stability of the hydrogel. GEN, with its smaller molecular weight and primary participation in Schiff base crosslinking, is released from the hydrogel to the wound surface, exerting an antibacterial effect. Simultaneously, it synergizes with the antioxidant and anti-inflammatory effects of ODop and the cell adhesion and migration-promoting effects of Gtn-HPA to jointly promote wound repair.

[0023] The dual cross-linked drug-loaded hydrogel of this invention exhibits excellent pH responsiveness, antibacterial activity, and multiple healing-promoting functions in in vitro experiments, providing a novel dressing material with both intelligent responsiveness and multiple bioactivities for the treatment of infected wounds, and offering an efficient and safe new strategy for the treatment of chronic infected wounds.

[0024] Beneficial effects: Compared with the prior art, the present invention has significant advantages:

[0025] I. The hydrogel of this invention combines the pH-responsive properties of Schiff base crosslinking with the mechanical enhancement properties of photocrosslinking. Schiff base crosslinking enables the material to respond to changes in environmental pH, achieving precise and controllable drug release, thereby reducing the frequency of dressing changes. The introduction of photocrosslinking significantly improves the mechanical properties of the hydrogel, compensating for the shortcomings of a single network structure. In the dual crosslinking system of this invention, Schiff base crosslinking undertakes part of the network construction function, thus reducing the amount of photocrosslinking agent used, effectively avoiding cytotoxicity caused by excessive use of photocrosslinking agent, ensuring good biocompatibility of the material, and making it a biosafe smart responsive dressing material.

[0026] II. The raw material used in this invention, oxidized Dendrobium polysaccharide, retains the active groups of natural Dendrobium polysaccharide, which can scavenge excess reactive oxygen species (ROS) in the wound microenvironment, reduce oxidative stress damage to fibroblasts and endothelial cells, and downregulate the expression of pro-inflammatory factors, accelerating the transition from the inflammatory phase to the proliferative phase, fundamentally solving the core problem of chronic wound inflammation and its protracted healing. The Gtn-HPA component can promote fibroblast proliferation and collagen synthesis, thereby promoting wound healing; finally, the amino-containing antibacterial drug loaded in this invention is stably bound to the hydrogel network through Schiff base bonds. It is targeted and released in the acidic microenvironment caused by bacterial infection, inhibiting bacterial growth and preventing and treating wound infection.

[0027] Third, the preparation method of the hydrogel of the present invention is simple. After mixing the raw materials in sequence, a double cross-linked drug-loaded hydrogel can be formed under blue light irradiation. The mechanical properties and pH response properties of the hydrogel can be adjusted by adjusting the concentration of photocrosslinking agent, oxidant and drug loading agent. The hydrogel can be adjusted according to the actual application.

[0028] Fourth, the hydrogel of the present invention has multiple effects such as antibacterial, antioxidant, anti-inflammatory and cell migration promotion when applied to chronic wounds, and can promote the healing of infected chronic wounds. Attached Figure Description

[0029] Figure 1 The above are characterization diagrams of the ODop of the present invention; wherein (a) is the measurement diagram of ultraviolet absorption spectrum, (b) is the measurement diagram of Fourier transform infrared spectrum, and (c) is the measurement diagram of nuclear magnetic resonance hydrogen spectrum.

[0030] Figure 2 This is the hydrogen nuclear magnetic resonance spectrum of the Gtn-HPA synthesized in this invention;

[0031] Figure 3 This is the standard curve for determining the grafting rate of Gtn-HPA synthesized in this invention;

[0032] Figure 4 This invention relates to the ODop / Gtn-HPA@GEN double crosslinked drug-loaded hydrogel.

[0033] Figure 5 The images show the photocrosslinking of the Gtn-HPA synthesized in this invention under different concentrations of Ru and SPS conditions.

[0034] Figure 6 The swelling curves of the ODop and Gtn-HPA synthesized in this invention, formed in different proportions as single cross-linked unloaded drug hydrogels at different pH values;

[0035] Figure 7 Swelling curves of double cross-linked drug-loaded hydrogels with different drug loading concentrations at different pH values;

[0036] Figure 8 The rheological properties of the double crosslinked drug-loaded hydrogel of the present invention are shown in the figure.

[0037] Figure 9 The swelling performance curve of the double cross-linked drug-loaded hydrogel of the present invention is shown.

[0038] Figure 10 The diagram shows the drug release performance test of the double cross-linked drug-loaded hydrogel of the present invention; wherein, (a) is the high performance liquid chromatography (HPLC) standard curve of gentamicin; and (b) is the drug release curve.

[0039] Figure 11 The diagram shows the antibacterial effect test of the dual-crosslinked drug-loaded hydrogel of the present invention; wherein, (a) is a plate diagram of the antibacterial effect of the dual-crosslinked drug-loaded hydrogel on E. coli and the bacterial survival rate; (b) is a plate diagram of the antibacterial effect of the dual-crosslinked drug-loaded hydrogel on S. aureus and the bacterial survival rate.

[0040] Figure 12 The diagram shows the cell migration promotion performance of the dual cross-linked drug-loaded hydrogel of the present invention; (a) shows the survival rate of HaCaT cells after co-incubation with different concentrations of dual cross-linked drug-loaded hydrogel for 24 h; (b) shows the healing of HaCaT cell scratches after treatment with dual cross-linked drug-loaded hydrogel for 0, 12, and 24 h.

[0041] Figure 13 This invention demonstrates the inhibitory effect of the double cross-linked drug-loaded hydrogel on NO release in RAW 264.7 cells. Detailed Implementation

[0042] Unless otherwise specified, the experimental methods described in the embodiments are conventional methods; unless otherwise specified, the reagents and materials are commercially available.

[0043] Example 1

[0044] Preparation of ODop: Crude polysaccharide was extracted from dried Dendrobium officinale by water extraction and alcohol precipitation. Proteins and other impurities in the crude polysaccharide were removed by Sevag reagent. Pure polysaccharide Dop was obtained by freeze drying. Oxidized Dendrobium officinale polysaccharide ODop was obtained by oxidizing Dop with sodium periodate and then dialysis purification.

[0045] Characterization of ODop: The UV absorption curves, IR absorption curves, and 1H NMR spectra of Dop and ODop were measured, and the results are as follows: Figure 1 (a) Figure 1 (b) Figure 1 As shown in (c), this indicates that ODop was successfully synthesized, the aldehyde group was successfully introduced, and the structure of ODop is more homogeneous than that of Dop. The titration curve of ODop was determined by hydroxylamine hydrochloride-potentiometric titration. After differentiation using Origin software, the volume of NaOH at the titration endpoint was found to be 11.4 mL. The value of V was calculated using the following formula. NaOH c is the volume of NaOH at the titration endpoint, in mL. NaOH The concentration of the NaOH solution used is given in mol / L; m ODOP The mass of ODop is expressed in mg; Mw Dop The value represents the molecular weight of Dop monosaccharide, expressed in g / mol.

[0046]

[0047] The calculated oxidation degree of this ODop is 19.8%.

[0048] Synthesis of Gtn-HPA: The carboxyl group on p-hydroxybenzoic acid is covalently linked to the amino group on Gtn to form an amide ester bond through a carbodiimide / active ester-mediated coupling reaction. After purification by dialysis, Gtn-HPA is obtained by lyophilization.

[0049] Characterization of Gtn-HPA: The proton NMR spectra of Gtn and Gtn-HPA were determined as follows: Figure 2 As shown, the grafting of HPA onto Gtn was successful. The grafting rate of Gtn-HPA was determined by the ninhydrin colorimetric method. Gtn aqueous solutions with concentrations of 0, 2.0, 3.0, 4.0, 5.0, 6.0, and 7.0 mg / mL were prepared. 1 mL of each concentration solution was pipetted into a 4 mL EP tube, and 1 mL of 1% ninhydrin-ethanol solution was added. After mixing thoroughly, the tubes were placed in a water bath at 85 °C for 30 min. After cooling, the absorbance at 565 nm was measured. Using the concentration of the Gtn solution as the x-axis and the absorbance as the y-axis, the optimal model for the curve was calculated using Origin as the Expdec1 function. A standard curve was then fitted using this function, as shown below. Figure 3As shown, the nonlinear regression equation is y = 0.02312*exp(x / 1.80296) + 0.02592, R0 2 = 0.99998. Then, 8 mg / mL of Gtn-HPA was prepared for the ninhydrin reaction, and the grafting rate was determined to be 39.7%.

[0050] Preparation of the bicrosslinked drug-loaded hydrogel (ODop / Gtn-HPA@GEN + light): A stock solution containing 30% Ru, 48% SPS, 15% ODop (pH 7.4), and 5% GEN (pH 7.4) was prepared using PBS. 80 μL of Gtn-HPA stock solution was added sequentially to 30 μL of GEN, 15 μL of Ru, and 15 μL of SPS stock solution. After vortexing, 160 μL of ODop stock solution was added, forming a mixed solution with the following concentrations: 8% ODop, 4% Gtn-HPA, 0.5% GEN, 1.5% Ru, and 2.4% SPS. The solution was vortexed and allowed to stand for 30 min to allow complete Schiff base crosslinking. Then, the solution was irradiated under blue light for 1 min, forming a bicrosslinked drug-loaded hydrogel with a total volume of 300 μL. The gel formation image is shown below. Figure 4 As shown, the solution forms a hydrogel that does not flow when inverted, and the colloid is yellow and transparent.

[0051] Comparative Example 1

[0052] Preparation of photocrosslinked hydrogels (Gtn-HPA + light) with different concentrations of photocrosslinking agent ODop / Gtn-HPA@GEN

[0053] The preparation steps were the same as in Example 1, except that the Ru to SPS mass ratio was fixed at 1:3.2. Photocrosslinking hydrogel solutions with different photocrosslinking agent concentrations (0.375%, 0.75%, 1.5%, 3%, and 6% Ru mass fraction) were prepared using PBS with a final Gtn-HPA concentration of 1%. These solutions were then irradiated with blue light for 1 min to form hydrogels. The gelation results are as follows... Figure 5 As shown, when the Ru concentration is 0.375%, 0.75%, and 1.5%, and the SPS concentration is 1.2%, 2.4%, and 4.8%, the photocrosslinked hydrogel forms normally and becomes more robust with increasing concentration. However, when the Ru concentration is 3% and 6%, and the SPS concentration is 9.6% and 19.2%, the hydrogel undergoes excessive crosslinking and dehydration shrinkage. Therefore, preferably, the Ru mass fraction is 0.375%-1.5%, and the SPS mass fraction is 1.2%-4.8%.

[0054] Comparative Example 2

[0055] Preparation of a single cross-linked drug-free hydrogel (ODop / Gtn-HPA hydrogel)

[0056] Prepare ODop and Gtn-HPA stock solutions with a mass fraction of 15% and a pH of 7.4 using PBS. Take 80 μL of Gtn-HPA stock solution, add 60 μL of PBS, vortex to mix, then add 160 μL of ODop stock solution, vortex to mix, and allow to stand to form a gel, resulting in an ODop / Gtn-HPA hydrogel with a total volume of 300 μL and mass fractions of 8% ODop and 4% Gtn-HPA, respectively.

[0057] Comparative Example 3

[0058] The preparation steps were the same as those in Comparative Example 2, except that 120 μL of Gtn-HPA stock solution and 120 μL of ODop stock solution were used to prepare ODop / Gtn-HPA hydrogels with mass fractions of 6% ODop and 6% Gtn-HPA, respectively.

[0059] Comparative Example 4

[0060] The preparation steps were the same as those in Comparative Example 2, except that 160 μL of Gtn-HPA stock solution and 80 μL of ODop stock solution were used to prepare ODop / Gtn-HPA hydrogels with mass fractions of 4% ODop and 8% Gtn-HPA, respectively.

[0061] Comparative Example 5

[0062] Preparation of a single cross-linked drug-loaded hydrogel (ODop / Gtn-HPA@GEN)

[0063] Prepare ODop and Gtn-HPA stock solutions according to Comparative Example 2. Prepare GEN stock solution with a mass fraction of 5% and a pH of 7.4. Take 80 μL of Gtn-HPA stock solution, add 12 μL of GEN stock solution and 48 μL of PBS, vortex mix, then add 160 μL of ODop stock solution, vortex mix, and let stand to form a gel, forming a single cross-linked drug-loaded hydrogel with a total volume of 300 μL and mass fractions of 8% ODop, 4% Gtn-HPA, and 0.2% GEN.

[0064] Comparative Example 6

[0065] The preparation steps were the same as those in Comparative Example 5, except that 30 μL of GEN stock solution and 30 μL of PBS were used to prepare single cross-linked drug-loaded hydrogels with mass fractions of 8% ODop, 4% Gtn-HPA, and 0.5% GEN, respectively.

[0066] Comparative Example 7

[0067] The preparation steps were the same as those in Comparative Example 5, except that Schiff base crosslinked drug-loaded hydrogels with mass fractions of 8% ODop, 4% Gtn-HPA, and 1% GEN were prepared by adjusting 60 μL of GEN stock solution and 0 μL of PBS.

[0068] Comparative Example 8

[0069] Preparation of bicrosslinked unloaded hydrogels (ODop / Gtn-HPA + light)

[0070] The preparation steps were the same as in Example 1, except that the GEN stock solution was replaced with an equal volume of PBS, resulting in a total volume of 300 μL and mass fractions of 8% ODop, 4% Gtn-HPA, 0% GEN, 1.5% Ru, and 2.4% SPS, respectively.

[0071] Test Example 1: Swelling Properties Test of Single-Crosslinked Unloaded Hydrogel

[0072] Take 2 mL sample vials, weigh the vials (m0), and prepare 300 μL of each of the following single-crosslinked hydrogels according to comparative examples 2, 3, and 4, with a total mass fraction of 12% for both ODop and Gtn-HPA and ODop:Gtn-HPA mass ratios of 2:1, 1:1, and 1:2, respectively, into the sample vials. Weigh the vials (m0). 初 Six parallel samples of each type were prepared and weighed. 1.2 mL of PBS buffer (pH 7.4 or pH 5.0) was added to each sample vial. At specific time intervals of 1 h, 2 h, 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h, the solution was aspirated and surface moisture removed. The sample vials were then weighed. t Then, PBS buffer was added again to allow it to swell until the experiment was completed. The swelling rate was calculated using the following formula:

[0073]

[0074] The swelling results are as follows Figure 6As shown, when the mass ratio of ODop to Gtn-HPA is 1:1 and 1:2, the ODop content is relatively low (4% and 6%), the total amount of aldehyde groups is low, the cross-linking density is low, and the network structure is loose. Therefore, the swelling rate is high at pH 7.4, reaching a maximum swelling rate of 156.0% and 154.6% at 24 h, and then it begins to degrade rapidly. At pH 5.5, due to the low cross-linking density, the electrostatic expansion caused by protonation is greater than the hydrolytic destruction of cross-linking, and no obvious pH-responsive degradation is observed. However, when the ODop content increases to 8%, the increase in aldehyde groups forms a dense cross-linked network, exhibiting better structural stability, with a slower swelling and degradation rate, avoiding rapid collapse in the physiological environment, and providing a structural basis for the stable and sustained release of drugs after loading. At the same time, due to the increase in Schiff base cross-linking, the pH responsiveness of the hydrogel increases, and the swelling rate at different pH values ​​reaches a maximum difference of 34.4% at 36 h. The rapid degradation of the hydrogel at pH 5.5 provides a basis for the rapid release of loaded drugs. The optimal ratio is 8% ODop mass fraction, 4% Gtn-HPA mass fraction, and a mass ratio of 2:1.

[0075] Test Example 2: Swelling Performance Test of Single-Crosslinked Drug-Loaded Hydrogels

[0076] According to Comparative Examples 2, 5, 6, and 7, 300 μL of each monocrosslinked hydrogel with GEN mass fractions of 0%, 0.2%, 0.5%, and 1% were prepared, and their swelling properties were determined according to Test Example 1.

[0077] Swelling curve as shown Figure 7 As shown, when the mass fraction of GEN is 0.2% and 0.5%, its swelling curve at pH 7.4 is similar to that of the untreated hydrogel, with little change. However, under weakly acidic conditions (pH 5.5), the degradation of the untreated hydrogel with single crosslinking is slower, while the degradation rate of the untreated hydrogel with single crosslinking increases with increasing GEN concentration. When the GEN concentration is 1%, due to the excessively high GEN concentration, it competes with Gtn-HPA for aldehyde sites on ODop, leading to instability of the crosslinking network and rapid hydrolysis of the hydrogel at pH 7.4. This indicates that the addition of an appropriate amount of GEN can increase the pH-responsive release of the hydrogel; preferably, the mass fraction of GEN is 0-0.5%.

[0078] Test Example 3: Rheological property testing of the double cross-linked drug-loaded hydrogel of the present invention.

[0079] The mechanical properties of 250 μL of the bicrosslinked drug-loaded hydrogel prepared in Example 1 and 250 μL of the Schiff base monocrosslinked drug-loaded hydrogel prepared in Comparative Example 6 were measured and compared using a TA rheometer. The conditions were 25 °C, 1 Hz, and strain range of 0.1% to 2000%.

[0080] like Figure 8 As shown, the storage modulus of the double crosslinked drug-loaded hydrogel prepared by this invention is 936.78244 Pa, which is much greater than the storage modulus of 429.32057 Pa of the single crosslinked drug-loaded hydrogel, proving that photocrosslinking enhances the mechanical properties of the hydrogel.

[0081] Test Example 4: Swelling Performance Test of the Double Crosslinked Drug-Loaded Hydrogel of the Present Invention

[0082] The swelling properties of the 300 μL cross-linked drug-loaded hydrogel prepared according to Example 1 and the 300 μL cross-linked unloaded hydrogel prepared according to Comparative Example 8 were tested according to Test Example 1.

[0083] Swelling curve as shown Figure 9 As shown, in PBS buffer at pH 7.4, the unloaded bicrosslinked hydrogel (ODop / Gtn-HPA + light) reached swelling equilibrium at 36 h with a swelling rate of 159%, and began to rapidly degrade to a swelling rate of 125% between 48 and 72 h. In contrast, the drug-loaded bicrosslinked hydrogel (ODop / Gtn-HPA@GEN + light) of this invention rapidly reached swelling equilibrium at 24 h with a swelling rate of 152%, and began to slowly degrade to a swelling rate of 143% between 48 and 72 h. This indicates that the stability of the bicrosslinked hydrogel increases after drug loading. Under weakly acidic conditions (pH 5.5), the swelling properties of the dual-crosslinked drug-loaded and unloaded hydrogels reflect their pH response. The unloaded hydrogel reached swelling equilibrium after 12 hours, with a swelling rate of 137%, and then began to degrade rapidly. In contrast, the drug-loaded hydrogel began to degrade slowly after 8 hours. At 72 hours, the swelling rates of the drug-loaded and unloaded hydrogels were 92% and 76%, respectively. The degradation rate of the drug-loaded hydrogel was significantly lower than that of the unloaded hydrogel. This characteristic allows the dual-crosslinked drug-loaded hydrogel to release the drug for a longer period under weakly acidic conditions, reducing the frequency of replacement. These data demonstrate the excellent swelling and pH response properties of the dual-crosslinked hydrogel of this invention.

[0084] Test Example 5: Drug Release Performance Test of the Double Crosslinked Drug-Loaded Hydrogel of the Present Invention

[0085] (1) Preparation of o-phthalaldehyde (OPA) derivatization reagent: First, weigh 100 mg of OPA, dissolve it in 100 μL of methanol, add 9.5 mL of borax buffer (pH 9.5), mix and dissolve, then adjust the pH to 10.4 with 8 mol / L NaOH solution, then add 300 μL of mercaptopropionic acid, mix well and adjust the pH to 10.4 with 8 mol / L NaOH solution to obtain the OPA derivatization reagent.

[0086] (2) Construction of GEN standard curve: GEN solutions with concentrations of 500, 250, 125, 62.5, 31.25, 15.625, and 7.8125 μg / mL were prepared. 100 μL of each GEN solution was added to 375 μL of borax buffer (pH 9.5), mixed thoroughly, and then 25 μL of OPA derivatization reagent was added. The mixture was reacted at 60 °C for 15 min, cooled to room temperature, filtered through a microporous membrane, and analyzed by HPLC. The HPLC conditions were as follows: mobile phase: water (A) – acetonitrile (B); flow rate: 1 mL / min; column temperature: 25 °C; elution gradient: 0–15 min, 70% A; 15–15.1 min, 10% A; 15.1–20 min, 10% A; 20–20.1 min, 5% A; 20.1–25 min, 90% A; measurement wavelength: 334 nm. Plotting GEN concentration-time on the x-axis and chromatographic peak area on the y-axis, a standard curve was obtained by linear fitting using Origin: y = 7097.78669x - 19220.8908, R0. 2 = 0.99968, the curve is as follows Figure 10 As shown in (a) of the diagram.

[0087] (3) Determination of drug release curve: Take 5 mL centrifuge tubes and prepare a 200 μL double cross-linked drug-loaded hydrogel according to the method in Example 1. Prepare 6 samples in parallel and divide them into two groups. Add 2 mL of PBS buffer at pH 7.4 to one group and 2 mL of PBS buffer at pH 5.0 to the other group. Incubate at 37 °C on a shaker. At specific time intervals of 1 h, 2 h, 4 h, 8 h, 12 h, 24 h, 36 h, 48 h, and 72 h, take 400 μL of sample solution from the centrifuge tube and add 400 μL of PBS buffer at the corresponding pH to the centrifuge tube. Take 100 μL of sample solution, process it in the same way as the standard solution and measure it. Substitute the peak area into the curve to calculate the drug concentration.

[0088] Release curves at different pH values ​​are as follows: Figure 10 As shown in (b) above, at pH 7.4, the drug reaches its maximum release rate of 25.01% after 12 hours; while at pH 5.0, the sustained release of the drug lasts for 72 hours, reaching a maximum of 54.99%. The release rate varies significantly under different pH conditions from t=8 to 72 hours, demonstrating the pH responsiveness of the drug-loaded hydrogel of this invention, which enables continuous release of antibacterial drugs in the bacterial microenvironment to treat wound infections.

[0089] Test Example 6: Antioxidant effect of the double cross-linked drug-loaded hydrogel of the present invention

[0090] The antioxidant properties of the double crosslinked drug-loaded hydrogel prepared in Example 1 were evaluated using ABTS free radical scavenging and hydroxyl free radical scavenging experiments.

[0091] (1) Determination of hydroxyl radical scavenging rate

[0092] 200 μL of the dual-crosslinked drug-loaded hydrogel prepared in Example 1 was placed in a centrifuge tube. 1 mL of deionized water, 0.5 mL of 2.5 mmol / L FeSO4 solution, and 0.5 mL of 2.5 mmol / L salicylic acid-ethanol solution were added. After rapid mixing, 0.5 mL of 2.5 mmol / L H2O2 solution was added. The mixture was reacted at 37 °C in the dark for 30 min. After centrifugation, the supernatant was collected, and the absorbance (A1) at 510 nm was measured. The negative control group consisted of 200 μL of the dual-crosslinked drug-loaded hydrogel with 2 mL of water and 0.5 mL of ethanol, and the absorbance was recorded as A2. The blank control group consisted of 1 mL of aqueous solution with 0.5 mL of FeSO4 solution, 0.5 mL of salicylic acid-ethanol solution, and 1 mL of H2O2 solution, and the absorbance was recorded as A3.

[0093] Each experiment was conducted in triplicate, and the average value was taken. The OH scavenging rate was calculated using the following formula.

[0094]

[0095] The measured values ​​A1 were 0.066, 0.064, and 0.065, A2 was 0 for all values, and A3 was 0.749, 0.751, and 0.767. Calculations using the formula show that the scavenging rate of the bicrosslinked drug-loaded hydrogel of this invention against hydroxyl radicals is 91.4% ± 0.13%. This indicates that the bicrosslinked drug-loaded hydrogel of this invention has a strong scavenging ability against hydroxyl radicals.

[0096] (2) Determination of ABTS free radical scavenging rate

[0097] 5 mL of a 2.45 mmol / L K₂S₂O₈ solution was mixed with a 7.0 mmol / L ABTS-methanol solution and reacted at room temperature in the dark. After 12 h, the mixture was diluted 15–20 times with methanol, and the absorbance at 734 nm was measured to be 0.70 ± 0.02, yielding the ABTS stock solution.

[0098] 200 μL of the double-crosslinked drug-loaded hydrogel prepared in Example 1 was placed in an EP tube, and 0.5 mL of deionized water and 1 mL of ABTS stock solution were added and mixed well. The mixture was then incubated at 37 °C in the dark for 30 min. After centrifugation, the supernatant was collected, and the absorbance (A1) at 734 nm was measured using a UV spectrophotometer. The negative control group consisted of 200 μL of double-crosslinked drug-loaded hydrogel with 0.5 mL of deionized water and 1 mL of methanol, and the absorbance was recorded as A2. The blank control group consisted of 0.5 mL of aqueous solution with 1 mL of ABTS solution, and the absorbance was recorded as A3.

[0099] Repeat the experiment three times, take the average value, and calculate the clearance rate of ABTS according to the following formula.

[0100]

[0101] The measured values ​​A1 were 0.103, 0.098, and 0.113, A2 was 0 for all values, and A3 was 0.360, 0.363, and 0.357. Calculations using the formula show that the scavenging rate of the bicrosslinked drug-loaded hydrogel of this invention against ABTS free radicals is 70.9% ± 2.12%. This indicates that the bicrosslinked drug-loaded hydrogel of this invention has a strong scavenging ability against ABTS free radicals.

[0102] In summary, the dual cross-linked drug-loaded hydrogel of the present invention possesses excellent antioxidant capacity.

[0103] Test Example 7: Antibacterial effect test of the double cross-linked drug-loaded hydrogel of the present invention

[0104] 200 μL of a double-crosslinked drug-loaded hydrogel was prepared according to Example 1, and 200 μL of a double-crosslinked unloaded hydrogel was prepared according to Comparative Example 8. Staphylococcus aureus was used to represent Gram-positive bacteria, and Escherichia coli was used to represent Gram-negative bacteria, respectively, for the experiments. The bacterial suspensions were diluted to 2 × 10⁻⁶. 6 CFU / mL, place the prepared drug-loaded / unloaded hydrogels into shake tubes, add 1 mL of bacterial suspension for incubation, and add 200 μL of PBS buffer to the blank control group. Incubate at 37℃ on a shaker until the absorbance of the control bacterial solution at 600 nm is 0.2~0.4, then dilute the bacterial suspension 10. 4 The bacterial culture was diluted 10-fold each time, for a total of 4 dilutions. 100 μL of the diluted bacterial culture was spread onto agar broth solid medium, labeled, and incubated at 37°C for 24 h. Photos were taken and the number of strains was counted.

[0105] The results of photographing the antibacterial effect of hydrogel on E. coli and the calculated bacterial survival rate are as follows: Figure 11As shown in (a) above, the results of photographing the antibacterial effect of S. aureus on the plate and the calculated bacterial survival rate are as follows: Figure 11 As shown in (b) of the figure. Compared with the blank control group, under the action of the unloaded double cross-linked hydrogel ODop / Gtn-HPA, the survival rate of E. coli was 27.76%, and the survival rate of S. aureus was only 6.18%. No bacteria survived in the drug-loaded hydrogel group ODop / Gtn-HPA@GEN. This result indicates that the double cross-linked drug-loaded hydrogel of the present invention has strong antibacterial and bactericidal effects against both Gram-positive and Gram-positive bacteria, and its effect on Gram-positive bacteria is stronger than that on Gram-positive bacteria.

[0106] Test Example 8: Cell Migration Promotion Performance Test of the Double Crosslinked Drug-Loaded Hydrogel of the Present Invention

[0107] (1) Biocompatibility test of hydrogels

[0108] The MTT assay was used to detect the in vitro cytotoxicity of the ODop / Gtn-HPA@GEN dual-crosslinked drug-loaded hydrogel prepared in Example 1 of this invention. Human immortalized epidermal cells (HaCaT, ATCC, USA) were seeded at a density of 3000 cells per well in 96-well plates, containing 100 μL of culture medium, and incubated at 37 °C with 5% CO2 for 24 h. The dual-crosslinked drug-loaded hydrogel mixture prepared in Example 1 was diluted 100-fold, and then serially diluted to obtain composite hydrogel solutions with ODop concentrations of 0, 50, 100, 200, 400, and 800 μg / mL. 100 μL of this solution was added to each well in a 96-well plate and cultured for another 24 h, with five replicates for each concentration. After 24 h, 20 μL of MTT reagent (Beyotime Biotechnology Co., Ltd.) (5 mg / mL) was added and incubation continued for 4 h. After removing the culture medium, 200 μL of dimethyl sulfoxide (DMSO) was added to dissolve the formed formazan crystals. The absorbance of each well at 490 nm was measured using a microplate reader (Sunrise™, Tecan, Austria). Cell viability was calculated using Origin software to assess the biocompatibility of the samples.

[0109]

[0110] Figure 12 (a) shows the survival rate of HaCaT cells after co-incubation for 24 h with different concentrations of double cross-linked drug-loaded hydrogels. After incubation for 24 h with hydrogel solutions of final concentrations from 0 to 400 μg / mL, the cell survival rate remained above 85%, indicating that the hydrogels have low cytotoxicity. Within the concentration range of drugs and cross-linking agents used in this invention, the hydrogels exhibit good biocompatibility.

[0111] (2) Cell scratch test

[0112] HACAT cells were seeded at a density of 10,000 cells per well in six-well plates, each containing 1 mL of culture medium, and incubated at 37 °C with 5% CO2 for 24 h. A double-crosslinked drug-loaded hydrogel mixture was prepared according to Example 1, diluted 100-fold, and crosslinked by irradiation with blue light for 1 minute to obtain a double-crosslinked drug-loaded hydrogel solution with an ODop concentration of 800 μg / mL (ODop / Gtn-HPA@GEN + light). 1 mL of this solution was added to six-well plates for further culture, while 1 mL of culture medium was added to the control group. Cell migration was calculated using ImageJ software at 0, 12, and 24 h after drug administration.

[0113] like Figure 12 As shown in (b), the cell migration rate under the treatment of the double-crosslinked drug-loaded hydrogel at 12 h was not significantly different from that of the blank control group, while the cell migration rate under the treatment of the double-crosslinked drug-loaded hydrogel at 24 h was significantly higher than that of the blank control group. The scratch healing rate was calculated as shown in Table 1. At 12 h, the migration rates of the double-crosslinked drug-loaded hydrogel group and the blank control group were 45.21% and 44.76%, respectively, with no significant difference. At 24 h, the healing rate of the double-crosslinked drug-loaded hydrogel was 95.32%, significantly higher than the 66.06% of the blank control group. This experiment demonstrates that the double-crosslinked drug-loaded hydrogel of the present invention promotes cell migration.

[0114] Table 1 Scratch healing rate

[0115] Healing rate (%) 12h 24h Blank control group (control) 44.76 66.06 ODop / Gtn-HPA@GEN + light 45.21 95.32

[0116] Test Example 9: Anti-inflammatory effect of the bi-crosslinked drug-loaded hydrogel of the present invention

[0117] The anti-inflammatory efficacy of the double-crosslinked drug-loaded hydrogel of the present invention was investigated by detecting the NO release level of mouse mononuclear macrophage leukemia cells (RAW 264.7). RAW264.7 cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) under the following incubation conditions: 37 °C, 5% CO2. When the cell growth density reached 80%~90%, RAW 264.7 cells were seeded at a density of 10,000 per well in 96-well plates, 50 μL per well, and incubated for 24 h. The dual-crosslinked drug-loaded hydrogel mixture prepared in Example 1 was diluted 100-fold and then serially diluted to obtain composite hydrogel solutions with ODop concentrations of 0, 50, 100, 200, 400, and 800 μg / mL. 100 μL of the composite hydrogel solution was added to each well of a 96-well plate and incubated for 2 h. Then, 50 μL of 40 μg / mL LPS solution was added and incubated for 24 h. After lysing cells with 20 μL of lysis buffer, the cells were centrifuged. 50 μL of supernatant from each concentration was added to each well of the 96-well plate, and the reaction was performed using reagents from a NO detection kit. The absorbance of each well at 560 nm was measured using a microplate reader. Based on the NaNO2 standard curve, the amount of NO released from RAW 264.7 cells at different concentrations was calculated, and the NO clearance rate was compared with the untreated group to evaluate the anti-inflammatory efficacy of the dual-crosslinked drug-loaded hydrogel.

[0118] The results are as follows Figure 13 As shown, the anti-inflammatory effect of the drug increases with increasing concentration. When the final concentration of ODop in the solution is 400 μg / mL, the NO scavenging rate reaches 82.49%. This indicates that the dual-crosslinked drug-loaded hydrogel has a good and concentration-dependent anti-inflammatory effect.

Claims

1. A multifunctional Schiff base-photocrosslinked drug-loaded hydrogel, characterized in that, The hydrogel comprises Dendrobium oxidized polysaccharide (ODop), gelatin-hydroxyphenylpropionic acid (Gtn-HPA), an amino-containing antibacterial drug, a photocrosslinking agent, and an oxidizing agent; wherein, Dendrobium oxidized polysaccharide (ODop) undergoes Schiff base crosslinking with the amino-containing antibacterial drug and gelatin-hydroxyphenylpropionic acid (Gtn-HPA), and gelatin-hydroxyphenylpropionic acid (Gtn-HPA) itself undergoes photocrosslinking to enhance mechanical strength. Under acidic pH conditions, the Schiff base crosslinking hydrolyzes, and the amino-containing antibacterial drug is released from the hydrogel.

2. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The amino-containing antibacterial drug is one of gentamicin sulfate (GEN) and neomycin sulfate; the concentration of the amino-containing antibacterial drug is 0~0.5%.

3. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The photocrosslinking agent is one of Ru(II) polypyridine complex and riboflavin.

4. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The oxidant is one of sodium persulfate (SPS) or ammonium persulfate.

5. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The concentration of the oxidized Dendrobium polysaccharide (ODop) is 4-8% (wt%).

6. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The mass ratio of the oxidized Dendrobium polysaccharide (ODop) to gelatin-hydroxyphenylpropionic acid (Gtn-HPA) is 4:8 to 8:

4.

7. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The pH value is 5.5~7.

4.

8. The multifunctional Schiff base-photocrosslinked drug-loaded hydrogel according to claim 1, characterized in that, The mass fraction of the photocrosslinking agent is 0.375%~1.5%, and the mass fraction of the oxidant is 1.2%~4.8%.

9. The method for preparing the multifunctional Schiff base-photocrosslinked drug-loaded hydrogel as described in claim 1, characterized in that, Includes the following steps: (1) At room temperature, Dendrobium officinale polysaccharide (ODop), gelatin-hydroxyphenylpropionic acid (Gtn-HPA), amino-containing antibacterial drugs, oxidants, and photocrosslinking agents were prepared into stock solutions using PBS buffer and the pH was adjusted to weakly alkaline. (2) After mixing gelatin-hydroxyphenylpropionic acid (Gtn-HPA) and amino-containing antibacterial drugs, add oxidant and photocrosslinking agent in sequence and mix evenly; (3) Add Dendrobium oxidized polysaccharide (ODop) to the mixture prepared in step (2), mix evenly, and then place it under a blue light irradiator for 5~180s for photocrosslinking, and finally form a double crosslinked drug-loaded hydrogel.

10. The application of the multifunctional Schiff base-photocrosslinked drug-loaded hydrogel as described in claim 1 in chronic wound medication.

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