Composite active dressing for nerve repair as well as preparation method and application of composite active dressing

Abstract

The invention provides a composite active dressing for neural restoration and a preparation method and application thereof, and relates to the technical field of medical materials. The composite active dressing comprises a transdermal inner layer and a small extracellular vesicle slow-release outer layer, the transdermal inner layer is of a membrane-shaped structure and comprises a composite active component and a hydrogel matrix material, and the composite active component is selected from one or more of an anti-inflammatory component, a microcirculation improving component, an antioxidant component and a neurotrophic component; the small extracellular vesicle sustained-release outer layer is of a three-dimensional network structure and comprises small extracellular vesicles and a hydrogel matrix material. According to the invention, stable loading and layered deep delivery and release of the small extracellular vesicles are realized, the inflammatory response and microcirculation state of an injured part are improved, proliferation migration and remyelination of Schwann cells can be promoted, and axon regeneration and effective repair of nerve injury can also be promoted.

Description

Technical Field

[0001] This invention relates to the field of medical materials technology, and in particular to a composite active dressing for nerve repair, its preparation method, and its application. Background Technology

[0002] Facial nerve injury is a common clinical condition that can be caused by various factors, including trauma, tumor surgery, inflammation, infection, iatrogenic injury, diabetes, and viral infections. Symptoms often include facial muscle paralysis, incomplete eyelid closure, mouth deviation, drooling, and facial expression disorders, commonly known as facial paralysis (peripheral facial nerve palsy). Facial paralysis not only affects a patient's appearance and emotional expression but can also lead to complications such as corneal exposure and difficulty swallowing, resulting in a significant decline in quality of life. Due to the slow regeneration rate of the facial nerve, persistent local inflammation, damage to Schwann cells, and an ischemic and hypoxic microenvironment unfavorable to axonal regeneration, current treatments (including hormones, neurotrophic drugs, physical therapy, and injection therapy) have limited efficacy, and some patients suffer from long-term functional impairment.

[0003] In recent years, mesenchymal stem cell-derived small extracellular vesicles (SMEVs) have been considered important biotransmitter carriers for promoting peripheral nerve regeneration. They can regulate Schwann cell proliferation, inhibit inflammation, and promote axonal growth by carrying active components such as proteins and miRNAs. However, SMEVs themselves are easily and rapidly eliminated, have a short local duration of action, and the injection method is highly invasive and difficult to maintain a stable and effective concentration, severely limiting their application in facial paralysis and facial nerve injury. Studies have shown that hypoxia-induced SMEVs have stronger neuroprotective, angiogenic, anti-inflammatory, and regenerative capabilities, making them a more promising type of SMEV. However, their delivery method also faces the problem of insufficient stability. In addition, during the recovery process of facial paralysis, abnormal local microenvironment (such as edema, inflammation, and microcirculatory disorders) can further hinder nerve repair. Traditional drug dressings or topical preparations cannot simultaneously provide comprehensive regulatory effects such as anti-inflammatory, blood-activating, and nerve regeneration-promoting effects, nor can they achieve sustained-release delivery of SMEVs.

[0004] Therefore, there is an urgent need for a therapeutic system that can stably load hypoxia-induced small extracellular vesicles and enable them to work synergistically with other complex active ingredients, while also possessing sustained-release properties, improving the local microenvironment, and promoting facial nerve regeneration. Summary of the Invention

[0005] This invention addresses the shortcomings of current systems or methods for treating facial nerve injuries using small extracellular vesicles, which suffer from poor delivery stability and the inability to achieve layered and deep delivery. It provides a composite active dressing for nerve repair, which achieves stable loading and delivery of active ingredients such as small extracellular vesicles through a specific composite structure, and can deliver and release the drug layered and deep to the nerve injury site, thus achieving long-term sustained release of the drug.

[0006] Another object of the present invention is to provide a method for preparing a composite active dressing for nerve repair.

[0007] Another object of the present invention is to provide an application of a composite active dressing for nerve repair in the preparation of nerve injury repair products.

[0008] In a first aspect, the present invention protects a composite active dressing for nerve repair, comprising a transdermal inner layer and a small extracellular vesicle sustained-release outer layer; The transdermal inner layer has a membrane structure, and the raw materials for its preparation include composite active ingredients and hydrogel matrix materials. The composite active ingredients are selected from one or more of anti-inflammatory ingredients, microcirculation-improving ingredients, antioxidant ingredients, and neurotrophic ingredients. The outer layer of the microcellular extracellular vesicle has a three-dimensional network structure. The raw materials for preparation include microcellular extracellular vesicles and hydrogel matrix materials, and the mass concentration of the microcellular extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL.

[0009] According to the present invention, a composite active dressing for nerve repair is preferably provided, wherein the extracellular vesicles are hypoxia-induced mesenchymal stem cell-derived extracellular vesicles, and preferably the extracellular vesicles are cup-shaped structures with an average diameter of 50-150 nm.

[0010] According to the present invention, a composite active dressing for nerve repair is preferably provided in which the raw materials for preparing the transdermal inner layer further include transdermal promoting components, preferably selected from one or more of fatty alcohols, fatty acid derivatives, and surfactants, and the amount added is 0.1~5wt%.

[0011] According to the composite active dressing for nerve repair protected by the present invention, preferably, the raw materials for preparing the transdermal inner layer further include a humectant and a thickener, preferably the humectant is selected from one or more of glycerin, propylene glycol or butylene glycol, and the amount of humectant added is 1~20wt%; The preferred thickener is selected from one or more of carboxymethyl cellulose, xanthan gum, and carbomer, and the amount of thickener added is 0.1~3wt%.

[0012] According to the composite active dressing for nerve repair protected by the present invention, preferably, the thickness of the transdermal inner layer is 0.1~0.5mm, and the thickness of the small extracellular vesicle sustained-release outer layer is 0.5~2.0mm.

[0013] According to the present invention, a composite active dressing for nerve repair is preferably prepared by a cross-linking agent in the raw materials for the preparation of the small extracellular vesicle sustained-release outer layer. The cross-linking agent is preferably selected from natural cross-linking agents or transglutaminase solution, and the amount of cross-linking agent added is 0.1~3wt%.

[0014] According to the present invention, a composite active dressing for nerve repair is preferably made of a hydrogel matrix material selected from one or more of sodium hyaluronate, sodium carboxymethyl cellulose, gelatin, polyvinyl alcohol, chitosan and its biocompatible modifiers.

[0015] According to the present invention, a composite active dressing for nerve repair is preferably further comprising an adhesive layer, wherein the adhesive layer and the transdermal inner layer are preferably a flexible support membrane or a medical polyurethane film.

[0016] Secondly, the present invention also provides a method for preparing a composite active dressing for nerve repair, comprising the following steps: S1. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% inner layer hydrogel matrix solution, then add the composite active ingredients to dissolve and disperse, and let stand to form a film to obtain the transdermal inner layer; S2. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% outer hydrogel precursor solution, then add small extracellular vesicles to uniformly disperse them in the outer hydrogel precursor solution through physical embedding. The mass concentration of the small extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL; S3. The outer hydrogel precursor solution of S2 is coated onto the surface of the transdermal inner layer to form a hydrogel structure at 25~37℃, resulting in a three-dimensional network structure of small extracellular vesicle sustained-release outer layer.

[0017] According to the method for preparing a composite active dressing for nerve repair protected by the present invention, preferably, before adding the composite active ingredients in S1, the method further includes one or more of the following steps: adding transdermal promoting ingredients, moisturizers, and thickeners to the inner layer hydrogel precursor solution. S2 may also include a step of adding a crosslinking agent.

[0018] Thirdly, the present invention also specifically protects the application of a composite active dressing for nerve repair or the composite active dressing for nerve repair prepared by the preparation method of the composite active dressing for nerve repair in the preparation of nerve injury repair products.

[0019] Beneficial effects: This invention provides a composite active dressing for nerve repair. The inner layer of composite active ingredients pre-regulates the microenvironment of local tissues, improving inflammation, enhancing microcirculation, softening the stratum corneum, and promoting penetration, thereby optimizing the nerve regeneration environment. Small extracellular vesicles are fixed in the outer hydrogel layer and stably supplied in a sustained-release form, continuously outputting nerve repair signals with pro-angiogenic, anti-inflammatory, and regenerative effects. This achieves continuous stable loading and layered deep delivery of small extracellular vesicles, improving inflammation and microcirculation, promoting Schwann cell function recovery, and repairing nerve cells, thus providing a new and efficient means for the treatment of facial nerve injury and facial paralysis. Attached Figure Description

[0020] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0021] Figure 1 Transmission electron microscopy images and size distribution diagrams of small extracellular vesicles.

[0022] Figure 2 The graph shows the cumulative drug release rate test results for Example 1 and Comparative Example 1.

[0023] Figure 3 The graph shows the drug penetration effect test results for Example 1 and Comparative Example 1.

[0024] Figure 4 The graph shows the degradation rate measurement results for Example 1 and Comparative Example 1.

[0025] Figure 5 This is a diagram showing the facial phenotype of a rat.

[0026] Figure 6 This is a graph showing the change in facial nerve function scores over time.

[0027] Figure 7 This is an electromyography (EMG) signal.

[0028] Figure 8 This is a histological assessment result of facial nerve injury. Detailed Implementation

[0029] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0030] This invention aims to address the current challenges in nerve injuries, such as peripheral nerve injuries (especially facial nerve injuries and facial paralysis), which often involve persistent local inflammation, significant microcirculatory disturbances, and a poor microenvironment for nerve regeneration. Furthermore, the application of microvesicle-based bioactive substances to nerve injuries often suffers from difficulties in stable and effective delivery to the lesion area, and microneedle loading also struggles to achieve stable loading of microvesicles. Therefore, in a specific embodiment, this invention provides a composite active dressing for nerve repair, comprising a transdermal inner layer and a microvesicle sustained-release outer layer. The transdermal inner layer has a membrane structure, and the raw materials for its preparation include composite active ingredients and hydrogel matrix materials. The composite active ingredients are selected from one or more of anti-inflammatory ingredients, microcirculation-improving ingredients, antioxidant ingredients, and neurotrophic ingredients. The outer layer of the microcellular extracellular vesicle has a three-dimensional network structure. The raw materials for preparation include microcellular extracellular vesicles and hydrogel matrix materials, and the mass concentration of the microcellular extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL.

[0031] It should be noted that: The present invention provides a composite active dressing for nerve repair comprising a transdermal inner layer and a microvesicle sustained-release outer layer. The transdermal inner layer contains a membrane structure with composite active ingredients, which can be absorbed transdermally to the site of action. The inner layer's composite active ingredients pre-treat and regulate local tissue, improving inflammation, enhancing microcirculation, softening the stratum corneum, and promoting penetration, thereby optimizing the nerve regeneration environment and pre-regulating the microenvironment of nerve damage. Furthermore, the microvesicles, fixed within the three-dimensional network structure of the microvesicle sustained-release outer layer, diffuse during use, providing a stable, sustained-release supply, reducing the risk of microvesicle clearance, increasing local concentration, and overcoming the shortcomings of injection methods such as short treatment time and large fluctuations. This continuously delivers nerve repair signals with pro-angiogenic, anti-inflammatory, and regenerative properties, providing a sustained source of nerve repair.

[0032] The present invention provides a composite active dressing for nerve repair, which offers a two-layer composite dressing structure with functional zones. This structure allows the composite active ingredients and hypoxia-induced extracellular vesicles to be spatially layered, temporally gradient-released, and graded-delivered, achieving synergistic effects in biological processes. Ultimately, this forms a local microenvironment regulation system that better conforms to the laws of nerve injury repair, thereby improving nerve repair efficiency and promoting functional recovery.

[0033] Furthermore, the dressing application method avoids the pain of skin puncture and injection, making it more suitable for patients with facial paralysis or facial nerve damage who require long-term intervention.

[0034] Small extracellular vesicles have been shown to have the potential to promote nerve regeneration. Compared to normoxic small extracellular vesicles, hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles exhibit stronger neuroprotective, anti-inflammatory, angiogenic, and Schwann cell proliferation and axonal regeneration-promoting abilities. In some specific embodiments, the small extracellular vesicles mentioned in this invention are hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles, preferably with a goblet-shaped structure, and an average diameter of 50-150 nm, for example, values ​​such as 50 nm, 67 nm, 90 nm, 100 nm, 120 nm, 150 nm, or any other range thereof.

[0035] In some specific embodiments, the hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles mentioned in this invention can be prepared by the following method: (1) Select mesenchymal stem cells (MSCs) obtained from human umbilical cord and culture them to P5-P7 (cells cultured in vitro to the 5th to 7th generation). Expand them to the logarithmic growth phase under normoxic conditions, and then transfer the culture system to a hypoxic environment (preferably oxygen concentration 1-5%) and continue to culture for 24-72 hours to induce MSCs to secrete small extracellular vesicles with enhanced biological activity; (2) After induction, the cell supernatant was collected and small extracellular vesicles were separated and enriched to obtain a precipitate or concentrate containing small extracellular vesicles.

[0036] The resulting small extracellular vesicles can be further resuspended in PBS and stored at 4°C or -80°C as needed.

[0037] In some specific embodiments, the composite active ingredient of the present invention may include, but is not limited to, plant extracts, small molecule anti-inflammatory agents, vitamins, or polypeptide factors, to improve the local microenvironment of the facial nerve injury area. The preferred addition amount, based on the mass of the hydrogel matrix solution, is 0.1~5 wt%, for example, it can be point values ​​such as 0.1 wt%, 1 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, or any range thereof. In some specific embodiments, in order to further enhance the system's ability to promote stratum corneum hydration and enhance transdermal penetration, and to promote the subsequent permeation and absorption of hypoxia-induced extracellular vesicles, the transdermal inner layer mentioned in this invention also includes transdermal promoting components, which can be selected from one or more of fatty alcohols, fatty acid derivatives, and surfactants, with an addition amount of 0.1~5wt%, for example, it can be point values ​​such as 0.1wt%, 1wt%, 2wt%, 3wt%, 4wt%, 5wt%, or any range of values.

[0038] In some specific embodiments, in order to improve skin adhesion and softness, the transdermal inner layer mentioned in this invention also includes a moisturizer and a thickener.

[0039] In some specific exemplary embodiments, the moisturizer mentioned in this invention may be selected from one or more of glycerin, propylene glycol or butylene glycol, and the amount of moisturizer added is 1 to 20 wt%, for example, it may be a point value of 1 wt%, 5 wt%, 8 wt%, 10 wt%, 15 wt%, 20 wt% or any range of values.

[0040] In some specific exemplary embodiments, the thickener mentioned in this invention may be selected from one or more of carboxymethyl cellulose, xanthan gum, and carbomer, and the amount of thickener added is 0.1~3wt%, for example, it may be a point value of 0.1wt%, 0.5wt%, 1wt%, 2wt%, 3wt%, or any range of values.

[0041] The amounts of transdermal promoting ingredients, moisturizers, and thickeners added to the transdermal inner layer mentioned in this invention are calculated based on the mass of the hydrogel matrix solution.

[0042] In some specific embodiments, in order to better match the drug release rate and normal treatment time (less than or equal to 30 minutes) and achieve better graded delivery effect, the thickness of the transdermal inner layer mentioned in this invention is 0.1~0.5 mm, and the thickness of the small extracellular vesicle sustained-release outer layer is 0.5~2.0 mm.

[0043] In some specific embodiments, in order to form a stable three-dimensional network structure, the small extracellular vesicle sustained-release outer layer mentioned in this invention also includes a cross-linking agent. Preferably, the cross-linking agent is selected from natural cross-linking agents or transglutaminase solution. The amount of cross-linking agent added is 0.1~3wt% (based on the mass of the hydrogel matrix solution), for example, it can be a point value or any range of values ​​such as 0.1wt%, 0.5wt%, 0.8wt%, 1.0wt%, 1.2wt%, 1.5wt%, 1.8wt%, 2.0wt%, 2.5wt%, 3.0wt%.

[0044] In some specific embodiments, the hydrogel matrix material mentioned in this invention is a biocompatible hydrogel material with good skin affinity and film-forming properties. For example, it can be selected from one or more of sodium hyaluronate, sodium carboxymethyl cellulose, gelatin, polyvinyl alcohol, chitosan and its biocompatible modifiers.

[0045] In some specific embodiments, the composite active dressing for nerve repair of the present invention can be obtained by preparing the transdermal inner layer and the small extracellular vesicle sustained-release outer layer separately, and then assembling them into a whole. To achieve stable overall performance, strong interlayer bonding, and convenient application, the following assembly method is preferred: Using the transdermal inner layer as the base structure, a small extracellular vesicle sustained-release outer layer hydrogel precursor solution is coated on the inner layer surface, allowing the two layers to fully contact and fuse before complete gelation, causing the outer layer hydrogel to cross-link or gel, forming a stable composite film (a composite active dressing for nerve repair).

[0046] The transdermal inner layer is placed in a sterile working environment, maintaining its surface smooth and moist. The inner layer typically takes the form of a flexible hydrogel film or mask, possessing high water content and good adhesion, serving as the base structure for a bilayer dressing. Subsequently, the prepared small extracellular vesicle sustained-release outer layer hydrogel precursor solution is uniformly spread onto the inner layer surface, allowing the two layers to fully contact and fuse before complete gelation. To avoid air bubbles or interlayer voids, light scraping, self-leveling spreading, or mold pressing methods can be used to form a uniform and continuous film structure of the outer layer on the inner layer. No additional barrier layer is required between the two layers, allowing the small extracellular vesicles to migrate from the outer layer to the inner layer via aqueous diffusion during subsequent use.

[0047] Subsequently, the coated bilayer structure was placed under mild conditions of 25–37°C to allow the outer hydrogel to crosslink or gel, forming a stable composite film. After gelation, the outer and inner layers are firmly bonded together through physical association, hydrogen bonding, or partial crosslinking bridging, forming a continuous bilayer dressing structure. The resulting bilayer composite active dressing for nerve repair is transparent or translucent, with clear internal layers, good softness, and can naturally conform to the facial contours.

[0048] Furthermore, a flexible support film or medical polyurethane film can be adhered to the back (opposite side of the outer layer) of the composite active dressing used for nerve repair to enhance the dressing's mechanical strength, facilitate its removal and handling, and improve its fixation at the application site. The resulting double-layer dressing can be cut into sheet, half-face, or full-face mask shapes, suitable for covering facial skin along the facial nerve pathway.

[0049] In some specific embodiments, the composite active dressing for nerve repair mentioned in this invention further includes an adhesive layer, wherein the adhesive layer and the transdermal inner layer are preferably a flexible support membrane or a medical polyurethane film.

[0050] In a specific embodiment, the present invention also provides a method for preparing a composite active dressing for nerve repair, comprising the following steps: S1. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% inner layer hydrogel matrix solution, then add the composite active ingredients to dissolve and disperse, and let stand to form a film to obtain the transdermal inner layer; S2. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% outer hydrogel precursor solution, then add small extracellular vesicles to uniformly disperse them in the outer hydrogel precursor solution through physical embedding. The mass concentration of the small extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL; S3. The outer hydrogel precursor solution of S2 is coated onto the surface of the transdermal inner layer to form a hydrogel structure at 25~37℃, resulting in a three-dimensional network structure of small extracellular vesicle sustained-release outer layer.

[0051] In some embodiments, the hydrogel matrix solutions mentioned in S1 and S2 of this aspect can be prepared by dissolving the hydrogel matrix material in deionized water or a buffer solution. To promote dissolution, for example, magnetic stirring for 20-30 minutes can be used. In some specific embodiments, the preparation method mentioned in this invention further includes, before adding the composite active ingredient in S1, one or more of the following steps: adding a transdermal promoting ingredient (0.1-5 wt% by mass of the hydrogel matrix solution), a moisturizer (1-20 wt% by mass of the hydrogel matrix solution), and a thickener (0.1-3 wt% by mass of the hydrogel matrix solution) to the inner hydrogel precursor solution.

[0052] In some specific embodiments, the preparation method mentioned in this invention further includes a step of adding a crosslinking agent in S2, wherein the amount of crosslinking agent added is 0.1~3wt% (based on the mass of the hydrogel matrix solution).

[0053] In a specific embodiment, the present invention also provides the application of a composite active dressing for nerve repair or the composite active dressing for nerve repair prepared by the preparation method of the composite active dressing for nerve repair in the preparation of nerve injury repair products.

[0054] The small extracellular vesicles mentioned in the following embodiments and comparative examples of this invention are hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles, prepared by the following method: (1) Select mesenchymal stem cells (MSCs) obtained from healthy human umbilical cords, culture them to P5-P7, and expand them to the logarithmic growth phase under normoxic conditions. Then, transfer the culture system to a hypoxic environment (preferably oxygen concentration 1-5%) and continue to culture for 24-72 hours to induce MSCs to secrete small extracellular vesicles with enhanced biological activity; (2) After induction, the cell supernatant was collected, and the cells were removed by centrifugation at 300–500g. Cell debris was removed by centrifugation at 2000g. Then, 0.22μm filtration was used to further remove large impurities. The filtered supernatant was separated and enriched by ultracentrifugation (e.g., 100000g, 70–120min) or ultrafiltration concentration to obtain a precipitate or concentrate containing small extracellular vesicles. The resulting small extracellular vesicles can be further resuspended in PBS and stored at 4°C or -80°C as needed; (3) Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM) were used to detect the characteristics of small extracellular vesicles. At the same time, Western blotting was used to detect markers such as CD63, CD81, and TSG101 to confirm the characteristics of small extracellular vesicles.

[0055] Figure 1 Transmission electron microscopy images and size distribution diagrams of small extracellular vesicles, such as... Figure 1 The small extracellular vesicles shown have a typical cup-shaped structure with an average diameter of 67 nm.

[0056] Example 1 A composite active dressing for nerve repair, comprising a transdermal inner layer and a small extracellular vesicle sustained-release outer layer; The transdermal inner layer has a membrane structure, including a complex active ingredient, a hydrogel matrix material, a moisturizer (glycerin), and a transdermal penetration enhancer (propylene glycol). The complex active ingredient is Centella asiatica extract. The outer layer of the small extracellular vesicle sustained-release structure is a three-dimensional network structure, comprising small extracellular vesicles, hydrogel matrix material, and cross-linking agent. The mass concentration of the small extracellular vesicles is 1×10⁻⁶. 9 Particles / mL; The thickness of the transdermal inner layer is 0.3 mm, and the thickness of the small extracellular vesicle sustained-release outer layer is 1.0 mm.

[0057] This embodiment also specifically provides a method for preparing the above-mentioned composite active dressing for nerve repair, which specifically includes the following steps: S1. Take 80 mL of deionized water into a clean container, add 1.0 g of sodium carboxymethyl cellulose (CMC-Na), and stir magnetically for 20 min at room temperature to allow it to swell fully and form a homogeneous viscous liquid. Then add 0.5 g of sodium hyaluronate (molecular weight 100~300 kDa), and continue stirring until completely dissolved to obtain a hydrogel matrix solution; Add 5g of glycerin to the above hydrogel matrix solution as a moisturizer to improve softness and adhesion, and add 2g of butylene glycol as a mild transdermal penetration enhancer to enable the system to promote stratum corneum hydration and enhance transdermal penetration. Then add 1.0g of Centella Asiatica extract powder (or an equal volume of solution) as the active ingredient for anti-inflammatory and microcirculation improvement, and stir continuously at room temperature for 10 minutes to completely dissolve and evenly disperse it. The resulting mixed solution is poured into a pre-prepared mask mold or flat mold, with the thickness controlled at 0.3 mm. The mold is left to stand for 6 hours to allow the gel structure to gradually form and stabilize into a film. After demolding, a transparent or translucent flexible hydrogel film, i.e., the transdermal inner layer, can be obtained. S2. Place 70 mL of deionized water in a sterile beaker and keep it at a constant temperature of 37°C in a water bath. Add 2.0 g of gelatin and slowly dissolve it under magnetic stirring until a clear and homogeneous solution is formed. Then add 0.5 g of sodium hyaluronate and continue stirring for 20 min to fully dissolve it, obtaining a hyaluronic acid-gelatin mixed hydrogel matrix solution. After the solution has cooled naturally to 25–30°C, add a pre-prepared solution of hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles (MSC-hyposEVs) resuspended in PBS, bringing the final concentration of small extracellular vesicles to 1 × 10⁻⁶. 9 To avoid damaging the activity of small extracellular vesicles, after adding small extracellular vesicles, only gentle stirring or light shaking for 4 minutes is used to ensure that they are evenly dispersed in the hydrogel matrix, avoiding vigorous stirring or foaming. After the small extracellular vesicles are uniformly dispersed, 0.1g of the mild crosslinking agent, natural crosslinking agent giridinium, is added to the above mixed solution and gently mixed to form a crosslinkable small extracellular vesicle hydrogel precursor solution. Slowly pour the precursor solution onto the surface of the formed transdermal inner layer hydrogel film, keeping the thickness at 1.0 mm, taking care to avoid generating air bubbles; S3. Place the mold coated with the small extracellular vesicle precursor solution at 25-37℃ and let it stand for 10 hours to allow the cross-linking reaction to proceed gradually, forming a transparent or semi-transparent small extracellular vesicle sustained-release hydrogel layer with certain mechanical strength and elasticity.

[0058] After gelation, the outer and inner layers are firmly bonded together through physical association, hydrogen bonding, or partial cross-linking bridging, forming a continuous, two-layer dressing structure. The resulting two-layer composite dressing is transparent or translucent, with clear internal layers, good softness, and can naturally conform to the contours of the face.

[0059] The inner membrane (transdermal inner layer) prepared in this embodiment has high water content and good adhesion. When applied to facial skin, it can form a moist microenvironment, soften the stratum corneum, and improve skin permeability. The Centella asiatica extract in the inner layer can be gradually released in the superficial layer of the skin to exert anti-inflammatory and blood flow-improving effects; glycerin and butylene glycol can further enhance the transdermal penetration efficiency of subsequent outer layer microcellular extracellular vesicles.

[0060] Furthermore, a flexible support film or medical polyurethane film can be laminated to the back of the composite dressing (the side opposite to the outer layer) to enhance the dressing's mechanical strength, facilitate its removal and handling, and improve its fixation at the application site. The resulting double-layer dressing can be cut into sheet, half-face, or full-face mask shapes, suitable for covering facial skin along the facial nerve pathway.

[0061] Example 2 A composite active dressing for nerve repair is basically the same as that in Example 1, except that the composite active ingredient in the transdermal inner layer is curcumin, and the amount added is the same as in Example 1.

[0062] Example 3 A composite active dressing for nerve repair is basically the same as in Example 1, except that the outer layer of the small extracellular vesicle does not contain a cross-linking agent and contains an equal amount of co-methylcellulose.

[0063] Example 4 A composite active dressing for nerve repair is basically the same as in Example 1, except that the thickness of the transdermal inner layer is 1.0 mm and the thickness of the small extracellular vesicle sustained-release outer layer is 2.0 mm.

[0064] Increasing the thickness of the transdermal inner layer and the small extracellular vesicle sustained-release outer layer will slow down the overall drug release rate. Comparative Example 1 A conventional gel, differing from Example 1 in that it does not contain a transdermal inner layer, is prepared as follows: Take 70 mL of deionized water and place it in a sterile beaker. Keep the temperature at 37°C in a water bath. Add 2.0 g of gelatin and slowly dissolve it under magnetic stirring until a clear and homogeneous solution is formed. Then add 0.5 g of sodium hyaluronate and continue stirring for 20 min to fully dissolve it, thus obtaining a hyaluronic acid-gelatin mixed hydrogel matrix solution. After the solution has cooled naturally to 25–30°C, add a pre-prepared solution of hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles (MSC-hyposEVs) resuspended in PBS, bringing the final concentration of small extracellular vesicles to 1 × 10⁻⁶. 9 To avoid damaging the activity of small extracellular vesicles, after adding small extracellular vesicles, only gentle stirring or light shaking for 4 minutes is used to ensure that they are evenly dispersed in the hydrogel matrix, avoiding vigorous stirring or foaming. After the small extracellular vesicles are uniformly dispersed, 0.1g of the mild crosslinking agent, natural crosslinking agent giridinium, is added to the above mixed solution and gently mixed to form a crosslinkable small extracellular vesicle hydrogel precursor solution. Slowly pour the precursor solution onto the mold surface, keeping the thickness to 1.0 mm, taking care to avoid generating air bubbles; S3. Place the mold coated with the small extracellular vesicle precursor solution at 25-37℃ and let it stand for 10 hours to allow the cross-linking reaction to proceed gradually, forming a transparent or semi-transparent small extracellular vesicle sustained-release hydrogel layer with certain mechanical strength and elasticity.

[0065] Comparative Example 2 An active dressing, differing from Example 1 in that it has no layered structure, and its specific preparation method is as follows: Take 70 mL of deionized water and place it in a sterile beaker. Keep the temperature at 37°C in a water bath. Add 2.0 g of gelatin and slowly dissolve it under magnetic stirring until a clear and homogeneous solution is formed. Then add 0.5 g of sodium hyaluronate and continue stirring for 15–20 min to fully dissolve it, thus obtaining a hyaluronic acid-gelatin mixed hydrogel matrix solution. After the solution has cooled naturally to 25–30°C, add 5g of glycerin as a moisturizer to the above hydrogel matrix solution to improve softness and adhesion, then add 2g of butylene glycol as a mild transdermal penetration enhancer, followed by 1.0g of Centella asiatica extract powder (or an equal volume solution) as an anti-inflammatory and microcirculation-improving active ingredient. Stir continuously at room temperature for 15 minutes to ensure complete dissolution and uniform dispersion. Finally, add a pre-prepared solution of hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles (MSC-hyposEV) resuspended in PBS to achieve a final MSC concentration of 1×10⁻⁶. 9 After adding small extracellular vesicles at a particle / mL ratio, gently stir or gently shake for 4 minutes to ensure uniform dispersion in the hydrogel matrix, avoiding vigorous stirring or foaming. After the small extracellular vesicles are uniformly dispersed, 0.1g of the natural cross-linking agent gelidinium is added to the above mixed solution and gently mixed to form a cross-linkable small extracellular vesicle hydrogel precursor solution. Slowly pour the precursor solution into the mold, keeping the thickness to 1.0 mm, taking care to avoid air bubbles; place the mold with the small extracellular vesicle precursor solution at 25-37°C and let it stand for 10 hours to allow the cross-linking reaction to proceed gradually, forming a small extracellular vesicle sustained-release hydrogel layer.

[0066] The complex active ingredients, moisturizers, and transdermal penetration enhancers in the active dressing interact with microvesicles, causing the microvesicle membranes to rupture and preventing the construction of an effective graded delivery system.

[0067] Comparative Example 3 A composite dressing comprising a gel inner layer and a microcellular extracellular vesicle sustained-release outer layer, differing from Example 1 in that the gel inner layer does not contain composite active ingredients.

[0068] This comparative example also provides a specific method for preparing the aforementioned composite active dressing for nerve repair, which includes the following steps: S1. Take 80 mL of deionized water into a clean container, add 1.0 g of sodium carboxymethyl cellulose (CMC-Na), and stir magnetically for 20-30 min at room temperature to allow it to swell fully and form a homogeneous viscous liquid. Then add 0.5 g of sodium hyaluronate (molecular weight 100-300 kDa), and continue stirring until completely dissolved to obtain a hydrogel matrix solution; The resulting mixed solution is poured into a pre-prepared mask mold or flat mold, with the thickness controlled at 0.5~1.0 mm. The mold is placed in a 4℃ refrigerated environment and left to stand for 4~8 hours to allow the gel structure to gradually form and stabilize into a film. After demolding, a transparent or translucent flexible hydrogel film can be obtained. S2. Place 70 mL of deionized water in a sterile beaker and keep it at a constant temperature of 37°C in a water bath. Add 2.0 g of gelatin and slowly dissolve it under magnetic stirring until a clear and homogeneous solution is formed. Then add 0.5 g of sodium hyaluronate and continue stirring for 15–20 min to fully dissolve it, obtaining a hyaluronic acid-gelatin mixed hydrogel matrix solution. After the solution temperature naturally cools to 25-30℃, add a pre-prepared solution of hypoxia-induced mesenchymal stem cell-derived small extracellular vesicles (MSC-hyposEV) that has been resuspended in PBS, so that the final concentration of small extracellular vesicles is 300 μg / mL. To avoid damage to the activity of small extracellular vesicles, after adding the small extracellular vesicles, only use gentle stirring or light shaking for 3-5 minutes to make them evenly dispersed in the hydrogel matrix, and avoid vigorous stirring or foaming. After the small extracellular vesicles are uniformly dispersed, a small amount of mild cross-linking agent, such as 0.1g of the natural cross-linking agent giridinium, is added to the above mixed solution and gently mixed to form a cross-linkable small extracellular vesicle hydrogel precursor solution. Slowly pour the precursor solution onto the surface of the formed transdermal inner layer hydrogel film, keeping the thickness between 0.5 and 1.0 mm, taking care to avoid generating air bubbles; S3. Place the mold containing the small extracellular vesicle precursor solution at 25-37°C and let it stand for 10 hours to allow the cross-linking reaction to proceed gradually, forming a transparent or semi-transparent small extracellular vesicle sustained-release hydrogel layer.

[0069] The inner layer structure of the flexible hydrogel film alone cannot open the skin surface in advance to promote the transdermal absorption of subsequent extracellular vesicles, nor can it optimize the nerve regeneration environment of extracellular vesicles. Instead, it forms a barrier to the sustained release of extracellular vesicles to the site of action, which is not conducive to the penetration of extracellular vesicles.

[0070] Result detection (1) Drug release effect detection Equal volumes of drug-loaded hydrogel samples were placed in the same release medium and incubated with shaking at 37°C. A certain volume of the released solution was collected at predetermined time points for analysis, and an equal volume of fresh release medium was added. After centrifugation or filtration, the drug concentration in the collected release solution was determined by HPLC. The cumulative release amount and cumulative release rate at each time point were calculated using the cumulative release calculation formula under replenishment conditions.

[0071] Figure 2 The graph shows the cumulative drug release rate test results for Example 1 and Comparative Example 1. It can be seen that in the short-time (60 min) release experiment, the composite gel exhibits a slower and more stable release kinetics, which contrasts with the rapid release of the conventional gel in Example 1, and is beneficial for achieving controllable drug release.

[0072] (2) Drug penetration effect detection Fresh pig skin was collected, subcutaneous fat was removed, and the skin was thoroughly cleaned. Fluorescently labeled extracellular vesicles were loaded onto a hydrogel and evenly applied to the stratum corneum of the pig skin. The gel was then incubated at 37°C for a specified time. After treatment, the pig skin was removed, rinsed with PBS to remove any residual sample, and then frozen sectioned. The skin sections were observed using a confocal laser scanning microscope to assess the transdermal permeability of the extracellular vesicles.

[0073] Figure 3 The graph shows the drug penetration effect test results for Example 1 and Comparative Example 1. Figure 3 The composite gel of the present invention shows that, compared with the conventional single gel of Comparative Example 1, the drug penetration rate is significantly improved, demonstrating superior transdermal drug delivery capability.

[0074] (3) Stability testing Hydrogel samples of identical shape and mass were placed in a lysozyme-containing medium and allowed to stand at 37°C. At predetermined time points, the samples were removed, excess solution was gently aspirated from the surface, and the samples were weighed, recording the mass change. The structural stability of the hydrogels in a simulated physiological environment was evaluated by comparing the rate of mass change at different time points.

[0075] Figure 4 The graph shows the degradation rate measurement results for Example 1 and Comparative Example 1. Figure 4 As can be seen, in the long-term (7-day) release experiment, the degradation rate of the composite gel of the present invention was significantly lower than that of the conventional single gel of Comparative Example 1 throughout the entire 7-day period, and it has a sustained release effect.

[0076] (4) Detection of nerve repair effect Healthy adult SD rats were randomly divided into a sham-operated group, a facial nerve injury group, and a facial nerve injury repair group. Under general anesthesia, the skin was incised in the postauricular region to expose the main trunk of the facial nerve. In the injury and repair groups, standardized injury treatment was performed on the facial nerve to establish a facial nerve injury model. In the sham-operated group, only the nerve was exposed without causing damage. After model establishment, the composite active dressing for nerve repair described in this invention was immediately applied to the skin surface of the repair group after the incision was closed, while the facial nerve injury group did not receive this dressing treatment.

[0077] Postoperatively, changes in facial phenotype in rats of each group were observed regularly, including whisker movement, eyelid closure, and facial symmetry. Facial nerve function was also assessed. Simultaneously, relevant electrophysiological signals were recorded at different postoperative time points to evaluate the recovery of facial nerve conduction and innervation muscle function. The results showed that rats in the facial nerve injury group exhibited significant facial asymmetry, limited whisker movement, and elevated facial nerve function scores. In contrast, rats in the repair group showed significant improvement in facial phenotype, with facial nerve function scores gradually decreasing over time, and were significantly better than the unrepaired injury group in the mid-to-late postoperative period. Further electrophysiological testing revealed significantly enhanced electromyographic responses in the repair group, indicating that the composite active dressing for nerve repair of this invention can effectively promote facial nerve regeneration and improve neuromuscular function recovery.

[0078] Figure 5 The facial phenotypes of rats in the three experimental groups are shown. The sham-operated group maintained normal symmetry, the facial nerve injury group showed obvious facial asymmetry and limited beard movement, and the repair group showed significant improvement in appearance.

[0079] Figure 6 This graph shows the change in facial nerve function score over time. The score is calculated on a 0-5 scale, with 0 representing a completely normal and healthy state, and higher scores indicating more severe nerve damage. The facial nerve injury group showed a rapid increase in score post-surgery and maintained a high level throughout the follow-up period. The facial nerve injury plus repair group showed a gradual decrease in score post-surgery, and the score was significantly lower than the non-repair group from days 7 to 14, indicating that the repair treatment effectively promoted the recovery of facial nerve function.

[0080] Figure 7 The results are electromyographic (EMG) signals. The sham-operated group recorded normal, high-amplitude EMG waveforms, while the facial nerve injury group showed almost no significant response, indicating severe nerve conduction impairment. The repair group recovered detectable EMG responses with significantly higher amplitudes than the injury group, further demonstrating the significant advantages of repair treatment in promoting facial nerve regeneration and restoring neuromuscular function.

[0081] Figure 8 For histological evaluation after facial nerve injury.

[0082] The top row shows the morphology of the facial nerve in a cross-section as revealed by hematoxylin-eosin staining. In the sham-operated group, regularly arranged nerve fiber structures with intact morphology were observed; in the facial nerve injury group, significant nerve fiber disorder, axonal degeneration, and lighter staining were observed; in the repair group, the nerve fiber arrangement was more regular than in the injury group, and the axonal morphology was improved, suggesting that repair treatment helps promote the recovery of nerve structure. The bottom row shows the Nissl staining results. In the sham-operated group, a large number of intact and clearly defined Nissl bodies were observed, indicating a normal neuronal state; in the facial nerve injury group, the number of Nissl bodies was significantly reduced and their distribution was uneven, indicating severe neuronal damage; in the facial nerve injury plus repair group, the number of Nissl bodies was significantly restored, and the staining was clearer and more uniform, indicating that repair treatment effectively promoted neuronal survival and regeneration.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composite active dressing for nerve repair, characterized in that, Including the transdermal inner layer and the small extracellular vesicle sustained-release outer layer; The transdermal inner layer has a membrane structure, and the raw materials for its preparation include composite active ingredients and hydrogel matrix materials. The composite active ingredients are selected from one or more of anti-inflammatory ingredients, microcirculation-improving ingredients, antioxidant ingredients, and neurotrophic ingredients. The outer layer of the microcellular extracellular vesicle has a three-dimensional network structure. The raw materials for preparation include microcellular extracellular vesicles and hydrogel matrix materials, and the mass concentration of the microcellular extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL.

2. The composite active dressing for nerve repair according to claim 1, characterized in that, The small extracellular vesicles are derived from hypoxia-induced mesenchymal stem cells, preferably with a cup-shaped structure and an average diameter of 50-150 nm.

3. The composite active dressing for nerve repair according to claim 1 or 2, characterized in that, The raw materials for preparing the transdermal inner layer also include transdermal penetration promoting components, preferably selected from one or more of fatty alcohols, fatty acid derivatives, and surfactants, with an addition amount of 0.1~5 wt%. And / or, the raw materials for preparing the transdermal inner layer also include a humectant and a thickener. Preferably, the humectant is selected from one or more of glycerin, propylene glycol or butylene glycol, and the amount of humectant added is 1 to 20 wt%. Preferably, the thickener is selected from one or more of carboxymethyl cellulose, xanthan gum or carbomer, and the amount of thickener added is 0.1 to 3 wt%.

4. The composite active dressing for nerve repair according to any one of claims 1 to 3, characterized in that, The thickness of the transdermal inner layer is 0.1~0.5mm, and the thickness of the small extracellular vesicle sustained-release outer layer is 0.5~2.0mm.

5. The composite active dressing for nerve repair according to any one of claims 1 to 4, characterized in that, The raw materials for preparing the sustained-release outer layer of the small extracellular vesicles also include a cross-linking agent, preferably selected from natural cross-linking agents or transglutaminase solution, and the amount of cross-linking agent added is 0.1~3wt%.

6. The composite active dressing for nerve repair according to any one of claims 1 to 5, characterized in that, The hydrogel matrix material is selected from one or more of sodium hyaluronate, sodium carboxymethyl cellulose, gelatin, polyvinyl alcohol, chitosan and their biocompatible modifiers.

7. The composite active dressing for nerve repair according to any one of claims 1 to 8, characterized in that, The composite active dressing for nerve repair also includes an adhesive layer, which is preferably a flexible support membrane or a medical polyurethane film, and is connected to the transdermal inner layer.

8. A method for preparing a composite active dressing for nerve repair, characterized in that, Includes the following steps: S1. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% inner layer hydrogel matrix solution, then add the composite active ingredients to dissolve and disperse, and let stand to form a film to obtain the transdermal inner layer; S2. Dissolve the hydrogel matrix material to obtain a 0.5~5wt% outer hydrogel precursor solution, then add small extracellular vesicles to uniformly disperse them in the outer hydrogel precursor solution through physical embedding. The mass concentration of the small extracellular vesicles is 1×10⁻⁶. 8 ~10 12 Particles / mL; S3. The outer hydrogel precursor solution of S2 is coated onto the surface of the transdermal inner layer to form a hydrogel structure at 25~37℃, resulting in a three-dimensional network structure of small extracellular vesicle sustained-release outer layer.

9. The method for preparing the composite active dressing for nerve repair according to claim 8, characterized in that, Before adding the complex active ingredients in S1, the process also includes adding one or more of the following to the inner hydrogel precursor solution: transdermal penetration enhancer, moisturizer, and thickener. S2 may also include a step of adding a crosslinking agent.

10. The application of a composite active dressing for nerve repair prepared by any one of claims 1 to 7 or by the preparation method of the composite active dressing for nerve repair according to claim 8 or 9 in the preparation of nerve injury repair products.

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