A responsive sustained-release bilayer film for treating oral ulcers and a preparation method thereof

Abstract

The application belongs to the field of oral ulcer film treatment, and specifically discloses a responsive sustained-release double-layer film for treating oral ulcer and a preparation method thereof. The double-layer film comprises an inner starch layer and an outer pectin layer, wherein the starch layer is loaded with pyrogallol-zinc nanozyme (PA-Zn), and the mass concentration of PA-Zn in the starch layer is 0.2%-0.6%. The outer pectin layer contains glycerol and a calcium chloride solution. In the preparation, a starch solution containing PA-Zn is cast into a film, and then the film is covered with a pectin solution after being semi-dried, and is subjected to drying and humidity balancing treatment to form the film. The film realizes the responsive sustained release of PA-Zn through the specific degradation of the starch layer by salivary amylase (in vitro simulation release time: 1-72 hours), has excellent mechanical properties (tensile strength: 0.5-13.1 N / mm, adhesive strength: 0.5-1.16 N), and high biological safety (no significant difference in organ pathology score compared with the healthy group), and is suitable for oral ulcer treatment drugs or medical devices.

Description

Technical Field

[0001] This invention belongs to the field of oral ulcer film treatment, and specifically discloses a responsive sustained-release bilayer film for treating oral ulcers and its preparation method. Background Technology

[0002] 1.1 Overview of Biofunctional Materials

[0003] Biofunctional materials are a class of special materials possessing bioactivity, biocompatibility, or the ability to interact with biological systems. Their functional design focuses on the needs of life sciences, medicine, and environmental protection. These materials play a crucial role in medicine, agriculture, and bioengineering by mimicking biological structures, responding to biological signals, or participating in biological processes. Their core characteristics revolve around biocompatibility, function orientation, and dynamic responsiveness, while simultaneously integrating the interdisciplinary needs of engineering and life sciences. They possess features such as biocompatibility, function orientation, dynamic responsiveness, biodegradability, biomimicry, multifunctional integration, intelligent interface characteristics, and green sustainability. Currently, research on biofunctional materials is booming, with numerous innovative achievements emerging globally. In the field of tissue engineering, researchers have successfully constructed highly biomimetic artificial organs and tissue models using biofunctional materials, such as 3D-printed heart valves and liver tissue, providing new possibilities for organ transplantation and disease treatment. Meanwhile, intelligent responsive biomaterials have become a research hotspot. These materials can sense changes in their surrounding environment and respond accordingly, such as changing their shape or releasing drugs when temperature or pH changes, greatly improving the accuracy of drug delivery and therapeutic efficacy. Furthermore, the application of biofunctional materials in environmental protection is becoming increasingly widespread. For example, the development of biodegradable plastics has effectively alleviated the environmental pollution problems caused by traditional plastics; while in the energy sector, artificial photosynthetic systems that mimic photosynthesis have opened up new paths for the development of clean energy. However, despite significant progress, biofunctional materials still face many challenges. Issues such as the long-term stability of materials, biosafety, and the controllability of large-scale production still need further resolution. In the future, with the continuous development of cutting-edge technologies such as synthetic biology and nanomedicine, research on biofunctional materials will become more in-depth, and their application prospects in fields such as medical health, environmental protection, and sustainable energy will be even broader, potentially bringing a more profound impact on human society. In conclusion, as an emerging interdisciplinary field, biofunctional materials are leading the future development direction of materials science with their unique charm and enormous potential. With the continuous deepening of research and technological innovation, it is believed that biofunctional materials will create a better life for humankind in the near future.

[0004] 1.2 Classification of Biofunctional Materials

[0005] 1.2.1 Film

[0006] Thin-film technology in the biomedical field represents a significant innovative direction at the intersection of materials science and medicine. Through functional material layers with thicknesses ranging from nanometers to micrometers, it enables core medical functions such as disease diagnosis, drug delivery, and tissue repair. Its core applications encompass four main areas: In drug delivery systems, starch-based biomaterials, with their biodegradability and biocompatibility, have become ideal alternatives to traditional synthetic polymers. For example, cross-linked starch films can achieve sustained release of protein drugs such as insulin by controlling the rate of starch hydrolysis; their porous structure can also load antibacterial agents (such as gentamicin) for the treatment of infected wounds. Transdermal patches combined with microneedle array films can overcome the skin barrier, providing a painless delivery solution for macromolecular drugs such as insulin. In the field of biosensors, electrochemical bioelectrodes constructed from graphene-modified indium tin oxide (ITO) films can monitor blood glucose and tumor markers in real time, while surface plasmon resonance (SPR) technology achieves highly sensitive detection of viral antigens through changes in film thickness. In tissue engineering, 3D printing combined with electrospinning produces collagen / chitosan porous membranes that mimic the extracellular matrix, promoting tissue regeneration. Bilayer artificial skin substitutes have been used for burn wound repair, and coatings containing antibacterial silver nanoparticles effectively prevent infection. In diagnostic technology, rapid test strips composed of nitrocellulose membranes and colloidal gold-labeled antibodies are widely used for early pregnancy and COVID-19 antigen self-testing, while PDMS microfluidic chips combined with antibody-coated membranes enable simultaneous multi-indicator analysis of minute samples. Key technological breakthroughs are reflected in the development of biomimetic films, such as erythrocyte membranes encapsulating nanoparticles to achieve immune escape; cationic polymer films serving as gene delivery carriers targeting specific cells; and titanium dioxide and silver nanoparticle composite coatings possessing both photocatalytic antibacterial and long-lasting bactericidal functions.

[0007] However, this field still faces challenges such as biocompatibility optimization, precise control of degradation cycles, and large-scale production of medical-grade films. Future trends will focus on intelligent response systems (such as magnetic field / photo-controlled drug release), organoid culture chips, and personalized film design based on patient data, including Abbott's non-invasive blood glucose monitoring sensor, Medtronic's drug-eluting scaffold, and MIT's flexible electronic skin. These innovations are driving the evolution of biomedical films from passive functions to intelligent and precise technologies, deeply integrating artificial intelligence and synthetic biology, and bringing revolutionary breakthroughs to personalized medicine, regenerative medicine, and point-of-care diagnostics.

[0008] 1.2.2 Application of film in the treatment of oral ulcers

[0009] In recent years, oral dispersible films (ODFs), as a personalized drug delivery system, have been gradually replacing traditional treatments for oral diseases. Their unique feature is their ability to exert effects both locally and systemically. ODFs are ultra-thin, postage stamp-sized, portable, patient-friendly, and hydrophilic films that dissolve and disintegrate within seconds on the tongue or in the oral cavity, without the need for water or swallowing. This method of administration is particularly common in pediatric, geriatric, and special patient groups with swallowing difficulties. The thin and small nature of ODFs results in a relatively low drug loading capacity.

[0010] 1.3 Scheme of the prior art closest to the present invention

[0011] Yujie Yan et al. successfully developed a thin, lightweight bilayer oral film based on FDA-approved excipients using a simple method, exhibiting excellent adhesion and outstanding biocompatibility under moist oral conditions. It consists of an adhesive layer for in-situ anchoring to deliver the drug and a hydrophobic layer for isolating the drug from saliva for unidirectional drug delivery. The bilayer oral film has an extremely thin profile (only 0.11 mm thick) and excellent adhesion (up to 150 minutes in oral ulcers in SD rats), which also matches its drug release time (87.47% release within 2 hours).

[0012] Shu-Yin Liu et al. reported a solvent casting technique for preparing eugenol and borax-containing ODF (EB-ODF) for the treatment of oral ulcers. EB-ODF is composed of vinylpyrrolidone / vinyl acetate copolymer (… The film-forming agent was composed of VA64 and hydroxypropyl methylcellulose (HPMC-K250), with eugenol and borax loaded in it. The resulting EB-ODF film had a thickness of 0.119±0.001 mm and a tensile strength of 13.1±1.1 N / mm (>0.05). The prepared film disintegrated in the oral cavity within 30 seconds, and more than 90% of the eugenol was released from the film within the first 5 minutes. It also showed significant synergistic antibacterial properties against both Gram-negative and Gram-positive bacteria. In a constructed rat model study, the EB-ODF treatment group showed a 100% reduction in ulcer area after 10 days of treatment (>0.05), and a 38.7% reduction in oral ulcer area compared to the control group (<0.0001). The EB-ODF treatment group showed minimal oral irritation, scoring only 1 point and having a 65% preference in the taste test (<0.0001).

[0013] Zhongchao Wang et al. developed a mussel-inspired adhesive hydrogel by grafting catechol onto hyaluronic acid (C-HA) and adding dopamine for oxidative prepolymerization to form modified hyaluronic acid (M-HA), which significantly increased the hydrogel's adhesiveness. The M-HA was then infiltrated into a gelatin methacryloyl (GelMA) network. Chlorhexidine gluconate (CHG) was then incorporated into the hydrogel, enhancing its usability and therapeutic efficacy through its sustained-release capability. The GelMA / M-HA hydrogel exhibited strong adhesion to wet tissues, antibacterial and anti-inflammatory properties, and good biocompatibility. In rat oral ulcers and infected wounds, the adhesive hydrogel significantly accelerated the healing of ulcers and infected wounds.

[0014] Mengyu Chen et al. developed an injectable, photoinduced, in-situ enhanced hydrogel for oral ulcer repair (named GIL2) by incorporating dynamic phenylboronic acid ester bonds and imidazole ions into a methacrylated gelatin matrix. GIL2 exhibits rapid gelation (3s), low swelling (1.07 g / g), strong tensile strength (56.83 kPa), and high adhesive strength (63.38 kPa), enabling it to effectively adhere to the ulcer surface. Furthermore, GIL2 exhibits inherent antibacterial and antioxidant properties. In a diabetic rat model of oral ulcers, GIL2 effectively alleviated oxidative stress and reduced inflammation in the ulcer wound, thereby significantly accelerating the healing process of these ulcers.

[0015] Xi Chen et al. formulated a viscous coagulant containing tea polyphenols (TP) based on the adhesive properties observed in *Sarcasmella fusarium*. The coagulant, composed of Pluronic F68 (F68) and TP, was synthesized via a coagulation reaction. The F68-TP coagulant readily adhered to pig skin. It also reduced bacterial activity and possessed the ability to scavenge reactive oxygen species. In animal ulcer models, these coagulants exhibited anti-inflammatory effects and enhanced collagen and muscle fiber synthesis.

[0016] Traditionally, ODFs have been primarily used for low-dose delivery of potent drugs for conditions such as asthma, cardiovascular problems, central nervous system disorders, and mental health. To maximize the advantages of ODFs in treating oral diseases, the drug dosage within the ODF can be increased by increasing the film size or thickness or by employing multilayer fabrication. However, this approach may lead to longer disintegration times and potential issues with patient compliance. There is a need to achieve higher drug absorption efficiency and faster disintegration times while maintaining a relatively low drug loading in the film. Summary of the Invention

[0017] To address the above-mentioned problems, this invention discloses a responsive sustained-release bilayer membrane for treating oral ulcers and its preparation method.

[0018] To achieve the above objectives, the present invention includes the following technical solutions:

[0019] A responsive sustained-release bilayer membrane for treating oral ulcers, comprising:

[0020] The inner starch layer is loaded with pyrogallol-zinc nanozyme (PA-Zn), wherein the mass concentration of PA-Zn is 0.2%-0.6% of the starch layer;

[0021] The outer pectin layer covers the surface of the starch layer;

[0022] The bilayer membrane achieves responsive sustained release of PA-Zn by degrading the starch layer with salivary amylase.

[0023] Furthermore, in the aforementioned responsive sustained-release bilayer membrane for treating oral ulcers, the preparation method of the PA-Zn includes:

[0024] Dissolve pyrogallol and zinc acetate dihydrate in water at a mass ratio of 1:0.5-1.5 (preferably 1.15:1);

[0025] Adjust the pH to 4.0-6.0 (preferably 5.0) using a 5-10 mol / L (preferably 8 mol / L) KOH solution;

[0026] The hydrothermal reaction is carried out at 100-150℃ (preferably 120℃) for 6-24 hours (preferably 12 hours).

[0027] Furthermore, in the aforementioned responsive sustained-release bilayer membrane for treating oral ulcers, the starch layer is prepared by gelatinizing a 5-10% (preferably 8%) starch solution in a water bath at 70-100°C (preferably 80°C) for 20-40 minutes (preferably 30 minutes).

[0028] Furthermore, in the aforementioned responsive sustained-release bilayer membrane for treating oral ulcers, the raw material for the pectin layer is a pectin solution: it is prepared by dissolving pectin in water, adding glycerin and calcium chloride solution, and then stirring until homogeneous.

[0029] Furthermore, the above-mentioned responsive sustained-release bilayer membrane for treating oral ulcers, wherein the preparation method of the bilayer membrane includes:

[0030] The starch solution containing PA-Zn was cast into a film and dried at room temperature until it was semi-dry.

[0031] A pectin solution is cast onto the surface of the starch layer and dried at room temperature for 12-48 hours (preferably 24 hours).

[0032] Equilibrate at 20-30℃ (preferably 25℃) and 40-60% humidity (preferably 50% RH) for 24-72 hours (preferably 48 hours).

[0033] Furthermore, in the aforementioned responsive sustained-release bilayer membrane for treating oral ulcers, the PA-Zn is released in simulated saliva over a period of 1-72 hours, and SOD enzyme activity reaches its peak within 1.5 hours (based on...). Figure 9 data).

[0034] Furthermore, the mechanical properties of the aforementioned responsive sustained-release bilayer membrane for treating oral ulcers meet the following requirements:

[0035] Tensile strength: 0.5-13.1 N / mm (based on...) Figure 4 data);

[0036] Adhesion strength: 0.5-1.16 N (tested using wet pigskin to simulate oral mucosa, based on...) Figure 4 data).

[0037] This invention also discloses the application of the above-mentioned double-layer membrane in the preparation of oral ulcer treatment drugs.

[0038] Furthermore, in the above applications, the double-layer membrane reduces the ulcer area by 38.7%-100% within 5 days (based on background technology and...). Figure 11 (Data); by downregulating TNF-α levels to 30-70% of the control group (based on...) Figure 13 (19 data).

[0039] The present invention also discloses a medicament / medical device for treating oral ulcers, comprising the double membrane described in any one of the above claims, and the medicament / medical device is configured as follows:

[0040] It adheres to the oral mucosal wound;

[0041] PA-Zn sustained release is triggered by salivary amylase;

[0042] Daily changeable design (based on) Figure 10 Animal experimental procedures).

[0043] Furthermore, the aforementioned drugs / medical devices possess the following quantifiable properties:

[0044] PA-Zn release time in in vitro simulated saliva ranges from 1 to 72 hours;

[0045] It reduces the expression of macrophage M1 marker IL-1β by 40-80% and increases the expression of M2 marker Arg-1 by 50-200%.

[0046] There was no significant difference in organ tissue pathology scores compared to the healthy control group.

[0047] Compared with the prior art, the present invention has the following outstanding advantages:

[0048] This invention successfully prepared a PZ / SP bilayer film using a casting method. Specifically, the bilayer film comprises an inner starch layer and an outer pectin layer. The starch layer acts as a carrier, providing a stable environment for the immobilization of PA-Zn, while the pectin layer adheres to the outer surface of the starch layer, further regulating the drug release rate. Since salivary amylase is present in the oral cavity, it can specifically degrade the starch layer. This design enables the slow release of PA-Zn, significantly prolonging the drug release time, thereby significantly reducing the levels of pro-inflammatory cytokines TNF-α and PGE2, inducing macrophage polarization from M1 to M2, and accelerating the regeneration and remodeling of oral mucosa. Through this innovative bilayer film structure design, this study provides a new strategy for the continuous treatment of oral diseases and expands the potential for new applications of biomedical materials in the field of oral medicine. Attached Figure Description

[0049] Figure 1 This is a schematic diagram illustrating the preparation of the PZ / SP bilayer thin film according to the present invention;

[0050] Figure 2 Schematic diagram of water contact angles of PZ / SP bilayer membranes with different concentrations;

[0051] Figure 3 Schematic diagram of stress-strain curves of PZ / SP bilayer films with different concentrations;

[0052] Figure 4 Schematic diagram of elongation at break, tensile strength, tensile strength and peel strength of PZ / SP bilayer films with different concentrations;

[0053] Figure 5 Schematic diagram of Fourier transform infrared spectra of PZ / SP bilayer films with different concentrations;

[0054] Figure 6 Schematic diagram of thermogravimetric curves of PZ / SP bilayer films with different concentrations;

[0055] Figure 7 A schematic diagram comparing the surface and cross-section of PZ / SP bilayer membranes with different concentrations one hour after digestion.

[0056] Figure 8 Schematic diagram of the release of different concentrations of PZ / SP bilayer membranes 1 hour after digestion;

[0057] Figure 9 Schematic diagram of SOD enzyme-like activity after immersion in simulated saliva for 1 h and 1.5 h with different concentrations of PZ / SP bilayer membranes;

[0058] Figure 10 Schematic diagram showing the changes in oral ulcer area in rats over 1-5 days under different treatment conditions;

[0059] Figure 11 Compared with the blank group, the injury group, and the PZ / SP double membrane treated rats, the relative area of ​​oral ulcers in the PZ / SP double membrane treated rats was as follows:

[0060] Figure 12 A schematic diagram illustrating the therapeutic effect of H&E and Masson staining on oral ulcers in rats;

[0061] Figure 13 Schematic diagram of ELISA analysis of rat pro-inflammatory cytokine TNF-α;

[0062] Figure 14 A schematic diagram of PGE2 expression levels;

[0063] Figure 15 Schematic diagram of immunofluorescence images of CD11b and IL-1β staining in rats after treatment with PZ / SP double membrane for oral ulcers;

[0064] Figure 16 Immunofluorescence images of IL-1β and Arg-1 in macrophages and a schematic diagram of representative analysis of IL-1β and (B)Arg-1 (C);

[0065] Figure 17 Schematic diagram of the expression levels of pro-inflammatory cytokines CD11b (A), IL-1β (B), and IL-1β / CD11b (C) in rats;

[0066] Figure 18 A schematic diagram illustrating the epidermal healing process of a rat wound.

[0067] Figure 19 Schematic diagram of the expression levels of TNF-α and PGE2 in rat wounds;

[0068] Figure 20 A schematic diagram of the staining of major organs (heart, liver, spleen, lung, and kidney) after PZ / SP double-layer film treatment using H&E staining. Detailed Implementation

[0069] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0070] Table 1 Experimental Reagents

[0071]

[0072]

[0073] Table 2 Experimental Instruments

[0074]

[0075] Test method:

[0076] 1. Field emission scanning electron microscope

[0077] The surface morphology of the PZ / SP bilayer film was observed using field emission scanning electron microscopy. The prepared PZ / SP bilayer film was flatly placed on a conductive adhesive to prepare the sample, allowed to air dry at room temperature, fixed to a copper plate stage with conductive adhesive, and then sputter-coated with gold. The sample was observed and photographed at an accelerating voltage of 20.0 kV.

[65] .

[0078] 2. Fourier Transform Infrared Spectroscopy (FT-IR)

[0079] The infrared spectrometer (Nicolet iS10, Thermo Fisher Scientific (China) Co., Ltd.) was used, with a scanning range of 4000 cm⁻¹. -1 -400cm -1 The resolution is 4cm. -1 PA-Zn and bilayer films were analyzed, with each spectrum acquired through 64 consecutive scans.

[0080] 3. Contact Angle Test

[0081] The hydrophilicity of the bilayer membrane was observed using a contact angle meter (Theta Bio-Aolin (Shanghai) Trading Co., Ltd.). The membrane was attached to the moving platform of the water contact angle analyzer, and approximately 5 μL of water was dropped onto the membrane surface before rapid imaging. At least three measurements were taken for each concentration group. The contact angle coefficient (CA) of the membrane was measured using the five-point fitted ellipse method with an accuracy of 0.01°, employing ImageJ (Image Processing and Analysis in Java).

[0082] 4. Mechanical property test

[0083] Following the method of Sarwar et al., tensile strength (TS), elongation at break (EB), and Young's modulus of bilayer films were tested using a universal testing machine (Instron 5300, Instron (Shanghai) Testing Equipment Trading Co., Ltd.). The bilayer films were cut into 20mm × 40mm strips and fixed to the testing machine fixtures with an initial clamping distance of 20mm. The tensile speed was 50mm / min until the film broke. At least three tests were performed for each concentration.

[0084] 5. Adhesion test

[0085] The adhesion strength of the bilayer film was tested using a universal testing machine (Instron 5300, Instron (Shanghai) Testing Equipment Trading Co., Ltd.). The bilayer film was cut into strips of 40mm × 50mm and applied to fresh pigskin. Before testing, a force of 1N was applied for 1 minute to establish good contact between the patch and the oral mucosa. One end of the pigskin was fixed to the testing machine clamp, and one end of the bilayer film was fixed to the other end of the clamp. The initial clamping distance was 20mm, and the tensile speed was 50mm / min until the bilayer film separated from the pigskin membrane. Each concentration was repeated at least five times, and the results were reported as mean ± standard deviation.

[0086] 6. Thermal Performance Analysis

[0087] Thermogravimetric analysis (TG209F3, Netzsch Scientific Instruments Trading (Shanghai) Co., Ltd.) was used. 0.3g-0.5g of the membrane was placed in the sample dish of the thermogravimetric analyzer, with an empty dish as a reference. The temperature was set to rise from 25℃ to 600℃ at a rate of 10℃ / min, and the nitrogen flow rate was controlled at 50mL / min.

[0088] 7. SOD enzyme activity assay and release experiment using PZ / SP double-layer membrane

[0089] The SOD activity of PA-Zn was determined by measuring its photocatalytic inhibition of nitroblue tetrazolium (NBT). Different concentrations of PZ / SP bilayer membranes were immersed in a digestion solution for 0, 5, 20, 40, 60, 90, 150, 510, and 990 min. A solution containing riboflavin (20 μM), methionine (0.013 M), and NBT (75 μM) was prepared using 25 mM phosphate buffer (pH 7.4) and added to the digestion solution. The mixture was irradiated with a constant intensity lamp at 25 °C for 10 min. Immediately after irradiation, the absorbance was measured at 560 nm. The entire reaction mixture was placed in a foil-lined box, and a tube identical to the reaction mixture was placed in the dark as a blank to measure the SOD activity of the PZ / SP bilayer membrane. The PZ / SP bilayer membrane was then immersed in simulated saliva for 72 h, and the absorbance at 300 nm was recorded periodically using a UV-Vis spectrometer to assess PA-Zn release.

[0090] 8. Rat wound healing experiment

[0091] Female SD rats aged 6-8 weeks and weighing 180-220g were anesthetized by intraperitoneal injection of urethane (30mg / kg). An oral ulcer model was induced using 70% acetic acid. Specifically, 5×5mm circular filter paper was soaked in acetic acid solution and applied to the oral mucosa of the rats for 3 minutes. The animals were randomly divided into six groups, with intervention starting two days after ulcer formation (day 0). The experimental group rats had a PZ / SP double-layer film applied to the ulcer site, while a control group without treatment was established. Patch treatment was performed on days 1, 3, and 5 after the first application (day 0 of the experiment), with the material changed daily. During the experiment, photographs of the ulcer site were taken before each treatment, and observations were conducted for 5 consecutive days. The ulcer closure rate was analyzed using ImageJ software.

[0092] 9. Anti-inflammatory experiment of rat tissue cells

[0093] 9.1 Detection of inflammatory cytokines

[0094] Cells were incubated with 0.3% Triton X-100 in PBS for 30 min using an enzyme-linked immunosorbent assay (ELISA), followed by two washes with PBS. Cells were then blocked with 1% BSA for 45 min. The cells were treated overnight at 4°C with the primary antibody, followed by treatment with the secondary antibody for 12 h. The resulting cells were stained with DAPI and observed under a confocal laser microscope.

[0095] 9.2 Immunofluorescence staining

[0096] Immunofluorescence staining was used to study pro-inflammatory cytokines that play a key role in the pathophysiology of ulcers.

[0097] The expression levels of TNF-α, PGE2, IL-1β, and the cell surface marker CD11b were determined. The specific steps were as follows: Tissue sections were fixed with 4% paraformaldehyde and washed with PBS. They were blocked with 1% BSA for 45 min. The sections were treated overnight at 4°C with the primary antibody. They were then treated with the secondary antibody for 12 h. The sections were stained with DAPI and observed under a confocal laser microscope.

[0098] 9.3 Macrophage Polarization Experiment

[0099] RAW264.7 cells were used to observe macrophage polarization in vitro. RAW264.7 cells were cultured to an appropriate density in culture dishes, and then treated with a PZ / SP bilayer membrane after labeling with inducible IL-1β and arginase-1 (Arg-1). The expression levels of markers for M1 and M2 macrophages were detected. Immunofluorescence staining was used to analyze the expression of these markers, assessing the effect of the PZ / SP bilayer membrane on macrophage polarization.

[0100] 9.4 Biocompatibility Experiment

[0101] Rats were randomly divided into a PZ / SP group and a control group. The PZ / SP group received intravenous injections of PZ / SP solution daily for 30 days; the control group received an equal volume of physiological saline. After 30 days, the rats were sacrificed, and the heart, liver, spleen, lungs, and kidneys were removed. Histopathological examination of these organs was performed to observe for pathological changes such as necrosis, inflammation, or hemorrhage. Platelet analysis was performed on rats in both the PZ / SP group and the control group, detecting platelet count (PLT), platelet specific volume (PCT), platelet distribution (PDW), and mean platelet volume (MPV). Simultaneously, liver function indicators (AST and ALT), kidney function indicators (BUN and CRE), white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MON), red blood cell count (RBC), hemoglobin marker (HGB), mean corpuscular hemoglobin content (MCH), red blood cell distribution width (RDW), and red blood cell distribution width coefficient of variation (RDWc) were measured in the PZ / SP group to explore the biocompatibility of the PZ / SP bilayer membrane.

[0102] 10. Statistical Analysis

[0103] All measurements were repeated in triplicate, and data are reported as mean ± standard deviation. Origin 2025 was used to create the figures. Statistical analysis of color difference, thermal stability, and texture characteristics was performed using IBM SPSS (version 22), with significance set at p < 0.05. One-way ANOVA with Duncan post-hoc test was used to determine significant differences, which were considered significant at p < 0.05.

[0104] Example 1

[0105] Preparation, characterization, and mechanical property testing of starch-pectin bilayer films composed of A-Zn nanoparticles:

[0106] This example relates to a method for preparing a starch-pectin bilayer membrane loaded with PA-Zn nanoparticles. The specific preparation steps are as follows: Figure 1 As shown:

[0107] (1) Dissolve 1.01g of pyrogallol and 0.878g of zinc acetate dihydrate in 40mL of deionized water and stir continuously;

[0108] (2) After controlling the pH value of the above mixed solution to 5.0 using 8.0 mol / L KOH solution, place it in an autoclave and heat it to 120℃ and maintain it for 12 h;

[0109] (3) The reaction solution was centrifuged to obtain the final product, washed several times with ultrapure water and anhydrous ethanol, and dried in a vacuum oven at 60°C to obtain PA-Zn nanoparticles.

[0110] (4) Dissolve a certain amount of starch in water to make an 8% starch solution. Heat the solution in an 80℃ water bath for 30 minutes with constant stirring. After the solution is finished, add PA-Zn and stir to disperse it evenly. Then take 5 mL of the mixed solution and pour it into a plate. Let it dry at room temperature until it is semi-dry to prepare PA-Zn slow-release film (SP, PZ2 / SP, PZ4 / SP, PZ6 / SP) containing 0%, 0.2%, 0.4%, and 0.6% respectively.

[0111] (5) Prepare a protective layer using pectin (P) as a matrix. Dissolve 2.6g of pectin in 200mL of water, add 2.34g of glycerol and 3.84g of 10% calcium chloride solution, and stir with a magnetic stirrer for 2h to disperse it evenly;

[0112] (6) Pour 5 mL of pectin solution onto the starch layer and dry the double film at room temperature for 24 h;

[0113] (7) Place the obtained double-layer film (PZ / SP) in a constant temperature and humidity incubator at 25℃ and equilibrate with 50% RH for 48h.

[0114] This example uses scanning electron microscopy (SEM), MilliQ Water microscopy, field emission scanning electron microscopy, water contact angle analyzer, and universal testing machine to study the unique properties of the PZ / SP bilayer film. The stability of the PZ / SP bilayer film was investigated using thermogravimetric analysis. The results are as follows: Figures 2-7 .

[0115] from Figure 2 Studies on the water contact angle of PZ / SP bilayer membranes with different concentrations showed that the water contact angle (CA) of the pure SP bilayer membrane was 43.03°, indicating relatively poor hydrophobicity. The addition of PA-Zn increased the CA of the PZ / SP bilayer membrane to 65.54°, significantly improving the membrane's hydrophobicity.

[0116] from Figure 3 , Figure 4 The mechanical and adhesive properties show that as the PA-Zn concentration gradually increases, both tensile stress and tensile strain gradually decrease. This may be related to PA-Zn overload, leading to uneven distribution of PA-Zn in the SP bilayer membrane. In the adhesion test, wet pig skin was used to simulate the oral environment. With the gradual increase of PA-Zn concentration, the adhesion strength of the membrane decreased significantly from 1.16 N to 0.54 N (p < 0.05), and the membrane's resistance to separation gradually weakened.

[0117] from Figure 5The Fourier transform infrared (FTIR) spectra show different PA-Zn peaks at 1584 cm⁻¹ (COO⁻) and 1282 cm⁻¹ (CO), while the carbohydrate fingerprint region remains unchanged. The increased band intensity at 3300 cm⁻¹ indicates that PA-Zn disrupts the hydrogen bonds of SP.

[0118] from Figure 6 The thermogravimetric analysis (TGA) plots show that all films exhibit three similar thermal decomposition stages. The first stage of weight loss occurs in the range of 50℃-140℃, mainly due to the evaporation of adsorbed water in the membrane. The second stage of weight loss occurs in the range of 150℃-300℃, and this is the main stage of weight loss for the membrane. The addition of PA-Zn did not change the maximum decomposition temperature of the SP bilayer membrane (220℃), but significantly reduced the weight loss in this stage. The third stage of weight loss occurs in the range of 300℃-450℃, mainly due to the decomposition of residues. After three stages of degradation, the residual weight of the PZ / SP bilayer membrane is higher than that of the pure SP bilayer membrane. The results indicate that the addition of PA-Zn has no significant effect on the thermal stability of the PZ / SP bilayer membrane.

[0119] from Figure 7 , 8 As can be seen, the structure of the PZ / SP bilayer membrane underwent substantial degradation after immersion in simulated saliva. To evaluate the potential for topical oral administration of the PA-Zn-loaded bilayer membrane, the in vitro release of PA-Zn from the PZ / SP bilayer membrane in simulated saliva containing α-amylase (pH 7) at 37°C was investigated. The amount of PA-Zn released from the membrane into the simulated saliva was as follows: Figure 8 As shown. Over time, in each membrane

[0120] The residual concentration of PA-Zn gradually decreased. PA-Zn release exhibited a rapid initial burst, followed by a slower, sustained release, consistent with release patterns observed in many controlled-release systems. The release rate of PA-Zn from simulated saliva increased with increasing PA-Zn concentration in the membrane matrix.

[0121] Example 2

[0122] SOD enzyme activity assay of PA-Zn.

[0123] from Figure 9 As can be seen, the SOD activity of PA-Zn was evaluated using PA-Zn enzyme solutions of different concentrations. PA-Zn exhibits strong O2 activity. 2·-The scavenging capacity was dose-dependent. To evaluate the SOD enzyme activity of PA-Zn-loaded PZ / SP bilayer membranes during simulated oral digestion, membranes containing different concentrations of PA-Zn were digested in simulated saliva. After 1 h of digestion, characteristic absorbance was observed at 600 nm for PZ / SP bilayer membranes with different PA-Zn concentrations, showing a clear concentration-dependent trend: the higher the concentration, the larger the absorbance peak. As the digestion time increased to 1.5 h, the absorbance peak gradually increased, and it was found that the catalytic efficiency was significantly positively correlated with both PA-Zn concentration and digestion time.

[0124] Example 3

[0125] Establishment of a mouse oral cavity model and testing of related indicators:

[0126] from Figure 10 As shown in Figure 11, the PZ6 / SP group healed the fastest, with the ulcer completely healed by day 5. Compared with SP, PZ2 / SP, and PZ4 / SP, PZ6 / SP had a more significant effect on wound healing.

[0127] from Figure 12 As can be seen, histological analysis using H&E and Masson trichrome staining further showed that PZ / SP double-layer membrane treatment accelerated ulcer healing and significantly reduced scar area by nearly 50% (n=3), indicating that...

[0128] PZ / SP double membrane can effectively promote ulcer healing.

[0129] from Figure 13 , 14 As shown in Figure 15, the expression of two representative inflammatory cytokines, tumor necrosis factor-α (TNF-α) and prostaglandin E2 (PGE2), was detected using ELISA. Compared with the blank group, serum group, oral mucosa group, and saliva group, the digestive fluid group and SP group showed the highest expression of inflammatory factors, indicating severe inflammation and prolonged inflammatory phase. In contrast, PA-Zn and PZ / SP double-layer membrane treatments significantly reduced the inflammatory response in the serum group, oral mucosa group, and saliva group, and the anti-inflammatory effect became more pronounced with increasing PA-Zn concentration. Furthermore, the expression of two other representative inflammatory cytokines, interleukin-1β (IL-1β) and integrin αM (CD11b), was assessed using immunofluorescence staining.

[0130] from Figure 16 To further investigate the reprogramming effect of the PZ / SP bilayer membrane on pretreated macrophages, the effect was assessed by labeling induced IL-1β (red, M1 labeled) and Arg-1 (green, M2 labeled).

[0131] In vitro polarization of RAW264.7 macrophages. LPS treatment produced a red signal, inducing M1 polarization.

[0132] from Figure 17 , 18 As shown in Figures 19, the expression levels of CD11b and IL-1β, as well as the IL-1β / CD11b ratio, were significantly decreased in the blank control group, the injury group, and the PZ / SP bilayer membrane treatment group. In the PZ / SP group, the thickness of newly formed epithelium was significantly reduced on day 5, while the expression levels of TNF-α and PGE2 in rats were significantly lower than those in the injury group (P<0.01). These findings suggest that the release of PA-Zn helps regulate and alleviate inflammation.

[0133] from Figure 20 As can be seen from the results, this section uses hematoxylin and eosin (H&E) staining to evaluate the potential organ toxicity of the PZ / SP double membrane (at a concentration 10 times that of ulcerated rats) in rats. Histological examination showed that, compared with untreated healthy tissue, rat organs treated with the PZ / SP double membrane did not show obvious signs of hemorrhagic necrosis or extensive infiltration of inflammatory cells.

[0134] The above are merely a few preferred embodiments of the present invention, described in a relatively specific and detailed manner, but they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A responsive sustained-release bilayer membrane for treating oral ulcers, characterized in that, include: The inner starch layer is loaded with pyrogallol-zinc nanozyme PA-Zn, wherein the mass concentration of PA-Zn is 0.2%-0.6% of the starch layer. The outer pectin layer covers the surface of the starch layer; The bilayer membrane achieves responsive sustained release of PA-Zn by degrading the starch layer with salivary amylase. The preparation method of PA-Zn includes: a) Dissolve pyrogallol and zinc acetate dihydrate in water at a mass ratio of 1:0.5-1.5; b) Adjust the pH to 4.0-6.0 using 5-10 mol / L KOH solution; c) Perform hydrothermal reaction at 100-150℃ for 6-24 hours; The starch layer is prepared by gelatinizing a 5-10 wt% starch solution in a water bath at 70-100°C for 20-40 minutes. The raw material for the pectin layer is a pectin solution: it is made by dissolving pectin in water, adding glycerin and calcium chloride solution, and then stirring until homogeneous. The method for preparing the bilayer membrane includes: 1) Cast the starch solution containing PA-Zn into a film and dry it at room temperature until it is semi-dry; 2) Cast pectin solution onto the surface of the starch layer and dry at room temperature for 12-48 hours; 3) Equilibrate at 20-30℃ and 40-60% humidity for 24-72 hours; The PA-Zn was released in simulated saliva in 1-72 hours, and the SOD enzyme activity reached its peak within 1.5 hours. The aforementioned responsive sustained-release bilayer membrane for treating oral ulcers meets the following mechanical properties: Tensile strength: 0.5-13.1 N / mm; Adhesion strength: 0.5-1.16 N.

2. The use of the responsive sustained-release bilayer membrane as described in claim 1 in the preparation of oral ulcer treatment drugs or medical devices.

3. A drug or medical device for treating oral ulcers, characterized in that, It comprises the responsive sustained-release bilayer membrane of claim 1, and is configured as follows: It adheres to the oral mucosal wound; PA-Zn sustained release is triggered by salivary amylase; A design that can be changed daily.

4. The drug or medical device according to claim 3, characterized in that, It has the following quantifiable properties: PA-Zn release time in in vitro simulated saliva ranges from 1 to 72 hours; It reduces the expression of IL-1β, a marker of M1 macrophages, by 40-80%, and increases the expression of Arg-1, a marker of M2 macrophages, by 50-200%. There was no significant difference in organ tissue pathology scores compared to the healthy control group.

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