A dual-enzyme response type double-network interpenetrating hydrogel film, a preparation method and application thereof

Abstract

The application discloses a kind of double enzyme response type double network interpenetrating hydrogel film and its preparation method and application, preparation method is first respectively synthesized terminal aldehyde group polyethylene glycol derivative and photosensitive carboxymethyl starch derivative, then two are mixed with chitosan acid solution according to specific proportion, after preliminary setting by Schiff base reaction, by ultraviolet light in situ construction second heavy crosslinking network, finally by casting, dry dehydration forms double enzyme response type double network interpenetrating hydrogel film.The application constructs double network interpenetrating structure, significantly improves the mechanical strength and toughness of hydrogel and its dry film, effectively overcome the defect that traditional polysaccharide material is big brittle, and the prepared hydrogel film has double response degradation characteristics of lysozyme and alpha-amylase, and the film can realize the bioresponsive controlled release of drug, and also can use the characteristics that chitosan can reversibly open oral mucosa epithelial cell tight junction, effectively promote the mucosal permeation absorption of encapsulated drug.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, specifically to a dual-enzyme responsive dual-network interpenetrating hydrogel film, its preparation method, and its application. Background Technology

[0002] Oral mucosal drug delivery has become a hot topic in biomedical research due to its ability to bypass the first-pass effect of the liver and gastrointestinal enzymatic degradation, its rapid onset of action, and its suitability for patients with dysphagia. Oral films, as a core dosage form, have great application prospects due to their convenience and good mucosal adhesion. However, the performance defects of existing matrix materials limit their development. Natural polysaccharide materials can be used to prepare oral films due to their excellent biocompatibility and biodegradability, but they have significant mechanical shortcomings: they are brittle and easily broken in the dry state, and their strength drops sharply and swelling becomes uncontrolled in the wet state, which can easily lead to film breakage and detachment before drug release.

[0003] Existing methods for improving the mechanical properties of polysaccharide-based hydrogels all have shortcomings. Chemical crosslinking agents, such as glutaraldehyde, are cytotoxic, while physical blending or single-network crosslinking offers limited strength gains. Although dual-network gels can achieve high strength and toughness, existing products are mostly based on non-degradable synthetic polymers, making them unsuitable for oral drug delivery. Furthermore, existing oral films have limited drug release mechanisms; fast-dissolving films cannot achieve long-term sustained release, and pH-responsive drug delivery systems are unstable due to pH fluctuations in saliva. The oral environment is rich in lysozyme and α-amylase, providing specific targets for intelligent drug delivery, but current technologies rarely produce dual-enzyme responsive materials that can simultaneously respond to both enzymes, making precise and controllable drug release difficult.

[0004] In addition, the tight junctions of oral mucosal epithelial cells form a drug permeation barrier, resulting in low bioavailability of macromolecular drugs. Existing chemical permeation enhancers are prone to irritating and damaging the mucosa. Chitosan can reversibly open the tight junctions of cells to achieve non-irritating permeation enhancement. However, in the existing preparation process, high-density cross-linking easily consumes its active amino groups, leading to a significant reduction in permeation enhancement function. It is difficult to balance high-strength cross-linking with the permeation enhancement activity of chitosan.

[0005] In summary, existing polysaccharide-based hydrogel films for oral mucosal drug delivery generally suffer from problems such as the contradiction between mechanical properties and biodegradability, lack of oral-specific intelligent response in drug release, and difficulty in balancing high-strength cross-linking and permeation-enhancing functions. There is an urgent need to develop a hydrogel film that combines high strength, dual-enzyme responsive drug release, efficient mucosal permeation enhancement, and good biocompatibility. Summary of the Invention

[0006] The purpose of this invention is to solve the above-mentioned technical problems. The purpose of this invention is to provide a dual-enzyme responsive dual-network interpenetrating hydrogel film, its preparation method and application. The film has the characteristics of high strength, high toughness, dual-enzyme responsive intelligent drug release, and efficient mucosal permeation enhancement. It also has good biocompatibility and can be effectively used as an oral mucosal drug delivery carrier to achieve controlled release and efficient permeation and absorption of drugs.

[0007] The above-mentioned objective of the present invention is achieved through the following technical solution:

[0008] The first aspect of this invention provides a method for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film, comprising the following steps:

[0009] (1) Polyethylene glycol (PEG) was esterified with p-aldehyde benzoic acid (CBA), and after purification, a terminal aldehyde-terminated polyethylene glycol derivative (PEG-CBA) was obtained.

[0010] (2) In the presence of an activator, carboxymethyl starch (CMS) reacts with a diamine or dihydrazide bridging agent to obtain a carboxymethyl starch derivative intermediate. After activation with lipoic acid (LA), the intermediate reacts with the carboxymethyl starch derivative, and the resulting product is purified to obtain a photosensitive carboxymethyl starch derivative (CMS-). g -LA);

[0011] (3) Dissolve chitosan (CS) in an acidic aqueous solution and adjust the pH of the system to 5.0-6.0 to obtain a chitosan solution; add the terminal aldehyde-modified polyethylene glycol derivative prepared in step (1) and the photosensitive carboxymethyl starch derivative prepared in step (2) to the chitosan solution in sequence to obtain a mixed solution;

[0012] (4) After the mixed solution obtained in step (3) is cast and spread, it is allowed to stand. The first cross-linking network is formed by Schiff base reaction, and then the disulfide bond cross-linking is initiated by ultraviolet light irradiation to construct the second cross-linking network, thus obtaining a dual-enzyme responsive dual-network interpenetrating hydrogel.

[0013] (5) The dual-enzyme responsive dual-network interpenetrating hydrogel obtained in step (4) is dried and dehydrated to obtain the dual-enzyme responsive dual-network interpenetrating hydrogel film.

[0014] This invention first synthesizes PEG-CBA and CMS- g-LA, then the two are mixed with an acidic chitosan solution in a specific ratio, and after preliminary shaping by Schiff base reaction, a second cross-linking network is constructed in situ by ultraviolet light irradiation. Finally, a dual-enzyme responsive dual-network interpenetrating hydrogel film is formed by casting and drying. This invention significantly improves the mechanical strength and toughness of the hydrogel and its dry film by constructing a dual-network interpenetrating structure, effectively overcoming the defect of high brittleness of traditional polysaccharide materials. The prepared hydrogel film has dual-response degradation characteristics of lysozyme and α-amylase. Lysozyme, which is common in the oral cavity, can recognize the acetylglucosamine residues retained on the chitosan molecular chain and achieve specific degradation of chitosan components. α-amylase can specifically degrade the low-substituted carboxymethyl starch component. Relying on this dual-enzyme responsive characteristic, the film can achieve bioresponsive and controllable release of drugs. At the same time, it can also utilize the property of chitosan to reversibly open the tight junctions of oral mucosal epithelial cells, effectively promoting the mucosal penetration and absorption of encapsulated drugs.

[0015] Further, in step (1), polyethylene glycol with a specific molecular weight and topological structure is selected as the backbone. The polyethylene glycol is selected from one or more of linear polyethylene glycol, four-arm polyethylene glycol (4-Arm-PEG), and eight-arm polyethylene glycol (8-Arm-PEG). The molecular weight of the linear polyethylene glycol is 2000-20000 Da, such as 2000 Da, 6000 Da, 10000 Da, 20000 Da, etc.; the molecular weight of the four-arm polyethylene glycol is 10000-40000 Da, such as 10000 Da, 20000 Da, 40000 Da, etc.; the molecular weight of the eight-arm polyethylene glycol is 10000-40000 Da, such as 10000 Da, 20000 Da, 40000 Da, etc.

[0016] Further, in step (1), the molar ratio of the terminal hydroxyl group of the polyethylene glycol to p-aldehyde benzoic acid is 1:(2-10), preferably 1:(5-10).

[0017] Further, in step (1), polyethylene glycol is dissolved in a solvent, p-aldehyde benzoic acid (CBA) and a catalyst are added, and the mixture is stirred at 20-30 °C for 24-72 h. The carboxyl group (-COOH) of p-aldehyde benzoic acid (CBA) is grafted onto the hydroxyl group (-OH) at the end of polyethylene glycol through a Steglich esterification reaction to obtain a terminal aldehyde-modified polyethylene glycol derivative.

[0018] Furthermore, the solvent is dichloromethane (DCM).

[0019] Furthermore, the catalyst is a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP), or a combination of N,N'-dicyclohexylcarbodiimide (DCC) and 4-dimethylaminopyridine (DMAP).

[0020] Further, in step (1), after the esterification reaction is completed, the product is purified. The specific operation is as follows: the reaction solution is concentrated by rotary evaporator, the concentrate is placed in ice-cold ether to precipitate, 5-10 mL of ice-cold ether is used for every 1 mL of concentrate, and the precipitate is collected by centrifugation; the precipitate is redissolved in dichloromethane and precipitated again, and this operation is repeated 3-5 times to remove unreacted p-aldehyde benzoic acid molecules and catalyst; the obtained precipitate is vacuum dried, then dissolved in water and dialyzed for 24-48 h, with the molecular weight cutoff of the dialysis bag being 500-1000 Da; finally, the terminal aldehyde-based polyethylene glycol derivative is obtained by freeze drying.

[0021] Step (2) involves grafting hydrophobic lipoic acid onto the side chain of hydrophilic carboxymethyl starch using a bridging method. The bridging agent molecule has a bifunctional structure, with its amino or hydrazide group at one end covalently coupled to the carboxyl group of carboxymethyl starch, while the exposed free amino or hydrazide group at the other end serves as an effective grafting site for lipoic acid. The resulting side-chain functionalized carboxymethyl starch derivative (i.e., photosensitive carboxymethyl starch derivative) has the following structural features: the carboxyl group of carboxymethyl starch is connected to the amino or hydrazide group at one end of the bridging agent via an amide bond, and the amino or hydrazide group at the other end of the bridging agent then forms an amide bond with the carboxyl group of lipoic acid. In this way, a dithiopentane structure with photocrosslinking activity is successfully introduced into the side chain of carboxymethyl starch. Taking adipic acid dihydrazide (ADH) as a bridging agent as an example, the carboxyl group of carboxymethyl starch forms an amide bond with the hydrazide group at one end of ADH, and the hydrazide group at the other end of ADH forms an amide bond with the carboxyl group of thioctic acid, thereby introducing a dithiopentane structure with photocrosslinking activity into the side chain of carboxymethyl starch.

[0022] Further, in step (2), the carboxymethyl starch is in the form of sodium salt with a degree of substitution (DS) of 0.1-0.3, and the apparent viscosity of 1% (w / v) sodium carboxymethyl starch aqueous solution measured at 25 °C is 100-500 mPa·s.

[0023] Further, in step (2), the diamine or dihydrazide bridging agent is 1,2-ethylenediamine (EDA), 1,6-hexanediamine (HDA), cystamine (CTA), 3,3'-dithiobis(propionylhydrazine) (DTPH) or adipic acid dihydrazide (ADH).

[0024] Further, in step (2), the molar ratio of the diamine or dihydrazide bridging agent to the carboxyl group of carboxymethyl starch is (4-6):1.

[0025] Further, in step (2), the activator is a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) and N-hydroxysuccinimide (NHS), or a combination of EDC·HCl and N-hydroxythiosuccinimide (Sulfo-NHS).

[0026] Further, in step (2), the molar ratio of the carboxyl group of the carboxymethyl starch, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide is 1:(1.5-2.5):(1.5-2.5); the molar ratio of the carboxyl group of the carboxymethyl starch, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide is 1:(1.5-2.5):(1.5-2.5).

[0027] Further, in step (2), carboxymethyl starch is dissolved in water, and an activator is added to activate the carboxyl groups of the CMS side chain. After activation for 0.5-8 h, a diamine or dihydrazide bridging agent is added, and the reaction is carried out at 15-25 °C for 12-48 h. After the reaction, the mixture is dialyzed in water for 72-120 h through a dialysis bag with a molecular weight cutoff of 3500 Da to remove unreacted bridging agents and small molecule byproducts. The mixture is then freeze-dried to obtain a CMS derivative intermediate with free amino or hydrazide groups (for example, using ADH as a bridging agent, the CMS derivative intermediate CMS- is obtained). g -ADH).

[0028] Further, in step (2), the method for activating lipoic acid is as follows: dissolve lipoic acid in dimethyl sulfoxide (DMSO), add a combination of EDC·HCl and NHS or a combination of EDC·HCl and Sulfo-NHS as an activator, and activate the carboxyl group at 4-20 °C for 2-8 h.

[0029] Further, the molar ratio of lipoic acid, EDC·HCl to NHS is 1:(1.5-2.5):(1.5-2.5); the molar ratio of lipoic acid, EDC·HCl to Sulfo-NHS is 1:(1.5-2.5):(1.5-2.5).

[0030] Further, in step (2), the molar ratio of thioctic acid to diamine or dihydrazide bridging agent is (1.5-2.5):1.

[0031] Further, in step (2), the carboxymethyl starch derivative intermediate is dissolved in phosphate buffer solution to obtain a carboxymethyl starch derivative intermediate solution, and the activated lipoic acid is added to the carboxymethyl starch derivative intermediate solution. The reaction is stirred for 24-48 h under light-protected conditions at 4-20 °C. After the reaction is completed, the reaction solution is purified and freeze-dried to obtain a photosensitive carboxymethyl starch derivative.

[0032] Furthermore, the concentration of the phosphate buffer solution is 0.5-1.5 mol / L, and the pH is 4.5-5.5.

[0033] Further, in step (2), the specific purification operation is as follows: the reaction solution is dialyzed through a dialysis bag with a molecular weight cutoff of 7000 Da under light-protected conditions and at 4~20℃ to remove solvent, unreacted small molecules and catalyst.

[0034] Furthermore, in step (2), the photosensitive carboxymethyl starch derivative needs to be stored at -20 °C and protected from light.

[0035] Further, in step (3), the degree of deacetylation of the chitosan is 70%-90%, and the apparent viscosity of its 1% (w / v) aqueous solution of 1% (v / v) acetic acid measured at 25 °C is 10-2000 mPa·s.

[0036] Further, in step (3), chitosan is dispersed in water, and 1 mol / L hydrochloric acid solution is added dropwise to aid dissolution. After the chitosan is completely dissolved, the pH of the system is adjusted to 5.0-6.0 with 1 mol / L sodium hydroxide (NaOH) solution. This pH range allows chitosan to maintain a stable dissolved state, and the degree of amino protonation is suitable, providing favorable conditions for the subsequent Schiff base reaction. If drug loading is required, the drug (such as small molecule drugs, peptides, proteins, etc.) can be uniformly dispersed and dissolved in the chitosan solution in this step.

[0037] Further, in step (3), the concentration of chitosan in the mixed solution is 0.5-5.0 wt%; the concentration of terminal aldehyde-modified polyethylene glycol derivative in the mixed solution is 0.1-15 wt%; and the concentration of photosensitive carboxymethyl starch derivative in the mixed solution is 0.1-3.0 wt%.

[0038] Further, in step (4), the mixed solution obtained in step (3) is poured into a mold, cast and spread to a predetermined thickness, and left to stand at 10-40 °C for 2-60 min; during this process, the aldehyde groups at the ends of PEG-CBA undergo nucleophilic addition reactions with the amino groups on the chitosan chains to form Schiff base bonds (-C=N-), the viscosity of the system increases sharply, forming the first layer of gel crosslinking network, while locking CMS-g - LA segment and distribution of the drug it carries.

[0039] Further, in step (4), the pre-shaped gel forming the first cross-linked network is placed under an ultraviolet (UV) curing lamp for irradiation under the following conditions: wavelength 365 nm, light intensity 10-50 mW / cm². 2 Irradiation time: 5-60 min; UV-excited CMS- g The dithiopentane structure of the LA side chain undergoes a ring-opening polymerization reaction to form a long-chain polydisulfide crosslinking network. This network interpenetrates within the first crosslinking network, forming a dual-enzyme responsive hydrogel with a double-network interpenetrating structure.

[0040] Further, in step (5), the dual-enzyme responsive dual-network interpenetrating hydrogel obtained in step (4) is placed in a constant temperature chamber at 15-35°C and dried by air drying or gradient temperature drying until constant weight is obtained to obtain the dual-enzyme responsive dual-network interpenetrating hydrogel film.

[0041] The second aspect of the present invention provides a dual-enzyme responsive dual-network interpenetrating hydrogel film prepared by the preparation method described in the first aspect.

[0042] The dual-enzyme responsive dual-network interpenetrating hydrogel film provided by this invention is prepared by drying and dehydration of a hydrogel with an interpenetrating polymer network structure. This interpenetrating polymer network structure comprises a first crosslinked network and a second crosslinked network. The first crosslinked network is formed by crosslinking chitosan with a polyethylene glycol derivative (PEG-CBA) terminally grafted with p-aldehyde benzoic acid via Schiff base bonds. The second crosslinked network is formed by linking a carboxymethyl starch derivative (CMS-CBA) grafted with lipoic acid via diamine or dihydrazide bridging. g -LA) is obtained by photocrosslinking disulfide bonds through ring-opening polymerization of dithiopentane under ultraviolet light.

[0043] The third aspect of this invention provides the application of the dual-enzyme responsive dual-network interpenetrating hydrogel film described in the second aspect in the preparation of oral mucosal drug delivery formulations.

[0044] Furthermore, the dual-enzyme-responsive dual-network interpenetrating hydrogel film is used as a drug delivery carrier for oral mucosa. The properties of chitosan that reversibly open the tight junctions of oral mucosal epithelial cells promote drug penetration and absorption. Furthermore, by utilizing its dual-responsive degradation properties to lysozyme and α-amylase in the oral environment, the controlled release of the drug in the oral cavity can be achieved.

[0045] In a specific embodiment, the method for loading drugs onto the dual-enzyme-responsive dual-network interpenetrating hydrogel film includes the following steps:

[0046] (1) PEG and CBA were esterified and purified to obtain PEG-CBA;

[0047] (2) In the presence of an activator, CMS reacts with a diamine or dihydrazide bridging agent to obtain a carboxymethyl starch derivative intermediate. LA is then activated and reacted with the carboxymethyl starch derivative intermediate, followed by purification to obtain CMS- g -LA;

[0048] (3) Dissolve CS in an acidic aqueous solution and adjust the pH of the system to 5.0-6.0 to obtain a chitosan solution; dissolve the drug in the chitosan solution to obtain a drug-containing chitosan solution, wherein the final drug concentration is 10. -3 - 10 mg / mL; Add the terminal aldehyde-modified polyethylene glycol derivative prepared in step (1) and the photosensitive carboxymethyl starch derivative prepared in step (2) to the chitosan solution in sequence to obtain a mixed solution, wherein the concentration of chitosan is 0.5-5.0 wt%, the concentration of the terminal aldehyde-modified polyethylene glycol derivative is 0.1-15 wt%, and the concentration of the photosensitive carboxymethyl starch derivative is 0.1-3.0 wt%;

[0049] (4) The mixed solution obtained in step (3) is cast and spread in a mold, and allowed to stand at 10-40 °C. The first cross-linked network is formed through Schiff base reaction, and then the second cross-linked network is constructed by initiating disulfide bond cross-linking under ultraviolet light irradiation, thus obtaining a drug-loaded dual-enzyme responsive dual-network interpenetrating hydrogel; the ultraviolet light wavelength is 365 nm and the irradiation intensity is 10-50 mW / cm. 2 The irradiation time is 5-60 minutes;

[0050] (5) The drug-loaded dual-enzyme responsive dual-network interpenetrating hydrogel obtained in step (4) is placed in a constant temperature oven at 15-35 ℃ to dry and dehydrate, thereby obtaining the drug-loaded dual-enzyme responsive dual-network interpenetrating hydrogel film.

[0051] The above-described technical solution of the present invention has the following beneficial effects:

[0052] 1. This invention constructs a dual-network interpenetrating structure combining a Schiff base dynamic network and a disulfide bond photocrosslinking network, abandoning the traditional single crosslinking mode of polysaccharide membranes and possessing an excellent toughening and strengthening mechanism. Specifically, the Schiff base network formed by chitosan and polyethylene glycol acts as a sacrificial network, preferentially breaking under stress to dissipate energy and imparting excellent toughness to the material; while the photocrosslinking network formed by modified carboxymethyl starch acts as a rigid framework to maintain the overall structural integrity. The synergistic effect of these two components makes the film flexible and brittle in a dry state and high-strength and undamaged in a wet state, solving the problem of high brittleness in traditional polysaccharide membranes.

[0053] 2. This invention designs an intelligent dual-enzyme-responsive drug delivery system, fully utilizing the abundant lysozyme and α-amylase in oral saliva to construct a dual biodegradation switch. The chitosan network can be specifically cleaved and degraded by lysozyme, and the carboxymethyl starch network can be specifically degraded by α-amylase. This response mechanism, which closely matches the oral physiological microenvironment, has higher specificity and controllability compared to ordinary diffusion release or pH response, enabling on-demand intelligent drug delivery.

[0054] 3. This invention simultaneously achieves the synergistic function of highly efficient penetration enhancement and bioadhesion. Through rational formulation design, the system forms a high-strength network while retaining sufficient unreacted amino groups on the chitosan chains. These positively charged amino groups can reversibly open the tight junctions between oral mucosal epithelial cells, significantly improving the mucosal penetration efficiency of drugs, especially macromolecular drugs. The hydrogel structure formed after the material absorbs water and swells also endows it with excellent mucosal adhesion ability, effectively prolonging the retention time of drugs at oral lesion sites.

[0055] 4. This invention features a flexible and adjustable structural design and a mild preparation process. By controlling the topology (linear / 4-arm / 8-arm) and molecular weight of polyethylene glycol, the gel mesh size and mechanical properties can be precisely controlled to meet the needs of different application scenarios. The entire preparation process is completed in an aqueous phase under mild conditions, avoiding residual organic solvents and harsh high-temperature conditions, maximizing the protection of the bioactivity of the loaded drug, and demonstrating excellent biocompatibility and promising prospects for clinical translation. Attached Figure Description

[0056] Figure 1 This is a schematic diagram of the chemical structure of chitosan and its terminal aldehyde-modified polyethylene glycol derivatives.

[0057] Figure 2 This is a schematic diagram of the process for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film according to the present invention.

[0058] Figure 3 Preparation of CMS- for Example 15 g -ADH and CMS preparation in Example 20- g -Schematic diagram of the synthesis route of LA.

[0059] Figure 4 CS / PEG was prepared for Comparative Example 10 6k -A physical image of the CBA hydrogel and a schematic diagram illustrating the construction principle of the hydrogel network.

[0060] Figure 5 The proton NMR spectrum of chitosan ( 1 (H-NMR) chromatogram; where (A) represents chitosan with low degree of deacetylation. 1 H-NMR spectrum, degree of deacetylation 72.7%; (B) shows high degree of deacetylation chitosan raw material.1 H-NMR spectrum, degree of deacetylation 85.3%.

[0061] Figure 6 PEG prepared in Example 1 2k -CBA 1 H-NMR spectrum.

[0062] Figure 7 The 4aPEG prepared in Example 6 20k of 1 H-NMR spectrum.

[0063] Figure 8 Sodium carboxymethyl starch and CMS prepared in Example 15 g -ADH 1 H-NMR spectrum.

[0064] Figure 9 The CMS- prepared in Example 20 g -LA 1 H-NMR spectrum.

[0065] Figure 10 Sodium carboxymethyl starch and CMS prepared in Example 15 g -ADH, CMS prepared in Example 20- g -FT-IR spectrum comparison of sodium carboxymethyl starch and CMS- g FT-IR spectrum comparison of -ADH, (B) sodium carboxymethyl starch and CMS- g - FT-IR spectrum comparison of LA.

[0066] Figure 11 The CMS- prepared in Example 20 g -Raman spectra of LA (bottom figure) and the lyophilized sample of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 (top figure).

[0067] Figure 12 The CMS- prepared in Example 20 g XPS full spectrum of lyophilized samples of LA and the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26; wherein, (A) is CMS- g - The XPS full spectrum of LA, (B) is the XPS full spectrum of the lyophilized sample of the dual-enzyme responsive dual-network interpenetrating hydrogel.

[0068] Figure 13The graphs show the gelation time data of hydrogels formed by crosslinking CS and PEG-CBA at different temperatures; where (A) is the gelation time data of Comparative Examples 7-10 at 25 ℃, and (B) is the gelation time data of Comparative Examples 22-25 at 37 ℃.

[0069] Figure 14 Rheological time-scan curves of CS / PEG-CBA hydrogels prepared for Comparative Examples 7-10 and 15-16 at 25 °C; where (A) is Comparative Example 7, (B) is Comparative Example 8, (C) is Comparative Example 9, (D) is Comparative Example 10, (E) is Comparative Example 15, and (F) is Comparative Example 16.

[0070] Figure 15 CMS- prepared for Comparative Example 3 g - Rheological self-healing performance characterization spectra of LA hydrogel and the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26; wherein, (A) is CMS- g -LA hydrogel, (B) is a dual-enzyme responsive dual-network interpenetrating hydrogel.

[0071] Figure 16 The images show a comprehensive characterization of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, as well as physical images of the hydrogels prepared in Comparative Example 16 and Example 26. Specifically, (A) is a physical image of the extruded gel, (B) is a physical image of the gel adhesion, (C) is an FT-IR image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, (D) is a SEM image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, (E) is a physical image of the CS / PEG-CBA hydrogel prepared in Comparative Example 16 in a square polyester mold, (F) is a physical image of the CS / PEG-CBA hydrogel prepared in Comparative Example 16 stably spread in the mold after being inverted, (G) is a physical image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 in a square polyester mold, and (H) is a physical image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 stably spread in the mold after being inverted.

[0072] Figure 17 The two enzyme-responsive dual-network interpenetrating hydrogel films prepared in Example 26, the CS films prepared in Comparative Example 1, and the CS / CMS films prepared in Comparative Example 4 are described. gThe mechanical properties and microstructure of the LA hydrogel film and the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 are comprehensively characterized. Among them, (A) is a physical picture of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 in the initial tensile state, (B) is a physical picture of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 after tensile testing, (C) is a cross-sectional SEM image (scale bar is 20 μm) of the dual-enzyme responsive dual-network interpenetrating hydrogel film prepared in Example 26 after being frozen and fractured by liquid nitrogen, and (D) is the tensile stress-strain curve of the hydrogel films prepared in Example 26, Comparative Example 1, Comparative Example 4 and Comparative Example 16.

[0073] Figure 18 The two enzyme-responsive dual-network interpenetrating hydrogel films prepared in Example 26, the CS films prepared in Comparative Example 1, and the CS / 4aPEG films prepared in Comparative Example 2 are as follows: 20k -CBA hydrogel film, CMS prepared in Comparative Example 3- g - Degradation curves of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 and Comparative Example 26 in simulated saliva; wherein, (A) is the degradation curve of the film of Comparative Example 1 and Comparative Example 16 in simulated saliva containing lysozyme, (B) is the degradation curve of the film of Comparative Example 2 and Comparative Example 16 in simulated saliva containing lysozyme, (C) is the degradation curve of the film of Comparative Example 3 in simulated saliva containing α-amylase and without α-amylase, and (D) is the degradation curve of the film of Comparative Example 3 in simulated saliva containing α-amylase, the film of Example 26 in simulated saliva containing α-amylase and simulated saliva containing both α-amylase and lysozyme.

[0074] Figure 19 The two-enzyme responsive double-network interpenetrating hydrogel film loaded with Cy5-BSA prepared in Example 36 and the CS / 4aPEG film loaded with Cy5-BSA prepared in Comparative Example 5 are compared. 20k -CBA hydrogel film drug cumulative release curves in different media; wherein, (A) is the drug cumulative release curve of the drug-loaded hydrogel film of Example 36 in simulated saliva containing α-amylase and lysozyme and the drug cumulative release curve of the drug-loaded hydrogel film of Comparative Example 5 in simulated saliva containing α-amylase and lysozyme and in an enzyme-free environment (enzyme-free simulated saliva), and (B) is the drug cumulative release curve of the drug-loaded hydrogel film of Example 36 in an enzyme-free environment (enzyme-free simulated saliva), simulated saliva containing only α-amylase and simulated saliva containing α-amylase and lysozyme. Detailed Implementation

[0075] 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.

[0076] Existing oral mucosal drug delivery carrier materials suffer from numerous technical defects. Traditional polysaccharide hydrogels struggle to balance mechanical properties and biodegradability, exhibiting insufficient strength and brittleness. Alternatively, non-degradable or toxic cross-linking agents are used to enhance strength. Furthermore, existing modified polysaccharide materials have relatively simple structural designs, often employing single cross-linking methods such as Schiff base reactions and photocross-linking. While Schiff base bonds possess dynamic reversibility, their poor hydrolytic stability leads to rapid in vivo degradation of the gel, while simple permanent chemical cross-linking results in a lack of material toughness. In addition, there is a lack of dual biological response mechanisms that can simultaneously respond to oral-specific markers such as lysozyme and amylase, hindering the achievement of intelligent degradation and drug release adapted to the oral physiological environment.

[0077] This invention uses modified polyethylene glycol, chitosan, and modified carboxymethyl starch as fully bio-based raw materials. It employs an interpenetrating polymer network (IPN) construction strategy of "chemical crosslinking + photocrosslinking". Through Schiff base covalent crosslinking and photo-initiated disulfide bond crosslinking, a double-network interpenetrating structure hydrogel film is constructed. This not only solves the contradiction between mechanical strength and biodegradability in traditional hydrogel materials, but also endows the film with dual specific response to lysozyme and α-amylase and efficient oral mucosal permeation function. While significantly improving mechanical strength and toughness, it achieves intelligent degradation and efficient drug penetration.

[0078] The method for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film of the present invention mainly includes the preparation and pretreatment of three key raw materials, as well as the in-situ construction and film formation of the dual network, specifically including the following steps:

[0079] (1) Polyethylene glycol (PEG) was esterified with p-aldehyde benzoic acid (CBA), and after purification, a terminal aldehyde-terminated polyethylene glycol derivative (PEG-CBA) was obtained.

[0080] (2) In the presence of an activator, carboxymethyl starch (CMS) reacts with a diamine or dihydrazide bridging agent to obtain a carboxymethyl starch derivative intermediate. After activation with lipoic acid (LA), the intermediate reacts with the carboxymethyl starch derivative, and the resulting product is purified to obtain a photosensitive carboxymethyl starch derivative (CMS-). g -LA);

[0081] (3) Dissolve chitosan (CS) in an acidic aqueous solution and adjust the pH of the system to 5.0-6.0 to obtain a chitosan solution; add the terminal aldehyde-modified polyethylene glycol derivative prepared in step (1) and the photosensitive carboxymethyl starch derivative prepared in step (2) to the chitosan solution in sequence to obtain a mixed solution;

[0082] (4) After the mixed solution obtained in step (3) is cast and spread, it is allowed to stand. The first cross-linking network is formed by Schiff base reaction, and then the disulfide bond cross-linking is initiated by ultraviolet light irradiation to construct the second cross-linking network, thus obtaining a dual-enzyme responsive dual-network interpenetrating hydrogel.

[0083] (5) The dual-enzyme responsive dual-network interpenetrating hydrogel obtained in step (4) is dried and dehydrated to obtain the dual-enzyme responsive dual-network interpenetrating hydrogel film.

[0084] In a specific embodiment, in step (1), polyethylene glycol with a specific molecular weight and topological structure is selected as the backbone. The polyethylene glycol is selected from one or more of linear polyethylene glycol, four-arm polyethylene glycol (4-Arm-PEG), and eight-arm polyethylene glycol (8-Arm-PEG). A schematic diagram of the chemical structure of chitosan and terminally aldehyde-modified polyethylene glycol derivatives is shown below. Figure 1 As shown.

[0085] The dual-enzyme responsive interpenetrating hydrogel film prepared by this invention has a unique microstructure, the core of which is an IPN structure: the photocrosslinking network (i.e., the second crosslinking network) formed by rigid carboxymethyl starch and poly(α-lipoic acid) blocks provides high tensile strength to the film material and can effectively prevent material deformation; while the Schiff base chemical crosslinking network (i.e., the first crosslinking network) formed by flexible and dynamically reversible chitosan and polyethylene glycol blocks serves as an energy dissipation component, which can endow the film material with excellent fracture toughness. The two work together to ensure the mechanical properties of the film.

[0086] In a specific embodiment, a schematic diagram of the process for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film is shown below. Figure 2As shown, (A) demonstrates the photocrosslinking mechanism of carboxymethyl starch grafted with lipoic acid undergoing ring-opening polymerization of its side chain dithiopentane structure to form a polydithiopentane crosslinking network under ultraviolet light at room temperature; (B) presents the stepwise construction process of the double-network interpenetrating hydrogel. After mixing chitosan, linear PEG derivatives and carboxymethyl starch grafted with lipoic acid, the first crosslinking network is formed by Schiff base chemical crosslinking, and then the second crosslinking network is constructed by ring-opening polymerization of lipoic acid under ultraviolet light irradiation, finally forming a double-network interpenetrating structure; (C) demonstrates the solvent casting film formation process. Through steps such as sol casting, mold filling, gelation, and water evaporation, the double-network interpenetrating hydrogel is prepared into a dense hydrogel film. After drying, the molecular chains are further entangled and the mechanical properties of the film material are improved. This intuitively confirms the feasibility and logic of the process of stepwise construction of the double-network interpenetrating structure by "chemical crosslinking + photocrosslinking" and then air drying to form a film.

[0087] The dual-enzyme responsive dual-network interpenetrating hydrogel film prepared by this invention has excellent mechanical properties. In the dry state, it has high tensile strength and high elongation at break, and can be rolled up arbitrarily without breaking. In the wet state (simulating the oral fluid environment), it can maintain the integrity of the membrane morphology and will not disintegrate instantly, but only swells moderately, which is suitable for the use of the moist physiological environment of the oral cavity.

[0088] The dual-enzyme-responsive degradation characteristics of the double-network interpenetrating hydrogel film prepared by this invention are as follows: in a medium containing lysozyme, the chitosan backbone undergoes specific hydrolytic breakage; in a medium containing α-amylase, the carboxymethyl starch backbone undergoes specific hydrolytic breakage; only under the combined action of the two enzymes, or in a real oral physiological environment, can the double-network interpenetrating structure achieve complete degradation and removal, thereby achieving precise and controllable drug release.

[0089] The application scenarios of the dual-enzyme responsive dual-network interpenetrating hydrogel film prepared by this invention are mainly focused on oral mucosa (including buccal mucosa and sublingual mucosa) drug delivery. It can be used for local or systemic drug delivery, and is especially suitable for large molecular protein drugs such as insulin, vaccines, and growth hormone. With the help of the film's excellent permeation-enhancing function, it can significantly improve the oral mucosal absorption efficiency and bioavailability of such drugs.

[0090] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0091] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, and the materials and reagents used are commercially available.

[0092] Linear PEGs with molecular weights of 2000, 6000, 10000, or 20000 Da in the following examples are named PEGs, respectively. 2k PEG 6k PEG 10k and PEG 20k Four-arm PEGs (4-Arm-PEG, or 4aPEG for short) with molecular weights of 10,000, 20,000, or 40,000 Da are named 4aPEG, respectively. 10k 4aPEG 20k and 4aPEG 40k Eight-arm PEGs (8-Arm-PEG, or 8aPEG for short) with molecular weights of 10,000, 20,000, or 40,000 Da are named 8aPEG, respectively. 10k 8aPEG 20k and 8aPEG 40k .

[0093] Examples 1-10

[0094] A method for preparing a terminally aldehyde-terminated polyethylene glycol derivative (PEG-CBA) includes the following steps:

[0095] PEG was dissolved in DCM, and CBA, EDC·HCl, and DMAP were added. The mixture was stirred at 25 °C for 72 h to allow for complete esterification of the hydroxyl groups (-OH) at the PEG terminus with the carboxyl groups (-COOH) of CBA. After esterification, the product was purified by concentrating the reaction solution using a rotary evaporator. The concentrate was then placed in excess ice-cold diethyl ether (frozen at -20 °C for 24 h), with 10 mL of ice-cold diethyl ether used for every 1 mL of concentrate. The precipitate was collected by centrifugation and dried under vacuum at 35 °C. The precipitate was redissolved in a small amount of DCM, with 10 mL of DCM used for every gram of precipitate, and then precipitated again. This precipitation process was repeated four times to remove unreacted small molecule CBA and catalyst. The precipitate was dried under vacuum, then dissolved in pure water and dialyzed for 24 h, with a molecular weight cutoff of 1000 Da. Finally, purified PEG-CBA was obtained by freeze-drying. (1H NMR spectroscopy was performed.) 1 H-NMR can confirm the appearance of characteristic peaks of benzene ring and aldehyde group, indicating that the grafting was successful.

[0096] The feed schedule for the preparation of PEG-CBA in Examples 1-10 is shown in Table 1:

[0097] Table 1

[0098]

[0099] Examples 11-15

[0100] A method for preparing a CMS derivative intermediate includes the following steps:

[0101] Sodium carboxymethyl starch powder (CMS, DS = 0.285, apparent viscosity 225 ± 40 mPa·s) was dissolved in water to prepare a 15 mg / mL solution. EDC·HCl and NHS were added, and the carboxyl groups of the CMS side chains were activated at room temperature for 48 h. Subsequently, a bridging agent was dissolved in a specific solvent (water or dimethylformamide DMF) and added to the above CMS reaction system, reacting at room temperature for 48 h. Based on the DS of CMS, 5 g of sodium carboxymethyl starch contained 8.8 mmol of carboxyl groups. The molar ratio of CMS carboxyl groups, EDC·HCl, and NHS was 1:2:2, therefore the amount of EDC·HCl and NHS added was 17.6 mmol each. The molar ratio of CMS carboxyl groups to the bridging agent was 1:5, therefore the amount of the bridging agent added was 44 mmol. After the reaction was completed, the reaction product solution was dialyzed in 0.9% (w / v) sodium chloride aqueous solution for 48 h, then transferred to pure water and dialyzed for another 24 h. The purified CMS derivative intermediate was obtained by freeze drying.

[0102] Examples 11-15 use EDA, HDA, CTA (cystamine dihydrochloride), DTPH, and ADH as bridging agents, respectively, and the corresponding CMS derivative intermediates are named CMS- g -EDA (Example 11), CMS- g -HDA (Example 12), CMS- g -CTA (Example 13), CMS- g -DTPH (Example 14) and CMS- g -ADH (Example 15).

[0103] The feed schedule for preparing CMS derivative intermediates in Examples 11-15 is shown in Table 2:

[0104] Table 2

[0105]

[0106] Table 3 shows the content of nitrogen, bridging agent, and carboxyl group conversion rate in the CMS derivative intermediates prepared in Examples 11-15.

[0107] Table 3

[0108]

[0109] Note: The bridging agent content (wt%) in the CMS derivative intermediate represents the mass fraction of the bridging agent grafted into the product, calculated from the nitrogen content (N, wt%) measured by an organic elemental analyzer. The carboxyl conversion rate (%) represents the proportion of carboxyl groups participating in the grafting reaction, reflecting the efficiency with which carboxyl sites in the raw material are replaced by the bridging agent.

[0110] Examples 16-20

[0111] A photosensitive carboxymethyl starch derivative (CMS- g The preparation method of (-LA) includes the following steps:

[0112] LA was dissolved in an appropriate amount of dimethyl sulfoxide (DMSO), and its carboxyl group was activated by adding EDC·HCl and NHS. Activation was carried out for 4 h in the dark at a reaction temperature of 4 °C, with the molar ratio of LA, EDC·HCl, and NHS being 1:2:2. 3 g of the CMS derivative intermediate prepared in Examples 11-15 was weighed and dissolved in 300 mL of phosphate buffer solution (PB, pH 5.0, concentration 1 mol / L). Subsequently, the activated lipoic acid solution was slowly added dropwise to the CMS derivative intermediate solution, and the reaction was stirred for 48 h in the dark at 4 °C. Because the bridging agent molecule has a bifunctional structure, one end of its amino group (or hydrazide group) has already been coupled to the carboxyl group of CMS, while the exposed free amino group (or hydrazide group) at the other end serves as an effective grafting site for LA. Based on stoichiometry and to ensure sufficient reaction, the molar ratio of LA to the bridging agent in the CMS derivative intermediate was 2:1. After the reaction was completed, the reaction solution was dialyzed at 4 °C under light-protected conditions (molecular weight cutoff 7000 Da) to remove DMSO, unreacted small molecules, and catalyst. Finally, it was freeze-dried to obtain CMS- g -LA, this product should be stored at -20 °C and protected from light.

[0113] Examples 16-20 Preparation of CMS- g - The material feeding table for LA is shown in Table 4:

[0114] Table 4

[0115]

[0116] Note: The number of bridging agents (mmol) is calculated based on the bridging agent content (wt%) of the carboxymethyl starch derivative intermediates in Table 3.

[0117] CMS- prepared in Examples 16-20 g - The LA content and its feed conversion rate are shown in Table 5:

[0118] Table 5

[0119]

[0120] Note: "LA feed amount per unit mass of intermediate" represents the number of moles of LA fed per unit mass of CMS derivative intermediate; "S content in product (wt%)" is determined by CMS- g - The LA product was measured by an organic elemental analyzer, and the "LA content in the product (wt%)" and "LA moles per unit mass of product (mmol / g)" could be calculated accordingly; the "LA feed conversion rate (%)" was obtained by dividing the "LA moles per unit mass of product (mmol / g)" by the "LA feed amount per unit mass of intermediate" and multiplying by 100%.

[0121] Figure 3 Preparation of CMS- for Example 15 g -ADH and CMS preparation in Example 20- g The schematic diagram of the synthetic route for -LA illustrates a molecular design strategy that uses carboxymethyl starch as a starting material and introduces photocrosslinking active groups into the side chain via a two-step process: The first step uses ADH as a bridging agent to introduce free hydrazide active ends into the carboxymethyl starch side chain, yielding CMS- g -ADH (Example 15); The second step utilizes CMS- g The free hydrazide group of the -ADH side chain undergoes an amidation reaction with the carboxyl group of LA, grafting thioctic acid onto the carboxymethyl starch side chain to obtain CMS- g -LA (Example 20). This two-step grafting design effectively solves the problem that lipoic acid is difficult to directly graft onto the starch backbone due to steric hindrance, ensuring that the side chains have sufficient flexibility and reactivity.

[0122] Examples 21-30

[0123] A method for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film includes the following steps:

[0124] Chitosan (CS) with a degree of deacetylation of 72.7% was dispersed in pure water, and 1 mol / L hydrochloric acid solution was added dropwise to aid dissolution. After complete dissolution, the pH was finely adjusted to 5.5 using 1 mol / L NaOH solution. Subsequently, the chitosan solution was concentrated to a concentration of 50 mg / g using a rotary evaporator (water bath temperature 50°C, vacuum degree 50 mbar, rotation speed 100 rpm), and then diluted to 45 mg / g with pure water to obtain a CS solution with a mass concentration of 45 mg / g and a pH of 5.5 (the chitosan solution was placed in a glass dish and dried to constant weight in a constant temperature drying oven at 105°C; the dry weight was then measured, and the mass concentration of the CS solution was calculated by the ratio of the obtained dry weight of CS to the corresponding initial solution mass). The PEG-CBA prepared in Examples 1-10 was dissolved in pure water to prepare a PEG-CBA solution of 90 mg / g. Subsequently, the CMS-CBA prepared in Example 20 was... g -LA was dissolved in pure water to prepare CMS solutions with a mass concentration of 30 mg / g. g -LA solution.

[0125] Add the PEG-CBA solution to the CS solution, mix thoroughly, and then immediately add CMS- g -LA solution, and mix thoroughly again, controlling the blending mass ratio of the three solutions to 1:1:1. Immediately pour 45 g of the mixed solution into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height), cast and spread into a liquid film of the predetermined thickness, and let it stand at 25 ℃ to form a gel. At this time, the aldehyde groups at the end of PEG-CBA undergo nucleophilic addition reaction with the amino groups on the chitosan chain to form Schiff base bonds (-C=N-), the viscosity of the system increases sharply, a gelation transition occurs, the first cross-linking network is formed, and CMS- is locked in. g - Distribution of the LA segment. The preliminarily shaped gel was irradiated under a UV curing lamp under the following conditions: UV wavelength of 365 nm and light intensity of 50 mW / cm². 2 The irradiation time was 10 minutes. During this process, CMS- was excited by ultraviolet light. g The dithiopentane structure of the -LA side chain causes it to undergo a ring-opening polymerization reaction, forming a second polydisulfide crosslinking network. The second crosslinking network formed by carboxymethyl starch and polythiooctanoic acid interpenetrates within the first crosslinking network, ultimately forming a two-enzyme responsive double-network interpenetrating hydrogel based on the three-component crosslinking of CS, PEG derivatives and CMS derivatives.

[0126] A polyester mold containing a dual-enzyme-responsive dual-network interpenetrating hydrogel was placed in a constant temperature incubator and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold to obtain the dual-enzyme-responsive dual-network interpenetrating hydrogel film.

[0127] The feed table for the preparation of dual-enzyme responsive dual-network interpenetrating hydrogel films in Examples 21-30 is shown in Table 6:

[0128] Table 6

[0129]

[0130] Note: The solutions referred to in "three-component solution mixing ratio (w / w / w)" are, in order, the initial PEG-CBA solution, the initial CS solution, and the initial CMS solution. g -LA initial solution.

[0131] Examples 31-36

[0132] A method for preparing a dual-enzyme-responsive, dual-network interpenetrating hydrogel film coated with sulfonyl Cy5 (Sulfo-Cy5) fluorescent dye-labeled bovine serum albumin (BSA) includes the following steps:

[0133] Sulfo-Cy5 fluorescent dye was added to an aqueous BSA solution to a concentration of 0.1% of the BSA mass. The mixture was protected from light and stirred at room temperature for 4 h. After the reaction was complete, the resulting Sulfo-Cy5-labeled BSA solution was purified by dialysis. After freeze-drying, Sulfo-Cy5-labeled BSA powder was obtained and named Cy5-BSA. Cy5-BSA serves as a model molecule for protein drugs.

[0134] A specific mass of Cy5-BSA was directly dissolved in a CS solution with a mass concentration of 45 mg / g (pH=5.5, degree of deacetylation 72.7%) to obtain a drug-containing CS solution with a specific Cy5-BSA drug concentration, in which the CS concentration and pH value remained unchanged. The 4aPEG prepared in Example 6 was then used... 20k -CBA was dissolved in pure water to prepare a 90 mg / g 4aPEG solution. 20k -CBA solution. Subsequently, the CMS- prepared in Example 20 was used. g -LA was dissolved in pure water to prepare CMS solutions with mass concentrations of 15, 24, and 30 mg / g, respectively. g -LA solution.

[0135] 4aPEG 20k - Add CBA solution to the drug-containing CS solution, mix well, and immediately add CMS- g -LA solution, and mix thoroughly again, controlling the blending mass ratio of the three solutions to 1:1:1.

[0136] 45 g of the mixed solution was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to form a liquid film of the predetermined thickness, and then allowed to stand at 25 ℃ to form a gel. After the first cross-linking network was formed, the preliminarily shaped gel was irradiated under a UV curing lamp with the following conditions: UV wavelength of 365 nm and light intensity of 50 mW / cm². 2 The irradiation time was 10 min. During this process, a second cross-linked network was formed, interpenetrating within the first cross-linked network, ultimately forming a two-enzyme-responsive, two-network interpenetrating hydrogel loaded with Cy5-BSA based on the three-component cross-linking of CS, PEG, and CMS derivatives.

[0137] A polyester mold containing a Cy5-BSA-enzyme-responsive dual-network interpenetrating hydrogel was placed in a constant temperature incubator and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold to obtain a Cy5-BSA-enzyme-responsive dual-network interpenetrating hydrogel film.

[0138] The feed table for the preparation of Cy5-BSA-loaded dual-enzyme responsive dual-network interpenetrating hydrogel films in Examples 31-36 is shown in Table 7:

[0139] Table 7

[0140]

[0141] Comparative Example 1

[0142] A method for preparing a CS thin film includes the following steps:

[0143] Take 20 g of a CS solution with a mass concentration of 30 mg / g and a pH of 5.5 (degree of deacetylation 72.7%), mix it with an equal mass of pure water, and then immediately pour 40 g of this mixture into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to cast and spread it into a liquid film of a predetermined thickness. Finally, place the polyester mold containing the CS aqueous solution in a constant temperature oven and slowly air-dry it at 30 ℃ until constant weight. Finally, remove the film material from the mold to obtain the CS film.

[0144] Comparative Example 2

[0145] A CS / 4aPEG 20k The preparation method of CBA hydrogel film includes the following steps:

[0146] Chitosan (CS) with a degree of deacetylation of 85.3% was dispersed in pure water, and 1 mol / L hydrochloric acid solution was added dropwise to aid dissolution. After complete dissolution, the pH was finely adjusted to 5.5 using 1 mol / L NaOH solution. Subsequently, the chitosan solution was concentrated to a concentration of 50 mg / g using a rotary evaporator (water bath temperature 50 ℃, vacuum degree 50 mbar, rotation speed 100 rpm), and then diluted to 30 mg / g with pure water. At this point, a CS solution with a mass concentration of 30 mg / g and a pH of 5.5 was obtained.

[0147] The 4aPEG prepared in Example 6 20k -CBA was dissolved in pure water to prepare a 60 mg / g 4aPEG solution. 20k -CBA solution; mix CS solution and 4aPEG 20k - The CBA solution was mixed at a mass ratio of 1:1, and then 40 g of the mixture was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to cast and spread into a liquid film of a predetermined thickness, and then allowed to stand at 25 ℃ to form a gel.

[0148] The polyester mold containing the hydrogel was placed in a constant temperature oven and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold to obtain a two-component crosslinked CS / 4aPEG of CS and PEG derivatives. 20k -CBA hydrogel film.

[0149] Comparative Example 3

[0150] A CMS- g The preparation method of -LA hydrogel film includes the following steps:

[0151] The CMS- prepared in Example 20 g -LA was dissolved in pure water to prepare a CMS solution with a mass concentration of 30 mg / g. g -LA solution. Add 15 g CMS- g - The LA solution was mixed with 30 g of pure water, and 45 g of the mixture was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to form a liquid film of the predetermined thickness. The liquid film was then irradiated under a UV curing lamp with the following conditions: wavelength 365 nm, light intensity 50 mW / cm². 2 The irradiation time was 10 minutes. During this process, CMS- was excited by ultraviolet light. gThe dithiopentane structure of the -LA side chain undergoes a ring-opening polymerization reaction, forming a cross-linked network of polydisulfide compounds. Therefore, a hydrogel ultimately formed is cross-linked through the ring-opening reaction of the dithiopentane structure of the CMS derivative side chain lipoic acid.

[0152] The polyester mold containing the hydrogel was placed in a constant temperature chamber and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold to obtain CMS- g -LA hydrogel film.

[0153] Comparative Example 4

[0154] A CS / CMS- g The preparation method of -LA hydrogel film includes the following steps:

[0155] A CS solution with a mass concentration of 30 mg / g (pH = 5.5) and a degree of deacetylation of chitosan of 72.7% was prepared. Subsequently, the CMS- prepared in Example 20 was... g -LA was dissolved in pure water to prepare a CMS solution with a mass concentration of 20 mg / g. g -LA solution.

[0156] CMS- g - The LA solution was added to the CS solution and mixed thoroughly, maintaining a 1:1 mass ratio between the two solutions. 40 g of the mixture was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to form a liquid film of the predetermined thickness, and allowed to stand at 25 °C to solidify. The mixture was then irradiated under a UV curing lamp with the following conditions: wavelength 365 nm, light intensity 50 mW / cm². 2 The irradiation time was 10 minutes. During this process, CMS- was excited by ultraviolet light. g The dithiopentane structure of the LA side chain allows it to undergo ring-opening polymerization, forming a cross-linked network of CMS and polydisulfide compounds, within which CS molecules are locked. Therefore, the final product is a two-component hydrogel based on CS and CMS derivatives.

[0157] The polyester mold containing the hydrogel was placed in a constant temperature chamber and slowly air-dried at 30 °C. Finally, the film material was removed from the mold to obtain CS / CMS- g -LA hydrogel film.

[0158] Comparative Example 5

[0159] A CS / 4aPEG encapsulated with Cy5-BSA 20k The preparation method of CBA hydrogel film includes the following steps:

[0160] Cy5-BSA was directly dissolved in a CS solution with a mass concentration of 30 mg / g (pH=5.5, degree of deacetylation 72.7%) to obtain a drug-containing CS solution with a Cy5-BSA concentration of 2 mg / g and a CS concentration of 30 mg / g. The 4aPEG prepared in Example 6 was then used... 20k -CBA was dissolved in pure water to prepare a 60 mg / g 4aPEG solution. 20k -CBA solution.

[0161] 4aPEG 20k - Add the CBA solution to the CS solution containing the drug, mix thoroughly, and control the blending mass ratio of the two solutions to 1:1. Immediately pour 40 g of the mixed solution into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height), cast and spread it into a liquid film of the predetermined thickness, and let it stand at 25 ℃ to form a gel.

[0162] The polyester mold containing the hydrogel was placed in a constant temperature chamber, protected from light, and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold, yielding CS / 4aPEG encapsulated with Cy5-BSA. 20k -CBA hydrogel film.

[0163] Comparative Example 6-21

[0164] A method for preparing a CS / PEG-CBA hydrogel film includes the following steps:

[0165] Chitosan (CS) with a degree of deacetylation of 72.7% was dispersed in pure water, and 1 mol / L hydrochloric acid solution was added dropwise to aid dissolution. After complete dissolution, the pH was finely adjusted to 5.5 using 1 mol / L NaOH solution. Subsequently, the chitosan solution was concentrated to a concentration of 50 mg / g using a rotary evaporator (water bath temperature 50℃, vacuum degree 50 mbar, rotation speed 100 rpm), and then diluted to 30 mg / g with pure water.

[0166] The PEG-CBA prepared in Examples 1-10 was dissolved in pure water to prepare a PEG-CBA solution of 15-60 mg / g. The CS solution and the PEG-CBA solution were mixed at a mass ratio of 1:1. Then, 40 g of the mixed solution was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to form a liquid film of a predetermined thickness, and allowed to stand at 25 °C to form a gel. At this time, the aldehyde group at the end of the PEG-CBA undergoes a nucleophilic addition reaction with the amino group on the CS chain to form a Schiff base bond (-C=N-). The viscosity of the system increases sharply, and a gelation transition occurs, forming a two-component hydrogel based on CS and PEG derivatives.

[0167] The polyester mold containing the hydrogel was placed in a constant temperature chamber and slowly air-dried at 30 °C until a constant weight was reached. Finally, the film material was removed from the mold, yielding the CS / PEG-CBA hydrogel film.

[0168] The feed table for preparing the CS / PEG-CBA hydrogel film in Comparative Example 6-21 is shown in Table 8:

[0169] Table 8

[0170]

[0171] Figure 4 CS / PEG was prepared for Comparative Example 10 6k - A physical image of the CBA hydrogel and a schematic diagram of its network construction principle are shown. Taking the linear PEG derivative of Comparative Example 10 as an example, the relationship between chitosan and PEG is illustrated. 6k -CBA forms a dynamic cross-linked network through Schiff base reaction. Meanwhile, the physical image of Comparative Example 10 shows the transformation process from a flowing "sol state" to a non-flowing "gel state" after the precursor solution is blended, which intuitively confirms the feasibility and gelation ability of constructing the first cross-linked network using Schiff base chemical cross-linking.

[0172] Comparative Examples 22-25

[0173] A CS / PEG 6k The preparation method of CBA hydrogel film includes the following steps:

[0174] Chitosan (CS) with a degree of deacetylation of 72.7% was dispersed in pure water, and 1 mol / L hydrochloric acid solution was added dropwise to aid dissolution. After complete dissolution, the pH was finely adjusted to 5.5 using 1 mol / L NaOH solution. Subsequently, the chitosan solution was concentrated to a concentration of 50 mg / g using a rotary evaporator (water bath temperature 50℃, vacuum degree 50 mbar, rotation speed 100 rpm), and then diluted to 30 mg / g with pure water.

[0175] The PEG prepared in Example 2 6k CBA is dissolved in pure water to prepare a PEG solution of 15-60 mg / g. 6k -CBA solution, CS solution and PEG 6k -CBA solution was blended at a mass ratio of 1:1, and then 40 g of this mixture was immediately poured into a square polyester mold (12 cm × 12 cm × 4 cm, length × width × height) to form a liquid film of predetermined thickness, and allowed to stand at 37 ℃ to form a gel. At this point, PEG 6k The aldehyde group at the end of -CBA undergoes a nucleophilic addition reaction with the amino group on the CS chain to form a Schiff base bond (-C=N-), which causes the viscosity of the system to increase sharply and undergo a gelation transition, forming a two-component hydrogel based on CS and PEG derivatives.

[0176] The polyester mold containing the hydrogel was placed in a constant temperature oven and slowly air-dried at 30 °C until constant weight was achieved. Finally, the film material was removed from the mold, yielding CS / PEG. 6k -CBA hydrogel film.

[0177] The feed table for the preparation of CS / PEG-CBA hydrogel films in Comparative Examples 22-25 is shown in Table 9:

[0178] Table 9

[0179]

[0180] Test Example 1

[0181] Using nuclear magnetic resonance hydrogen spectroscopy (NMR) 1 ¹H-NMR spectrophotometry was used to examine chitosan, PEG-CBA, CMS derivative intermediates, and CMS- g The chemical structure of -LA was characterized; the relationship between CMS derivative intermediates and CMS- was analyzed by Fourier transform infrared spectroscopy (FT-IR). g The chemical composition and structural changes of LA were investigated; and Raman spectroscopy and X-ray photoelectron spectroscopy (XPS) were used to study CMS- g-LA and lyophilized samples of dual-enzyme responsive dual-network interpenetrating hydrogels were systematically characterized. Scanning electron microscopy (SEM) was used to observe the cross-sectional microstructure of the dual-enzyme responsive dual-network interpenetrating hydrogels and their films. The hydrogel cross-section characterization method was as follows: The prepared hydrogel was cut into 5-10 mm pieces, fully swollen and equilibrated in deionized water, and then excess surface moisture was quickly wiped off with filter paper. The sample was then immersed in liquid nitrogen for complete freeze-embrittlement and rapidly fractured along a predetermined direction in a liquid nitrogen environment to obtain a fresh and flat cross-section. The fractured sample was immediately transferred to a freeze dryer for freeze-drying to remove internal moisture and maintain the original microstructure. After freeze-drying, the sample cross-section was fixed on the sample stage with the cross-section facing upwards and sputtered with gold to improve conductivity. Finally, the hydrogel cross-sectional morphology was observed and photographed under an accelerating voltage of 3-10 kV. The characterization method for the thin film cross-section is as follows: the dried thin film sample is cut into a size of 5 mm × 10 mm and quickly immersed in liquid nitrogen for complete freeze-embrittlement; the thin film is broken or cut in liquid nitrogen environment to obtain a clear and flat cross-section; the brittle film sample cross-section is vertically fixed on the sample stage and sputtered with gold to enhance conductivity; finally, the cross-sectional thickness and internal microstructure of the thin film are observed and analyzed under an accelerating voltage of 3-10 kV.

[0182] Figure 5 Chitosan 1 H-NMR spectrum; where (A) represents chitosan with low degree of deacetylation. 1 H-NMR spectrum, degree of deacetylation 72.7%; (B) shows high degree of deacetylation chitosan raw material. 1 The 1H-NMR spectrum showed a degree of deacetylation of 85.3%. The test employed a mixed deuterated reagent (CF3COOD and D2O in a 1:9 volume ratio) to dissolve the sample. The degree of deacetylation of the two raw materials was accurately determined by calculating the integral area ratio of acetyl methyl protons to backbone protons. This data confirms the presence of sufficient free amino groups (-NH2) on both chitosan chains, providing ample active sites for the subsequent Schiff base reaction with the terminal aldehyde groups of PEG-CBA, and also providing a crucial structural basis for the specific recognition and degradation by lysozyme. Subsequent test examples further compared the specific lysozyme-responsive degradation behavior of hydrogel films prepared from these two chitosan raw materials with different degrees of deacetylation in a simulated saliva environment.

[0183] Figure 6 PEG prepared in Example 1 2k -CBA 1 H-NMR spectrum, Figure 7 The 4aPEG prepared in Example 6 20k of 1The H-NMR spectrum shows that the deuterated reagent used in the test was deuterated chloroform (CDCl3). Figure 6 The characteristic peak of aldehyde protons appeared near 10 ppm, and the characteristic peak of benzene ring protons appeared at 7.5-8.5 ppm. The integral ratio of the two peaks was in high agreement with the theoretical design, which fully confirms that p-aldehyde benzoic acid (CBA) has been successfully grafted onto linear PEG. 2k The molecular terminus marks the successful synthesis of a bifunctional macromolecular crosslinking agent. Figure 7 Spectral features and Figure 6 The high degree of similarity clearly demonstrates that the modification method of this invention can achieve efficient modification of aldehyde groups at the ends of multi-arm topological PEG structures. The above results indicate that this modification method has good universality and can precisely adjust the crosslinking density and mechanical properties of the final double-network interpenetrating hydrogel film by controlling the topological structure (number of arms) of the PEG.

[0184] Figure 8 Sodium carboxymethyl starch and CMS prepared in Example 15 g -ADH 1 The 1H-NMR spectrum was obtained. The deuterated reagent used in the test was D2O, and the test temperature was 70 °C. Figure 8 Comparison of sodium carboxymethyl starch and CMS- g -ADH 1 H-NMR characteristics, CMS- g The spectrum of ADH showed a characteristic proton peak belonging to the methylene group of ADH, directly confirming that the bridging agent ADH had been successfully covalently coupled to the carboxyl group of the side chain of carboxymethyl starch, thus constructing the necessary reaction "bridge" for the subsequent grafting of lipoic acid.

[0185] Figure 9 The CMS- prepared in Example 20 g -LA 1 The 1H-NMR spectrum was obtained. The deuterated reagent used in the test was D2O, and the test temperature was 70 °C. Figure 9 The characteristic proton peaks (such as the dithiopentane and methylene peaks) belonging to the lipoic acid side chain can be seen, directly confirming that lipoic acid has been successfully grafted onto the carboxymethyl starch backbone. This CMS- g -LA is the core material basis for constructing the second cross-linked network, providing the material with a high-strength framework and endowing it with specific responsive degradation characteristics to α-amylase.

[0186] Figure 10 Sodium carboxymethyl starch and CMS prepared in Example 15 g -ADH, CMS prepared in Example 20- g -FT-IR spectrum comparison of sodium carboxymethyl starch and CMS- gFT-IR spectrum comparison of -ADH, (B) sodium carboxymethyl starch and CMS- g -FT-IR spectrum comparison of LA. The tests were performed using the potassium bromide (KBr) pellet method at room temperature, with a wavenumber scan range of 400-4000 cm⁻¹. -1 By comparing the infrared characteristic peaks of sodium carboxymethyl starch and the product, the chemical composition and structural evolution of the sample were characterized. Figure 10 The successful functionalization modification of carboxymethyl starch was further verified. Compared with sodium carboxymethyl starch, the products of Examples 15 and 20 showed better performance at 1600-1750 cm⁻¹. -1 The characteristic absorption peaks of amide bonds in the wavenumber range were significantly enhanced, corroborating the formation of amide bonds during the grafting reaction. These infrared characterization results corroborate the conclusions of the proton NMR spectroscopy analysis, confirming that the bridging grafting of ADH and the subsequent coupling with lipoic acid were successfully completed.

[0187] Figure 11 The CMS- prepared in Example 20 g Raman spectra of -LA (bottom image) and the lyophilized sample of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 (top image). Tests were performed at room temperature using a 785 nm laser as the excitation source, with a scanning range of 50-4000 cm⁻¹. -1 Raman spectroscopy can specifically detect the presence state of sulfur, CMS- g -LA in the low wavenumber region (1000 cm⁻¹) -1 Below, especially 500-700 cm -1 The spectrum (range) exhibits multiple sharp characteristic scattering peaks, corresponding to the SS and CS bond vibrations of the high-strength dithiopentane structure in the lipoic acid side chain, strongly confirming that the active lipoic acid groups have been completely grafted onto the starch chain. However, in the cross-linked double-network interpenetrating hydrogel, these sharp characteristic peaks completely disappear. This spectroscopic change indicates that under ultraviolet light irradiation, the dithiopentane structure of the lipoic acid side chain undergoes ring-opening polymerization, the original cyclic structure is destroyed and transformed into a linear polydisulfide cross-linked network. The disappearance of the monomer characteristic peaks directly confirms the successful construction of the second cross-linked network, rather than simple physical doping.

[0188] Figure 12 The CMS- prepared in Example 20 g XPS full spectrum of lyophilized samples of LA and the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26; wherein, (A) is CMS- g- The XPS full spectrum of LA is shown in Figure 1. (B) is the XPS full spectrum of the lyophilized sample of the dual-enzyme responsive dual-network interpenetrating hydrogel. The tests were conducted under ultra-high vacuum conditions, using Al Kα rays as the excitation source. The binding energy was corrected using C 1s (284.8 eV) as the reference, and was used to analyze the elemental composition and chemical valence state of the sample surface. The tested elements included C, N, O, and S. Obvious S 2p and S 2s characteristic signal peaks were detected in the spectra of Examples 20 and 26, qualitatively and quantitatively confirming that sulfur was successfully introduced into the carboxymethyl starch and the final hydrogel system through the lipoic acid component. It should be noted that... Figure 12 The Na and Cl signal peaks appearing in B are due to residual sodium chloride generated during the neutralization reaction when dilute hydrochloric acid and dilute sodium hydroxide solution were used to adjust the pH of the system during the preparation of chitosan aqueous solution.

[0189] Test Example 2

[0190] The gelation time of hydrogels formed by crosslinking CS and PEG-CBA at different temperatures was tested to investigate the regulatory effect of temperature on hydrogel formation kinetics. All experimental groups (Comparative Examples 7-10 and 22-25) were performed in three independent replicates. Data are expressed as mean ± standard deviation (n=3). The gelation time was determined using the inverted observation method: the precursor solutions were mixed at a set mass ratio and incubated at the corresponding temperature. The time when no solution flow was observed immediately after inverting the sample tube was recorded as the gelation time.

[0191] Figure 13 The graphs show the gelation time data for the crosslinking of CS and PEG-CBA to form hydrogels at different temperatures. (A) shows the gelation time data for Comparative Examples 7-10 at 25 °C, and (B) shows the gelation time data for Comparative Examples 22-25 at 37 °C. The comparison shows that, at the same PEG-CBA concentration, the gelation time at 37 °C is significantly shorter than that at 25 °C. This indicates that the Schiff base crosslinking reaction rate has a significant temperature dependence: higher temperatures promote molecular chain segment movement, accelerate the nucleophilic addition reaction of amino and aldehyde groups, and thus rapidly construct a three-dimensional crosslinked network. This characteristic endows the film preparation process with excellent process controllability—the longer gelation time at room temperature (25 °C) provides a sufficient operating window, ensuring uniform spread of the precursor solution and avoiding uneven film thickness due to premature curing; while the rapid gelation rate simulating physiological temperature or the drying film-forming stage (37 °C and above) can effectively fix the film morphology and improve production efficiency. This confirms that the system of the present invention can precisely balance the processing life and curing efficiency of hydrogels by adjusting the ambient temperature.

[0192] Test Example 3

[0193] The gelation kinetics and self-healing properties of the hydrogel were characterized using a Thermo Fisher HAAKE MARS-40 rotational rheometer.

[0194] The gelation kinetics test method was as follows: the "modulus-time scan" method in oscillation mode was used to monitor the changes in storage modulus (G') and loss modulus (G') during the transition of the precursor solution from "sol state" to "gel state". The test conditions were: flat plate fixture (diameter 20 mm), fixture spacing 500 μm, temperature 37 ℃, oscillation frequency 1 Hz, strain 1%, and sample volume 250 μL.

[0195] The self-healing performance testing method involves performing a modulus-time scan on the fully gelled hydrogel. An alternating strain-time scan test is used, applying alternating low strain (1%) and high strain (1000%), recording the changes in G' and G” over time. Through multiple cyclic loading-unloading processes, the structural recovery ability and self-healing performance of the hydrogel after large deformation failure are evaluated. Test conditions are: a flat plate fixture (20 mm diameter), a fixture spacing of 500 μm, a temperature of 37 ℃, and a cylindrical sheet with a diameter of 2 cm and a thickness of 500 μm as the test sample. G' characterizes the material's elastic response, and G” characterizes the material's viscous response. The high strain stage is used to disrupt the internal network structure, while the low strain stage characterizes the network's recovery process.

[0196] Figure 14 The rheological time-scan curves of the CS / PEG-CBA hydrogels prepared in Comparative Examples 7-10 and 15-16 at 25 °C are shown. This test monitors the dynamic changes of G' and G" over time during the transition of the samples from the "sol state" to the "gel state", and intuitively reflects their gelation kinetics. Figure 14 The study revealed the sol-gel transition process of the hydrogels, showing that the G' of each sample rapidly surpassed G” over time and eventually reached a stable plateau. Simultaneously, the final equilibrium modulus of the hydrogels showed a significant increasing trend with increasing crosslinking agent concentration. These results, from a microscopic viscoelastic perspective, fully demonstrate that the Schiff base reaction can drive the formation of a stable three-dimensional crosslinked network, and that the strength of the hydrogel network can be precisely controlled by adjusting the amount of crosslinking agent in the formulation.

[0197] Figure 15 CMS- prepared for Comparative Example 3 g - Rheological self-healing performance characterization spectra of LA hydrogel and the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26; wherein, (A) is CMS- g -LA hydrogel, (B) is a dual-enzyme responsive dual-network interpenetrating hydrogel. From Figure 15 As can be seen from the data, the CMS- prepared in Comparative Example 3... g-LA hydrogel, even under ultra-high shear strain of 1000%, did not exhibit a sharp decrease in G' and G" due to "liquefaction," but rather showed an upward trend, maintaining a gel state where G' > G" throughout, demonstrating a significant "strain hardening" phenomenon. This result confirms that the second cross-linked network possesses excellent structural integrity and toughness, maintaining its three-dimensional topological structure without collapse under extreme tensile conditions, providing a solid skeletal support for the dual-network material. The dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 exhibited excellent cyclic self-healing behavior: when a large strain of 1000% was applied, the hydrogel modulus dropped sharply, and G' and G" tended to overlap or approach each other, indicating that the first cross-linked network and some dynamic disulfide bonds preferentially dissociated as sacrificial units, confirming the necessity of the second cross-linked network in maintaining the overall morphology of the material; and when the strain recovered to 1%, G' and G" could quickly rebound to their initial levels, and this "damage-recovery" process was highly repeatable. This behavior manifests macroscopically as a reversible transformation where the material exhibits a "sol state" under large deformation conditions and reverts to a "gel state" under small deformation conditions. A comprehensive comparison reveals that the dual-network interpenetrating design of this invention cleverly combines the dynamic bonding characteristics of Schiff base bonds and disulfide bonds: under external force, the dynamic bonds dissipate energy through reversible breakage, protecting the material matrix; after the external force is removed, the dynamic bonds rapidly recombine in situ to achieve structural repair. This mechanism enables the film to achieve high structural strength through the second cross-linking network and possess excellent cyclic self-healing capabilities through the synergistic effect of the dual dynamic networks, perfectly adapting to the complex mechanical movements in the oral cavity environment.

[0198] Figure 16 The images show a comprehensive characterization of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, as well as physical images of the hydrogels prepared in Comparative Example 16 and Example 26. Specifically, (A) is a physical image of the extruded gel, (B) is a physical image of the gel adhesion, (C) is an FT-IR image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, (D) is a SEM image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26, (E) is a physical image of the CS / PEG-CBA hydrogel prepared in Comparative Example 16 in a square polyester mold, (F) is a physical image of the CS / PEG-CBA hydrogel prepared in Comparative Example 16 stably spread in the mold after inversion, (G) is a physical image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 in a square polyester mold, and (H) is a physical image of the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 stably spread in the mold after inversion. Figure 16 As can be seen from (A) and (B), the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 exhibits excellent compression resistance, which allows it to quickly recover its shape under external pressure, and strong adhesion in a humid environment, demonstrating its durability and fit in physiological environments such as the oral cavity. Figure 16(C) further confirmed the chemical composition and structure of the hydrogel; from Figure 16 As can be seen in (D), the hydrogel exhibits a regular, interconnected honeycomb-like porous structure. This three-dimensional interconnected channel provides a crucial physical pathway for the loading, diffusion, and controlled release of macromolecular drugs. Figure 16 As can be seen from (E), (F), (G), and (H), the gelation and spreading behavior of the CS / PEG-CBA hydrogel prepared in Comparative Example 16 and the dual-enzyme responsive dual-network interpenetrating hydrogel prepared in Example 26 in a square polyester mold are both stable in the mold and do not fall off when inverted, which confirms that the dual-network interpenetrating structure has good moldability and structural stability.

[0199] Test Example 4

[0200] The dual-enzyme responsive dual-network interpenetrating hydrogel film prepared in Example 26, the CS film prepared in Comparative Example 1, and the CS / CMS- prepared in Comparative Example 4 were compared. g Mechanical properties were tested on the LA hydrogel film and the CS / PEG-CBA hydrogel film prepared in Comparative Example 16, and the microstructure of the dual-enzyme responsive dual-network interpenetrating hydrogel film prepared in Example 26 was characterized. The tests were performed using a room temperature universal testing machine and SEM. For the mechanical tensile test, rectangular film sample strips with a length of 8 cm, a width of 4 cm, and a thickness of 80-120 μm were selected and tested at a tensile rate of 4 mm / min. Photographs of the hydrogel film of Comparative Example 16 in the initial tensile state and the tensile state were taken, and tensile stress-strain curves of four sets of samples were plotted.

[0201] Figure 17 The two enzyme-responsive dual-network interpenetrating hydrogel films prepared in Example 26, the CS films prepared in Comparative Example 1, and the CS / CMS films prepared in Comparative Example 4 are described. g The mechanical properties and microstructure of the CS / PEG-CBA hydrogel film prepared in Example 26 and Comparative Example 16 are comprehensively characterized. (A) is a photograph of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 in its initial tensile state; (B) is a photograph of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 after tensile testing; (C) is a cross-sectional SEM image of the dual-enzyme responsive dual-network interpenetrating hydrogel film prepared in Example 26 after liquid nitrogen freezing and brittle fracture; (D) is the tensile stress-strain curve of the hydrogel films prepared in Example 26, Comparative Example 1, Comparative Example 4, and Comparative Example 16. Figure 17As can be seen in (D), compared with the hydrogel films prepared in Comparative Examples 1, 4 and 16, the dual-enzyme responsive dual-network interpenetrating hydrogel film prepared in Example 26 has excellent tensile strength and elongation at break, and the dense structure shown in the cross-sectional SEM image further confirms the material densification and mechanical enhancement effect brought about by the dual-network interpenetrating structure.

[0202] Test Example 5

[0203] The two enzyme-responsive dual-network interpenetrating hydrogel film prepared in Example 26, the CS film prepared in Comparative Example 1, and the CS / 4aPEG film prepared in Comparative Example 2 were compared. 20k -CBA hydrogel film and CMS prepared in Comparative Example 3- g -LA hydrogel films were subjected to in vitro degradation tests to evaluate their degradation behavior in a simulated oral enzymatic environment. The in vitro degradation test method was as follows: Fusayama-Meyer simulated saliva was prepared according to the pharmacopoeia formulation. First, 0.40 g potassium chloride (KCl), 0.40 g sodium chloride (NaCl), 0.795 g calcium chloride dihydrate (CaCl2·2H2O), and 0.69 g sodium dihydrogen phosphate monohydrate (NaH2PO4·H2O) were added sequentially to 800 mL of deionized water, and magnetic stirring was used to ensure complete dissolution of all salts. Subsequently, the pH was precisely adjusted to 6.8 using 0.1 mol / L dilute hydrochloric acid solution and 0.1 mol / L dilute NaOH solution. Finally, the volume was accurately adjusted to 1000 mL using deionized water. α-Amylase was added to fresh simulated saliva at a concentration of 20 U / mL to obtain simulated saliva containing α-amylase. Lysozyme was added to fresh simulated saliva at a concentration of 20 μg / mL to obtain simulated saliva containing lysozyme. α-Amylase and lysozyme were then added sequentially to fresh simulated saliva at concentrations of 20 U / mL and 20 μg / mL, respectively, to obtain dual-enzyme simulated saliva containing both α-amylase and lysozyme. Fresh simulated saliva (without enzymes) and simulated saliva containing enzymes were used as degradation solutions for hydrogel films. The films were cut into rectangular strips (length × width = 2 cm × 1 cm, thickness 100 μm), and the strips were immersed in the degradation solution in small glass vials, using 5 mL of degradation solution per 0.2 g of film. At set time points, all degradation solution was removed from the small glass vials used in the film degradation experiment, and the remaining weight was weighed. A degradation curve was plotted based on the percentage of the remaining weight of the initial film. The degradation experiment was conducted in a constant temperature shaking chamber at 37°C and a shaking rate of 100 rpm. Each experimental group was repeated three times independently, and the data are expressed as mean ± standard deviation (n = 3).

[0204] Figure 18The two enzyme-responsive dual-network interpenetrating hydrogel films prepared in Example 26, the CS films prepared in Comparative Example 1, and the CS / 4aPEG films prepared in Comparative Example 2 are as follows: 20k -CBA hydrogel film, CMS prepared in Comparative Example 3- g - Degradation curves of the CS / PEG-CBA hydrogel film prepared in Comparative Example 16 and Comparative Example 26 in simulated saliva; wherein, (A) is the degradation curve of the film of Comparative Example 1 and Comparative Example 16 in simulated saliva containing lysozyme, (B) is the degradation curve of the film of Comparative Example 2 and Comparative Example 16 in simulated saliva containing lysozyme, (C) is the degradation curve of the film of Comparative Example 3 in simulated saliva containing α-amylase and without α-amylase, and (D) is the degradation curve of the film of Comparative Example 3 in simulated saliva containing α-amylase, the film of Example 26 in simulated saliva containing α-amylase and simulated saliva containing both α-amylase and lysozyme. Figure 18 This confirms the unique dual-enzyme responsiveness of the hydrogel film of the present invention, as well as the tunable nature of the response mechanism and degradation rate of the first and second cross-linking networks to specific enzymes. From... Figure 18 As can be seen in Figure (A), in simulated saliva containing lysozyme, the film of Comparative Example 16 exhibits significant mass loss and degradation behavior, while the pure CS film degrades slowly under the same conditions. This indicates that by introducing hydrophilic PEG segments to construct a cross-linking network, the hydration state of chitosan segments and the accessibility of enzyme contact are effectively improved, giving it good lysozyme responsiveness. Figure 18 In (B), the regulatory mechanism of this responsiveness was further explored. By comparison, it was found that the hydrogel film prepared with chitosan of high deacetylation degree (85.3%) (Comparative Example 2) had a significantly lower degradation rate than the film prepared with chitosan of lower deacetylation degree (72.7%) in Comparative Example 16. This phenomenon is highly consistent with the mechanism of action of lysozyme specifically hydrolyzing acetylglucosamine residues, which strongly confirms that the present invention can precisely regulate the biodegradation cycle of the first cross-linking network by changing the deacetylation degree of chitosan raw material. Figure 18 (C) Independent validation was performed on the second crosslinking network, and the results showed that the CMS- of Comparative Example 3... g The LA hydrogel film maintained a relatively constant mass without significant degradation in simulated saliva without α-amylase for 12 hours, demonstrating excellent structural stability. However, upon the addition of α-amylase, the hydrogel film experienced a precipitous decrease in mass and complete degradation within a short period. This "stability-mutation" binary behavior convincingly demonstrates the structural stability of CMS- g The -LA network exhibits highly specific and sensitive α-amylase-responsive switching function, providing another layer of protection for the intelligent degradation of the dual-network system in the complex oral environment. Figure 18 (D) reveals the synergistic degradation mechanism of the dual-network interpenetrating structure (Example 26). In simulated saliva containing only α-amylase, although the carboxymethyl starch component undergoes hydrolysis, the first cross-linked network is insensitive to the enzyme and acts as a "skeleton" to effectively prevent the overall disintegration of the material. As a result, its degradation rate and degree are significantly lower than those of the single-network comparative example 3, exhibiting a stable state of "semi-degradation". Only when α-amylase and lysozyme are present in the system at the same time (i.e., simulating the real oral environment) is the dual-network skeleton simultaneously sheared and destroyed, the interpenetrating structure is unlocked, and the material exhibits rapid and thorough degradation behavior (mass residue rate approaches 0). This confirms that a tight topological interpenetration and locking effect is formed between the first and second cross-linked networks.

[0205] Test Example 6

[0206] The two-enzyme responsive double-network interpenetrating hydrogel film loaded with Cy5-BSA prepared in Example 36 and the CS / 4aPEG film loaded with Cy5-BSA prepared in Comparative Example 5 were compared. 20k The drug-release capacity of the Cy5-BSA hydrogel film was tested using the following method: Enzyme-free and enzyme-containing simulated saliva prepared in Test Example 5 were used as release solutions for the drug-loaded hydrogel film. The drug-loaded hydrogel film was cut into rectangular strips (length × width = 2 cm × 1 cm, thickness 100 μm). The strips were immersed in the release solution in small glass vials, with 5 mL of release solution used for every 0.2 g of film. At set time points, 200 μL of release solution was collected, and the same volume of fresh release solution was added. The collected Cy5-BSA release solution was subjected to fluorescence emission spectroscopy to calculate the cumulative drug release amount, and Cy5-BSA release curves of the drug-loaded hydrogel film in different media were plotted. All release experiments were conducted in a constant-temperature shaking chamber at 37 ℃ and a shaking rate of 100 rpm. Each experimental group underwent three independent replicate experiments, and the data are expressed as mean ± standard deviation (n = 3).

[0207] Figure 19 The two-enzyme responsive double-network interpenetrating hydrogel film loaded with Cy5-BSA prepared in Example 36 and the CS / 4aPEG film loaded with Cy5-BSA prepared in Comparative Example 5 are compared. 20k-CBA hydrogel film drug cumulative release curves in different media; wherein, (A) is the drug cumulative release curve of the drug-loaded hydrogel film of Example 36 in simulated saliva containing α-amylase and lysozyme, and the drug cumulative release curve of the drug-loaded hydrogel film of Comparative Example 5 in simulated saliva containing α-amylase and lysozyme and in an enzyme-free environment (enzyme-free simulated saliva); (B) is the drug cumulative release curve of the drug-loaded hydrogel film of Example 36 in an enzyme-free environment (enzyme-free simulated saliva), simulated saliva containing only α-amylase, and simulated saliva containing α-amylase and lysozyme. From Figure 19 As shown in Figure (A), comparing the release curves of the drug-loaded hydrogel films of Example 36 and Comparative Example 5 under a dual-enzyme environment, the cumulative drug release rate of the dual-network film is significantly higher than that of the single-network system. This phenomenon indicates that the construction of the interpenetrating dual networks is not a simple physical superposition, but rather a significant improvement in the gel's response and release sensitivity to the complex enzymatic environment of the oral cavity through the synergistic sensing of the two components by the two enzymes, overcoming the shortcomings of the single-network system such as sluggish response and incomplete release. Figure 19 As can be further seen in (B), this stable structure undergoes a rapid mutation from "physical restriction" to "enzymatic disintegration" upon the introduction of two enzymes, causing the release rate of the macromolecular drug (Cy5-BSA) to surge from an extremely low level (approximately 20%) in the enzyme-free state to over 90% within 12 hours. This significant difference from "highly locked-in state in an enzyme-free environment" to "explosive release in a dual-enzyme environment" not only demonstrates that the present invention possesses a "smart erosion-release" mechanism triggered by specific enzymes, but also highlights the significant technological advancements of the dual-network interpenetrating structure in achieving precise drug delivery, strict control of unnecessary release, and improved biological response efficiency.

[0208] This invention confirms the relationship between the key intermediate PEG-CBA and CMS- through structural characterization using methods such as proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, Raman spectroscopy, and X-ray photoelectron spectroscopy. gThe successful synthesis and precise structural design of LA strongly validated the chemical mechanism of dithiopentane ring-opening polymerization to form a second cross-linked network. Based on the temperature dependence of the Schiff base reaction, flexible control of hydrogel gelation kinetics can be achieved, providing good controllability for the molding and processing of oral films. Combined with rheological and mechanical property analysis, it is evident that the dynamic Schiff base network, acting as a sacrificial unit, effectively dissipates external force energy and forms an interpenetrating synergistic effect with the highly stable photocrosslinked network. This results in materials possessing excellent cyclic self-healing properties, outstanding tensile strength and toughness, significantly improving the technical problems of brittleness and poor mechanical properties in traditional polysaccharide-based films. Further degradation behavior and drug release studies show that this system exhibits typical "structural interlocking" and dual-enzyme response characteristics: it can maintain structural stability under a single enzyme environment, reducing non-specific drug leakage; under a dual-enzyme environment simulating oral lesions, the dual-network framework can synchronously respond to degradation, achieving sensitive and controllable drug release. This invention fully demonstrates through multi-dimensional characterization and performance testing that the dual-enzyme responsive dual-network interpenetrating hydrogel film has high structural stability and high response sensitivity, and possesses significant technical advantages and broad application prospects in the field of biomedical materials.

[0209] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. Those skilled in the art should understand that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a dual-enzyme-responsive dual-network interpenetrating hydrogel film, characterized in that, Includes the following steps: (1) Polyethylene glycol was esterified with p-aldehyde benzoic acid, and after purification, a terminal aldehyde-modified polyethylene glycol derivative was obtained. (2) Under the condition of activator, carboxymethyl starch reacts with diamine or dihydrazide bridging agent to obtain carboxymethyl starch derivative intermediate. After activation with lipoic acid, the carboxymethyl starch derivative intermediate is reacted and purified to obtain photosensitive carboxymethyl starch derivative. The carboxymethyl starch is in sodium salt form with a degree of substitution of 0.1-0.

3. The diamine or dihydrazide bridging agent is 1,2-ethylenediamine, 1,6-hexanediamine, cystamine, 3,3'-dithiobis(propionylhydrazine) or adipic acid dihydrazide. The activator is 1-(3-dimethylamine) The lipoic acid is prepared by using a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide, or a combination of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxythiosuccinimide. The method for activating lipoic acid is as follows: lipoic acid is dissolved in dimethyl sulfoxide, and a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxysuccinimide or a combination of 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and N-hydroxythiosuccinimide is added as an activator. The carboxyl group is activated at 4-20 °C for 2-8 h. The molar ratio of lipoic acid to diamine or dihydrazide bridging agent is (1.5-2.5):

1. (3) Dissolve chitosan in an acidic aqueous solution and adjust the pH of the system to 5.0-6.0 to obtain a chitosan solution; add the terminal aldehyde-modified polyethylene glycol derivative prepared in step (1) and the photosensitive carboxymethyl starch derivative prepared in step (2) to the chitosan solution in sequence to obtain a mixed solution; the concentration of chitosan in the mixed solution is 0.5-5.0 wt%; the degree of deacetylation of the chitosan is 70%-90%; the concentration of the terminal aldehyde-modified polyethylene glycol derivative in the mixed solution is 0.1-15 wt%; the concentration of the photosensitive carboxymethyl starch derivative in the mixed solution is 0.1-3.0 wt%; (4) After the mixed solution obtained in step (3) is cast and spread, it is allowed to stand. The first cross-linking network is formed by Schiff base reaction, and then the disulfide bond cross-linking is initiated by ultraviolet light irradiation to construct the second cross-linking network, thus obtaining a dual-enzyme responsive dual-network interpenetrating hydrogel. (5) The dual-enzyme responsive dual-network interpenetrating hydrogel obtained in step (4) is dried and dehydrated to obtain the dual-enzyme responsive dual-network interpenetrating hydrogel film.

2. The preparation method according to claim 1, characterized in that, In step (1), the polyethylene glycol is selected from one or more of linear polyethylene glycol, tetra-arm polyethylene glycol, and octa-arm polyethylene glycol; the molecular weight of the linear polyethylene glycol is 2000-20000 Da; the molecular weight of the tetra-arm polyethylene glycol is 10000-40000 Da; the molecular weight of the octa-arm polyethylene glycol is 10000-40000 Da; and the molar ratio of the terminal hydroxyl group of the polyethylene glycol to p-aldehyde benzoic acid is 1:(2-10).

3. The preparation method according to claim 1, characterized in that, In step (1), polyethylene glycol is dissolved in a solvent, p-aldehyde benzoic acid and a catalyst are added, and the mixture is stirred at 20-30 °C for 24-72 h to obtain a terminal aldehyde-modified polyethylene glycol derivative.

4. The preparation method according to claim 1, characterized in that, In step (2), the molar ratio of the diamine or dihydrazide bridging agent to the carboxyl group of carboxymethyl starch is (4-6):1; the molar ratio of the carboxyl group of carboxymethyl starch, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 1:(1.5-2.5):(1.5-2.5); the molar ratio of the carboxyl group of carboxymethyl starch, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxythiosuccinimide is 1:(1.5-2.5):(1.5-2.5).

5. The preparation method according to claim 1, characterized in that, In step (4), the pre-shaped gel that forms the first cross-linked network is placed under a UV curing lamp for irradiation. The irradiation conditions are a wavelength of 365 nm and a light intensity of 10-50 mW / cm². 2 Irradiation time: 5-60 min.

6. A dual-enzyme responsive dual-network interpenetrating hydrogel film prepared by the preparation method according to any one of claims 1-5.

7. The use of the dual-enzyme responsive dual-network interpenetrating hydrogel film of claim 6 in the preparation of oral mucosal drug delivery formulations.

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