Film dressing for promoting wound healing and preparation method and application thereof

Abstract

The invention provides a film dressing for promoting wound healing and a preparation method and application thereof, and belongs to the technical field of wound dressings. A natural product beta-acid is used as an active substance to prepare a beta-acid / hydroxypropyl-beta-cyclodextrin inclusion compound, and the beta-acid / hydroxypropyl-beta-cyclodextrin inclusion compound is loaded in a CS / PVA polymer to prepare the film dressing. Due to the addition of the beta-acid / hydroxypropyl-beta-cyclodextrin inclusion compound, more movable areas are formed in a CS / PVA polymer network, so that permeation of water molecules is facilitated, the flexibility of the film dressing is enhanced, and the film dressing has good physical properties, stability, oxidation resistance and antibacterial activity; the healing of MRSA infected skin wounds can be promoted.

Description

Technical Field

[0001] The invention relates to the technical field of wound dressings, and in particular to a film dressing for promoting wound healing, and a preparation method and application thereof. Background Art

[0002] As the largest organ in the human body, the skin can protect internal organs from external damage. However, due to the interference of external physical and chemical factors, the skin is broken or damaged, resulting in wound formation. The repair process of damaged skin is a complex process including hemostasis, anti-inflammation, cell proliferation and tissue remodeling.

[0003] Since the wound surface is exposed to the complex external environment for a long time, the wound injury site is easily invaded by microorganisms, leading to wound infection and delaying the wound healing process. For example, many chronic burns, ulcers and diabetes-related wounds are extremely susceptible to microbial infection. These types of wounds may lead to serious economic burdens and high mortality rates, which is a major public health challenge facing clinicians.

[0004] Among traditional microbial infections, coccal infections and bacillus infections are common hospital infections. Patients with clinical MRSA infection often experience chronic inflammatory symptoms, which worsen wound healing and can lead to sepsis and even organ failure in severe cases. Therefore, the development of ideal wound dressings is crucial for the healing of bacterially infected wounds. Decades of clinical use have shown that traditional wound dressings (bandages and gauze) are very effective in absorbing moisture from wounds, but may lead to insufficient fluid in the wound site. When patients change dressings, traditional dressings adhere to the wound, causing discomfort and pain to the patient. Therefore, in-depth research and development of advanced biomaterials for wound healing is an urgent issue to be addressed in the biomedical field.

[0005] Suitable wound dressings can replace the skin, form the first line of defense against microbial invasion, effectively manage wound exudate, and reduce wound infection. In addition, ideal wound dressings should be highly biocompatible, non-toxic, easily available, and have moderate mechanical strength. Studies have shown that film materials are ideal wound dressings because the raw materials used to prepare natural films have biocompatibility, flexibility, suitable strength, and the ability to accelerate wound healing. Selecting different polymers to form films can improve the shortcomings of the original single material.

[0006] Chitosan (CS) is a derivative of chitin, composed of N-acetyl-D-glucosamine and D-glucosamine. A large number of studies have confirmed that CS is biocompatible, non-toxic, widely available, and gel-forming. In existing medical products, CS has been used as a hemostatic agent in wound bandages. In the pharmaceutical industry, CS has been used in wound dressing formulations and drug release. However, the low tensile strength of single-component CS films limits their medical applications. Polyvinyl alcohol (PVA) is a synthetic water-soluble polymer that has been widely used in society due to its advantages such as non-toxicity, high tensile strength, and good biocompatibility. Although PVA has ideal wound dressing properties, it cannot provide therapeutic effects (no free radical scavenging or antibacterial properties), which limits its medical application. In addition, studies have shown that synthetic polymers cannot degrade, but penetrate into the system and release toxic substances. Biodegradable polymers are biocompatible, but generally have poor mechanical strength. To overcome the shortcomings of single films, researchers have combined synthetic polymers with biopolymers. The physical and chemical properties of the composite film made of biopolymer (CS) and synthetic polymer (PVA) have become controllable, and it has gradually become one of the biomaterials with bright prospects. Researchers have found that PVA and CS can be mixed to prepare films to improve their respective shortcomings. Bano's (2019) group reported that different ratios of CS / PVA may be effective burn wound healing agents with better wound healing effects.

[0007] With the subsequent research on wound treatment, natural extracts were introduced into polymer materials as active ingredients, giving specific functions to biomaterials and can be used as wound dressings. Different studies have confirmed that extracts from natural sources, with their antioxidant, antibacterial and anti-inflammatory effects, help wounds recover quickly by accelerating the processes of wound contraction, angiogenesis, epithelialization and granulation. Researchers have added some phytochemicals to the polymer matrix to treat wounds. Tran et al. (2024) extracted Piper betle L. extract and added it to CS / PVA film. The experimental results showed that the single active ingredient added film had good wound healing potential. Morgado's team (2017) added ibuprofen / CD inclusion complex to CS / PVA film. They found that the film could prevent scabs and excessive inflammation, allowing the skin to heal faster. Ascorbyl palmitate / HP-β-CD inclusion complex was selected to be combined with the film material, and the researchers found that it could effectively promote wound healing.

[0008] β-acid is an active product extracted from hops, which has significant antibacterial and antioxidant properties. Previous studies have found that β-acid has good antioxidant and antibacterial effects. Since it comes from hops, β-acid has been widely used in food. However, due to its low water solubility, β-acid is mainly added to food cling film.

[0009] β-Cyclodextrin (β-CD) is a cyclic oligosaccharide composed of seven cyclic oligosaccharides connected by α-1,4-glycosidic bonds, with a surprisingly hydrophobic cavity and a hydrophilic outer surface. As one of the famous derivatives, 2-hydroxypropyl-β-cyclodextrin (HP-β-CD) is widely used as a drug carrier for encapsulating various water-insoluble molecules. CD inclusion complexes in medical dressings have been achieved by preparing different types of materials. Although the properties of membrane materials such as good biocompatibility and non-toxicity have been emphasized, the question of whether CD inclusion complexes combined with membrane materials for wound dressings can help MRSA wound healing needs to be solved urgently.

[0010] The current patent for a nanofiber dressing for skin wound repair and its preparation method (application number: 202410689873.1) uses curcumin as an active ingredient in the preparation of a dressing for skin wound repair, and loads curcumin into a ZIF-8 chitosan / polyvinyl alcohol nanofiber membrane. However, harmful chemical solvents such as methanol and glutaraldehyde are used in its preparation process, and the complex preparation process leads to increased costs, which may limit its popularity in large-scale applications. In addition, curcumin has poor stability under environmental factors such as light and temperature, and cannot play a lasting role. It has not been proven whether the dressing can promote the healing of MRSA-infected skin wounds.

[0011] Therefore, how to prepare a dressing that can be used for MRSA wound healing is crucial to help MRSA wound healing. Summary of the invention

[0012] The object of the present invention is to provide a film dressing for promoting wound healing, a preparation method and application thereof, using natural product β-acid as an active substance, preparing a β-acid / hydroxypropyl-β-cyclodextrin inclusion compound, and loading the compound in chitosan / polyvinyl alcohol to prepare a film dressing. The film dressing of the present invention has good physical properties, stability, antioxidant properties and antibacterial activity, and can promote the healing of MRSA-infected skin wounds.

[0013] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0014] The present invention provides a film dressing for promoting wound healing, the dressing comprising the following raw materials in parts by weight:

[0015] 0.1 to 1 part of β-acid, 0.1 to 1 part of hydroxypropyl-β-cyclodextrin, 1 to 3 parts of chitosan, and 1 to 5 parts of polyvinyl alcohol.

[0016] The present invention also provides a method for preparing a film dressing for promoting wound healing, comprising the following steps:

[0017] (1) Preparation of β-acid / hydroxypropyl-β-cyclodextrin inclusion complex: hydroxypropyl-β-cyclodextrin and β-acid are mixed, ground, and dried to obtain β-acid / hydroxypropyl-β-cyclodextrin inclusion complex;

[0018] (2) Preparation of film: β-acid / hydroxypropyl-β-cyclodextrin inclusion complex is dissolved in water to obtain β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution, the PVA solution is mixed with the CS solution, glycerol and β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution are added, stirred, allowed to stand, poured into a culture dish, and dried to obtain a film.

[0019] Preferably, in step (1), the grinding time is 0.5-4h.

[0020] Preferably, in step (2), the mass volume ratio of the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex to water in the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution is 0.5-2 g:2-20 mL;

[0021] Preferably, in step (2), the preparation method of the PVA solution is: dissolving 1-4 g of polyvinyl alcohol in 30-100 mL of distilled water at a temperature of 60-100° C. by stirring to obtain a PVA solution;

[0022] Preferably, in step (2), the CS solution is prepared by: dissolving 0.5-3 g of chitosan in 30-100 mL of acetic acid aqueous solution at a temperature of 20-100° C. to obtain a CS solution;

[0023] Preferably, in step (2), the volume ratio of the polyvinyl alcohol solution, chitosan solution and glycerol is 30-100 mL: 30-100 mL: 0.1-5 mL.

[0024] Preferably, in step (2), the stirring time is 0.5-2 hours; and the standing time is 1-16 hours.

[0025] The present invention also provides an application of the film dressing prepared by the method for preparing the film dressing for promoting wound healing in promoting wound healing.

[0026] Preferably, the wound is a wound infected with MRSA.

[0027] The beneficial effects of the present invention compared with the prior art are:

[0028] (1) The present invention uses natural product β-acid as an active substance to prepare a β-acid / hydroxypropyl-β-cyclodextrin inclusion compound, and loads the inclusion compound into a CS / PVA polymer to prepare a film dressing. Due to the addition of the β-acid / hydroxypropyl-β-cyclodextrin inclusion compound, more mobile areas are formed in the CS / PVA polymer network, which helps the penetration of water molecules and enhances the flexibility of the film dressing, so that the film dressing has good physical properties, stability, antioxidant properties and antibacterial activity, and can promote the healing of MRSA-infected skin wounds.

[0029] (2) The film dressing of the present invention can slowly release active ingredients, exert antioxidant and antibacterial activities for a long time, effectively promote enzyme repair, promote metabolic processes, and promote wound healing. The film-carrying β-acid can penetrate the bacterial cell membrane, cause bacterial cells to rupture and shrink through interaction with bacteria, and eventually lead to bacterial death, showing excellent healing ability in the healing of MRSA-infected skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0031] Figure 1 The test results of β-acid, HP-β-CD, PM and IC in Experimental Example 1 of the present invention are shown in Figure 1, wherein A is a SEM image, B is UV absorbance, C is a FTIR spectrum, and D is an XRD diffraction pattern;

[0032] Figure 2 Characterization of the HP-β-CD@CS / PVA film in Experimental Example 1 of the present invention, (A) SEM image, (B) FTIR spectrum, (C) XRD diffraction pattern;

[0033] Figure 3 These are the properties of the film dressing in Test Example 1 of the present invention, wherein A is tensile strength (TS), B is elongation at break (EAB), C is thickness, D is contact angle, E is moisture content, F is water solubility, G is water vapor permeability (WVP), H is weight loss, and I is derivative thermogravimetry (DTG).

[0034] Figure 4The experimental result diagram of Experimental Example 2 of the present invention, wherein A is the cumulative release of β-acid in PBS (pH 1.2, 6.8, 7.4); B is a partial enlarged view; C is IC; D is the scavenging activity of HP-β-CD@CS / PVA membrane on DPPH free radicals; E is the antibacterial image of IC and HP-β-CD@CS / PVA membrane; F is a scanning electron microscope of MRSA treated with PBS and HP-β-CD@CS / PVA;

[0035] Figure 5 This is the effect of the membrane in Experimental Example 3 of the present invention on wound healing in MRSA-infected mice, wherein A is a photo of the wound on the back of the mouse; B is the wound surface change model; C is the relative wound surface area treated with Control, CS / PVA and 0.9IC@CS / PVA.

[0036] Figure 6 This is an evaluation of the healing effect of tissue sections treated with control, CS / PVA and 0.9HP-β-CD@CS / PVA in Experimental Example 3 of the present invention, where A is H&E staining of wound tissue. Scale bars: 100 μm and 50 μm; B is an immunofluorescence staining image of regenerated skin tissue after 15 days of treatment with different samples, marked with IL-6 (red), TNF-α (red), VEGF (red) and cell nuclei (blue). Scale bar: 100 μm; C is a quantitative analysis of the relative fluorescence intensity of regenerated skin tissue stained with IL-6, TNF-α, and VEGF. DETAILED DESCRIPTION

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] Example 1

[0043] Embodiment 1 of the present invention provides a method for preparing a film dressing for promoting wound healing, and the specific steps are as follows:

[0044] Preparation of β-acid / hydroxypropyl-β-cyclodextrin inclusion complex:

[0045] (1) Accurately weigh 0.2 g of hydroxypropyl-β-cyclodextrin and 0.12 g of β-acid, and add 2 mL of distilled water. Grind the mixture at room temperature and 50 Hz for 1 h using a ball mill. After centrifugation, freeze-dry the supernatant to obtain the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex (IC), which is stored at -20°C.

[0046] (2) Preparation of thin film: accurately weigh 2.0 g PVA, stir and dissolve it in 50 mL distilled water at 95°C to obtain a PVA solution; dissolve 1.0 g CS in 50 mL acetic acid solution and stir at 70°C to obtain a CS solution; mix the PVA solution and CS solution, add 1.0 mL glycerol, and dissolve 0.9 g β-acid / hydroxypropyl-β-cyclodextrin inclusion complex in 10 mL H2O and add it to the CS / PVA mixed solution, stir the mixture for 1 hour, and let it stand for 12 hours to remove bubbles. Pour the bubble-free film solution into a polystyrene culture dish with a diameter of 9 cm and dry it at 37°C for 24 hours to obtain a thin film dressing.

[0047] Test Example 1

[0048] Test Example 1 of the present invention characterized the physical properties of the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex prepared in Example 1, and the specific steps are as follows:

[0049] 1. Characterization of inclusion complexes:

[0050] (1) Solubility

[0051] In order to determine the effect of β-acid / hydroxypropyl-β-cyclodextrin inclusion complex (IC) on the solubility of β-acid, the method and addition amount of Example 1 were adopted to measure the phase solubility of β-acid at different HP-β-CD concentrations. It was found that HP-β-CD had a solubilizing effect on β-acid. The results are shown in Table 1.

[0052]

[0053]

[0054] (2) SEM technology to analyze surface structure and morphology

[0055] Use SEM technology to analyze the surface structure and morphology of IC ( Figure 1 A). In the SEM images, β-acid appears as an amorphous lamellar crystal structure, while HP-β-CD appears as spherical structures of different sizes with pores on the surface.

[0056] The morphology of β-acid and HP-β-CD was tested after mixing by physical mixing method (PM). After mixing by physical mixing method, β-acid and HP-β-CD continued to maintain their original morphology. Compared with the ball milling method, the morphology of the inclusion complex prepared by ball milling method was very different from that of the inclusion complex prepared by physical mixing method. IC showed an irregular block structure, and this morphological change was direct evidence of the formation of inclusion complex.

[0057] (3) Ultraviolet spectrum detection

[0058] UV spectroscopy is a fast, simple and effective method to study whether inclusion complexes are formed. The UV spectra of β-acid, HP-β-CD, PM and IC are as follows: Figure 1 B. HP-β-CD has almost no absorption between 280 and 500 nm. β-acid has obvious UV absorption at a wavelength of 356 nm. The UV spectrum of PM is similar to that of β-acid, indicating that physical mixing does not change the position of its characteristic absorption peak in the UV spectrum.

[0059] Figure 2 C is the characteristic FTIR spectra of HP-β-CD, β-acid, IC and PM. The results show that HP-β-CD has a peak at 3382 cm -1 and 2930cm -1 The absorption peak at 1033-1156cm corresponds to the stretching vibration absorption of -OH and hydrocarbons. -1The absorption peaks near represent the CC / CO stretching vibration and antisymmetric stretching vibration of the COc glycosidic bridge in the HP-β-CD structure. The main absorption peaks of β-acid (-OH, CH and CO) are located at 3431 cm -1 、1973cm -1 and 1758cm -1 After characterizing the prepared inclusion complex by mechanical milling, it was found that the FTIR absorption peak of IC was different from that of β-acid and HP-β-CD. The -OH absorption peak appeared at 3393 cm -1 , indicating that there is a hydrogen bond between β-acid and HP-β-CD. β-acid is at 1758cm -1 The absorption peak shifted to 1642 cm -1 The absorption peak of HP-β-CD at 1033~1156cm-1 shifted to 1161~1029cm-1 -1 Nearby. Through simple physical mixing, the characteristic absorption peaks of each component appeared, which is a simple spectral superposition. Therefore, the FTIR results further confirmed the interaction between β-acid and HP-β-CD, reporting the successful formation of IC.

[0060] XRD data of β-acid, HP-β-CD, PM, and IC are shown in Figure 1 D. β-acid has obvious characteristic absorption peaks at 2θ=9.6° and 18.1°, representing the crystalline structure. For the amorphous structure of HP-β-CD, its absorption peak shows a broad peak mode near 2θ=18°, confirming its amorphous structure. PM can observe the overlap of β-acid and HP-β-CD spectra. It is worth noting that IC is aligned with HP-β-CD and has no obvious characteristic peaks, which strongly proves that β-acid is encapsulated by HP-β-CD.

[0061] 2. Thin film characterization:

[0062] (1) Scanning electron microscopy morphology observation

[0063] The morphology of the prepared membrane was observed using scanning electron microscopy ( Figure 2 A). The surface of the film composed only of CS and PVA is uniform and neat without cracks. IC was dissolved in H2O and added to the film solution. The film was crack-free after drying. The reason for the lack of cracks in the film may be that there was not enough surface tension to destroy the structure of the film during the drying process. After adding IC, SEM results showed that the active ingredients aggregated to varying degrees on the surface of the film containing the inclusion complex. Low concentrations of IC aggregated on the film surface, while high concentrations of IC were evenly dispersed in the film matrix. In the actual pictures, it was found that with the increase of IC content, the film became smooth and obviously harder.

[0064] (2) Characterization

[0065] The material composition of the film was characterized by FTIR ( Figure 2 B). CS / PVA film at 3277cm -1 The characteristic peaks at 2914 cm-1 are attributed to the stretching vibration of -OH groups in the PVA and CS structures. -1 The nearby peaks are related to the asymmetric and symmetric stretching vibrations of the polymer structure on the -CH2 groups. -1 and 1573cm -1 Nearby are the stretching vibrations of C=O and the asymmetric stretching vibrations of NH. -1 The absorption peaks appearing near 1418-1324cm belong to the asymmetric and symmetric stretching vibrations of the polymer structure on the -CH2 group. -1 The peak is attributed to COH bending vibration. It appears at 1030 cm -1 and 843cm -1 The characteristic bands of CS / PVA blends are related to the structure of COC stretching and CH bending vibration. After adding the inclusion complex, the FTIR characteristic peaks of HP-β-CD@CS / PVA shifted. The stretching vibration of the -OH group shifted to 3287 cm -1 , the stretching vibration of the NH group shifts to 1581 cm -1 , the stretching vibration of the COC group shifts to 1026 cm -1 The shift of the characteristic peaks of the above groups may be due to the formation of intermolecular / intramolecular hydrogen bonds between CS / PVA and IC. Importantly, the addition of IC creates a new chemical environment in the film.

[0066] The XRD spectrum of the thin film material shows the diffusion characteristics of the amorphous phase. Figure 2 As shown in Figure C, the CS / PVA film shows a clear amorphous state. After the addition of IC, a displacement peak of the characteristic peak of IC (2θ = 10°) appeared near 2θ = 8.8°. There are interactions between the molecules of CS (-OH / -NH groups), PVA (-OH groups) and IC (-OH groups). Different studies have shown that due to these interactions, the length of intermolecular / intramolecular hydrogen bonds is reduced and the rigidity of the film is increased.

[0067] It was found that IC in CS / PVA films had a significant effect on the mechanical properties ( Figure 3 A, Figure 3B). The tensile strength (TS), elongation at break (EAB) and Young's modulus (YM) of the CS / PVA film were 7.20 MPa, 41.00% and 21.73 MPa, respectively. When the IC content in the film increased, the TS of the film showed an increasing trend (7.60-35.90 MPa), and the EAB showed a trend of first increasing and then decreasing (83.30-212.20-90.60%). This phenomenon can be explained by the intermolecular interaction between the molecules of the substances forming the film, which leads to an increase in the rigidity of the film and limits the mobility of the polymer chains. The final film (0.9HP-β-CD@CS / PVA) showed greater flexibility, which was mainly due to the inclusion complex enhancing the flexibility of the film. This property is beneficial for wound healing, because an ideal wound dressing should have good resistance to mechanical wear and excellent elasticity. The addition of different contents of β-acid / hydroxypropyl-β-cyclodextrin inclusion complexes had no significant effect on the film thickness, which may be due to the good mixing of the inclusions with the film matrix ( Figure 3 C).

[0068] (3) Water contact angle measurement

[0069] The hydrophilicity and hydrophobicity of wound dressings are important when in contact with human skin, and the surface wettability of the membrane is evaluated by measuring the water contact angle.

[0070] The addition of IC increased the membrane water contact angle from 75.68° to 109.59°. Figure 3 D. The hydrophilicity of CS / PVA film is due to the presence of polar hydrophilic functional groups such as -OH groups, which makes the water contact angle of the film smaller. However, with the increase of IC content in the film, the content of β-acid in the film increases, and β-acid acts as a hydrophobic molecule, resulting in an increase in the water contact angle in the film. Compared with CS / PVA film, the water content of the film with the addition of inclusion complexes showed a downward trend (32.05±0.81%) ( Figure 3 E). It can be seen that the CS / PVA membrane loses water faster when placed for a long time, while the membrane loaded with the inclusion complex can effectively retain moisture over a period of time. Maintaining the moisture content of the wound is essential for healing, which can avoid wound dehydration and reduce cell death. Reducing the frequency of changing wound dressings can improve patient satisfaction. The water solubility of wound dressings is also an important property. The effective dissolution of the dressing also helps to release the active ingredients during long-term contact with biological fluids (such as wound exudate, blood or water) during the long-term placement of the wound. The solubility of CS / PVA (10.17±2.82%) is significantly lower than that of the membrane loaded with IC (12.69±1.15%-26.59±0.59%) ( Figure 3F). This difference may be related to the water solubility of IC, which has a high water solubility and easily forms hydrogen bonds with water molecules. Ideally, wound dressings should be able to absorb wound exudate, retain wound moisture, and allow water vapor to permeate. Therefore, this study tested the WVP ( Figure 3 G). The results show that WVP has an increasing trend, which is mainly due to the fact that the increase in IC forms more mobile areas in the polymer network of CS / PVA membrane, which facilitates the penetration of water molecules.

[0071] (4) Thermogravimetric analysis

[0072] Thermogravimetric analysis is an effective method to study the thermal stability of polymer films. Figure 3 H and Figure 3 I). The experimental results show that the addition of IC improves the thermal stability of the film. The improvement in thermal stability may be due to the increase in the number of hydrogen bonds between the molecules forming the film, which requires more energy to decompose. The TGA curve of the composite film shows three distinct weight loss zones. The first weight loss zone occurs between 30-150°C, which is caused by the evaporation of water present in the polymer film, and the mass decreases by about 10% with increasing temperature. The second weight loss zone, spanning 150-450°C, is related to the degradation of the polymer (mainly PVA) and the inclusion structure, resulting in a weight loss of about 70-80%. The third weight loss zone is between 450-600°C and corresponds to the decomposition of the chitosan chains. Figure 3 H and Figure 3 I shows that the thermal stability of the film is enhanced after adding IC.

[0073] Wound dressings are used to block harmful light from wounds and protect cells from light damage. Therefore, the transmittance of the film was detected in the visible wavelength range of 280nm, 350nm, 400nm, 500nm, 600nm, 700nm, and 800nm ​​(Table 1). As can be seen from Table 1, the transmittance of the film decreases with the increase of the inclusion complex content. In the visible light band (400-800nm), as the wavelength increases, the transmittance of the film with IC added gradually becomes equivalent to CS / PVA. In addition, the opacity value increases with the addition of IC (Table 1), and the better the light-shielding function. The opacities of 0.5HP-β-CD@CS / PVA, 0.7HP-β-CD@CS / PVA, and 0.9HP-β-CD@CS / PVA films are 0.97±0.05, 0.98±0.01, and 0.95±0.04, respectively. This phenomenon can be explained by the interaction between the inclusion complex and the CS / PVA matrix, which leads to light scattering and reflection. The results show that the IC@CS / PVA film has the effect of blocking ultraviolet rays and has the potential to be used as a UV-protective wound dressing.

[0074] Test Example 2

[0075] Test Example 2 of the present invention detected the drug release, antioxidant and antibacterial activities of the film dressing prepared in Example 1, and the specific steps were as follows:

[0076] (1) In vitro release assay

[0077] Normal skin has an acidic physiological environment, a natural barrier that effectively counteracts microbial colonization. Chronic wounds and bacterially infected wounds are characterized by pH values ​​above 7.3. When the film is used as a medical dressing, the rapid release of the drug may lead to high local drug concentrations, while the slow continuous release provides a steady delivery of the therapeutic drug, which may lead to better wound healing. Therefore, this study investigated the in vitro release of β-acid from 0.9HP-β-CD@CS / PVA film under different pH conditions. Figure 4 A and Figure 4 As shown in Figure B, the release of β-acid in the 0.9HP-β-CD@CS / PVA membrane is first rapid release and then gradually turns to sustained release. The maximum release of β-acid within 6 hours can reach about 12%. After 12 hours, the release of β-acid in the membrane gradually turns to sustained release, and the release amounts at 12 hours reach 20% (pH 7.4), 17% (pH 6.8) and 12% (pH 2), respectively. The rapid release of β-acid in the membrane may be due to the high solubility of IC in the membrane, which promotes the rapid release of β-acid. The maximum release at 24 hours remains at about 24%, and not all β-acid is released, which may explain why the membrane has an action time of more than 24 hours and can be used as a wound repair material.

[0078] (2) Antioxidant capacity test

[0079] When the skin is damaged to varying degrees, a large number of free radicals appear, causing oxidative stress on the wound surface. The DPPH method was used to determine IC( Figure 4 C) and thin films ( Figure 4 D) antioxidant capacity. Figure 5 C It can be seen that the antioxidant capacity of IC is proportional to the concentration, reaching 61.15% (8 mg / mL). When the antioxidant capacity of the membrane was tested, the antioxidant capacity of the membrane was significantly improved (5.56% to 67.27%). Previous studies have confirmed that CS has a high positive charge density and has antioxidant activity. Using CS and PVA as membrane matrices, HP-β-CD as a solubilizer, and β-acid as an antioxidant, the membrane has good antioxidant activity. When the skin is damaged, a large amount of reactive oxygen species will be produced during the inflammatory period, leading to biological damage. These wound dressings have antioxidant activity and can effectively promote enzyme repair, promote metabolic processes, and promote wound healing.

[0080] (3) Antibacterial activity detection

[0081] MRSA is one of the most troublesome bacterial strains for doctors in clinical practice at this stage. It is associated with wound infection and may be serious enough to endanger life. Therefore, determining the antibacterial activity of the membrane is one of the basic properties of wound dressings. The antibacterial activity of the composite membrane against Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Escherichia coli) was evaluated by measuring the inhibition zone. First, the microorganisms were placed in a 37°C incubator for 24 hours, and then 50 μL of the bacterial suspension was applied to the surface of the solid culture medium, and then a film with a diameter of 6 mm was attached to the culture medium. After culturing at 37°C for 24 hours, the growth of bacteria was observed, and the size of the antibacterial area was measured with an electronic vernier caliper with an accuracy of 0.01 mm. Then, the bacterial solution was cultured at 37°C to the logarithmic growth phase and the film sample was added for 12 hours. The treated bacteria were collected by centrifugation at 8000 rpm for 5 minutes, and after being fixed with 2.5% glutaraldehyde for 24 hours, they were gradiently dehydrated in 25%, 50%, 75%, 90%, and 100% ethanol solutions for 10 minutes. The bacterial samples were taken by freeze-drying method, and the bacterial structure was observed by scanning electron microscopy.

[0082] The antibacterial activities of the prepared inclusion complex and membrane against MRSA were shown in Figure 4 E. The antibacterial effect of the inclusion complex is due to the interaction of β-acid as a non-polar molecule with Gram-positive bacteria (MRSA), which presents hydrophobicity on the surface, resulting in cell rupture and the appearance inhibition zone of IC (11.64±0.09mm-7.57±0.12mm, Figure S3). After blending the inclusion complex with CS / PVA, the composite film exhibited a unique antibacterial effect. The results showed that the antibacterial activity of the composite film depends on the amount of IC added. With the increase of the inclusion complex content, the inhibition zone of the film against MRSA increased to 12.80±0.08mm. This is because the film-loaded β-acid can penetrate the bacterial cell membrane, cause bacterial cell rupture and shrinkage through interaction with bacteria, and ultimately lead to bacterial death ( Figure 4 F).

[0083] Test Example 3

[0084] Test Example 3 of the present invention detects the effect of the film dressing prepared in Example 1 on promoting wound healing, and the specific steps are as follows:

[0085] Mouse experiments

[0086] All mice were adapted to feeding for 1 week. They were randomly divided into 3 groups, 3 mice in each group: control group, CS / PVA group, and 0.9IC@CS / PVA group. The animals were grouped in order and received food and water regularly. On the day of animal surgery, the mice were intraperitoneally injected with 10% chloral hydrate (0.1 mL / 10 g). The back skin of the mice was scraped before modeling and disinfected with alcohol. A biopsy punch was used to make a circular full skin defect wound on the back (wound diameter 10.0 mm), and 100 μL of methicillin-resistant Staphylococcus aureus (1×10 7 CFU / mL) to establish the MRSA infection model. After MRSA infection, mice were kept in a single cage and the wound was not sutured to evaluate the healing process. After 24 hours of continuous infection, they were divided into groups as described above and given different treatments. After treatment, they were covered with sterile gauze for 15 consecutive days (all materials were replaced at specific time points 3, 6, 9, 12, and 15 days). The mice were photographed on days 0, 3, 6, 9, 12, and 15 ( Figure 5 A) to quantify the wound area. The mice with vertebral dislocation were killed on day 16, and the healed skin tissue was removed. The skin of the remaining 6 mice was stored at -80℃ and fixed with 4% formaldehyde for pathological sections.

[0087] Figure 5 It was shown that the 0.9HP-β-CD@CS / PVA membrane, which showed good performance in in vitro experiments, was used for wound healing. After 15 days of treatment, the relative wound area of ​​the control group was 7.11%. After CS / PVA covered the wound, the relative wound area was reduced to 6.87%, which was not statistically different from the control group. After treatment with 0.9HP-β-CD@CS / PVA, the wound area was reduced to 0.63%. The above results are helpful to evaluate the ability of 0.9HP-β-CD@CS / PVA to accelerate wound healing and promote wound contraction. HP-β-CD was selected to encapsulate β-acid, and β-acid was released faster and more fully in the wound exudate. During the granulation tissue formation stage, IC can promote cell adhesion, growth and differentiation, accelerate wound healing, and improve the ability to promote healing. Simulation of wound dynamic changes such as Figure 5 As shown in B.

[0088] CS has a great influence on promoting wound healing. CS has the ability to stop bleeding, inhibit microbial growth, promote cell proliferation, accelerate wound healing, regulate fibroblast activity, and control fibrosis and scar formation at the healing site. The wound healing speed of the 0.9HP-β-CD@CS / PVA group was the fastest, which was conducive to the absorption of wound exudate and the maintenance of a moist environment. After 15 days, the wound healing rate was 99.37%. Figure 6As shown in Figure C. Since IC has obvious antibacterial properties, its addition can effectively reduce bacterial infection in the process of chronic wound healing and prevent the occurrence of wound sepsis. In addition, the high content of reactive oxygen in MRSA-infected wounds will hinder the function of endogenous stem cells and the process of wound tissue regeneration. Due to the antioxidant activity of IC, it can remove ROS in the wound in a timely manner.

[0089] H&E staining further analyzed the repair process of MRSA-infected wounds from a histological perspective ( Figure 6 A). The upper epidermis of the control group was thinner than that of the other groups, and there were obvious pores between it and the lower epidermis, indicating that complete tissue had not yet formed on the wound surface. Although the skin tissue treated with CS / PVA and 0.9HP-β-CD@CS / PVA membranes has been developed, tissue regeneration still takes time. In contrast, the wound inflammatory cells decreased, fibroblasts increased, epidermal regeneration was complete, and dermal gaps decreased. These effects were attributed to the anti-inflammatory and fibroblast-promoting properties of β-acid. Interleukin-6 (IL-6) and tumor necrosis factor α (TNF-α), as cytokines secreted by macrophages, are closely related to inflammatory responses and have been shown to regulate pro-inflammatory activity. In addition, vascular endothelial growth factor (VEGF) promotes endothelial cell migration and angiogenesis. Therefore, immunofluorescence staining was used to evaluate the expression levels of IL-6, TNF-α, and VEGF ( Figure 6 B and 6C).

[0090] After the mice were euthanized, the treated skin samples were stained. Compared with the control group and the CS / PVA group, the expression levels of IL-6 and TNF-α in the 0.9HP-β-CD@CS / PVA group were reduced, and the expression level of VEGF was significantly increased. These results indicate that β-acid can downregulate the expression of IL-6 and TNF-α and upregulate the expression of VEGF, thereby regulating the inflammatory response of the wound and promoting skin regeneration. The results show that the 0.9HP-β-CD@CS / PVA film has antibacterial, antioxidant and anti-inflammatory properties, and has good wound healing ability.

[0091] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A film dressing for promoting wound healing, characterized in that: The dressing comprises the following raw materials in parts by weight: 0.1 to 1 part of β-acid, 0.1 to 1 part of hydroxypropyl-β-cyclodextrin, 1 to 3 parts of chitosan, and 1 to 5 parts of polyvinyl alcohol.

2. A method for preparing the film dressing for promoting wound healing according to claim 1, characterized in that: The steps include: (1) Preparation of β-acid / hydroxypropyl-β-cyclodextrin inclusion complex: hydroxypropyl-β-cyclodextrin and β-acid are mixed, ground, and dried to obtain β-acid / hydroxypropyl-β-cyclodextrin inclusion complex; (2) Preparation of thin film: dissolve β-acid / hydroxypropyl-β-cyclodextrin inclusion complex in water to obtain β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution, mix the PVA solution with the CS solution, continue to add glycerol and β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution, stir, let stand, pour into a culture dish, and dry to obtain a thin film dressing.

3. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (1), the grinding time is 0.5-4h.

4. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (2), the mass volume ratio of the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex to water in the β-acid / hydroxypropyl-β-cyclodextrin inclusion complex solution is 0.5-2 g:2-20 mL.

5. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (2), the preparation method of the PVA solution is: 1-4g of polyvinyl alcohol is dissolved in 30-100mL of distilled water by stirring at a temperature of 60-100°C to obtain a PVA solution.

6. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (2), the CS solution is prepared by dissolving 0.5-3 g of chitosan in 30-100 mL of acetic acid aqueous solution at a temperature of 20-100° C. to obtain a CS solution.

7. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (2), the volume ratio of the polyvinyl alcohol solution, chitosan solution and glycerol is 30-100 mL: 30-100 mL: 0.1-5 mL.

8. The method for preparing the film dressing for promoting wound healing according to claim 2, characterized in that: In step (2), the stirring time is 0.5-2 hours; the standing time is 1-16 hours.

9. Use of the film dressing prepared by the method for preparing the film dressing for promoting wound healing according to any one of claims 2 to 8 in promoting wound healing.

10. The use of the film dressing in promoting wound healing according to claim 9, characterized in that: The wound is a wound infected by MRSA.

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