An artificial skin with antibacterial and inflammation regulating functions and a preparation method thereof

Abstract

The application particularly relates to an artificial skin with antibacterial and inflammation regulating functions and a preparation method thereof, and belongs to the technical field of artificial skin. The artificial skin is obtained by combining a dermis layer formed by doping silver sulfadiazine and loading simvastatin polylactic acid microspheres in collagen as a carrier material with a silicone epidermis layer. The silver sulfadiazine loaded in the collagen matrix can effectively avoid bacterial infection, the slow-release function of the polylactic acid microspheres can release simvastatin in the later stage of skin regeneration, promote the conversion of macrophages to M2 type, participate in the anti-inflammatory and repair processes, and accelerate skin regeneration. The silicone epidermis layer can prevent the loss of body fluid and bacterial invasion.

Description

Technical Field

[0001] This invention belongs to the field of artificial skin technology, and specifically relates to an artificial skin with antibacterial and anti-inflammatory functions and its preparation method. Background Technology

[0002] The skin is the body's natural protective layer, isolating the body from direct contact with the external environment and preventing harm from harmful substances. However, because a large area of ​​the skin is exposed to the external environment, it is susceptible to damage. Common skin injuries in daily life include trauma, burns, and bacterial infections. Currently, biomaterials are being used for skin regeneration, and various types of artificial skin have been successfully developed. These artificial skins possess good cytoplasmic proliferation activity, a certain degree of mechanical strength, and are inexpensive. However, safety risks such as bacterial infection and inflammatory reactions still exist.

[0003] As the body's first line of defense, the skin's barrier function is compromised when damaged, making it easier for bacteria to invade and increasing the risk of infection. Bacterial infections can hinder skin regeneration and, in severe cases, even endanger life. The most common treatments for bacterial infections are oral or intravenous medications, which have drawbacks such as low absorption and utilization efficiency and unnecessary systemic side effects. Topical medications, on the other hand, are simple to administer and have fewer systemic side effects, making them a very promising option.

[0004] Skin regeneration after injury mainly involves four processes: inflammatory response, wound contraction, granulation tissue proliferation and scar formation, and regeneration of the epidermis and other tissues. Inflammation plays a dual role. On the one hand, the inflammatory response facilitates the recruitment of macrophages to phagocytose tissue debris, a crucial step in skin regeneration. On the other hand, long-term, repeated inflammatory responses may lead to the formation of non-healing chronic wounds. Macrophages play a vital role in inflammation regulation. There are two macrophage phenotypes: pro-inflammatory (M1) and anti-inflammatory (M2). Under different pathophysiological conditions, macrophages can switch between these two phenotypes. M1 macrophages can release substances such as IL-1β and IL-6 to promote the inflammatory process, while M2 macrophages can release anti-inflammatory factors such as IL-10 and TGF-β to promote wound healing. Summary of the Invention

[0005] The purpose of this application is to provide an artificial skin with antibacterial and anti-inflammatory functions and a method for preparing the same, in order to solve the problems of bacterial infection and inflammatory response that exist after treatment with artificial skin.

[0006] The artificial skin with antibacterial and anti-inflammatory functions provided in this embodiment of the invention uses collagen as a carrier material. The dermis is formed by doping silver sulfadiazine and polylactic acid microspheres loaded with simvastatin. The resulting dermis is then combined with a silicone epidermis to obtain the artificial skin.

[0007] The dermis layer was obtained through the following method:

[0008] (1) Dissolve polylactic acid and simvastatin in dichloromethane, then slowly add polyvinyl alcohol solution, stir the solution for a period of time, centrifuge, pour off the supernatant, take the remaining precipitate, dry it to obtain polylactic acid microspheres loaded with simvastatin.

[0009] (2) Collagen was dissolved in dilute acetic acid solution, silver sulfadiazine powder and polylactic acid microspheres loaded with simvastatin were added, and after stirring evenly, 1-ethyl-(3-dimethylaminopropyl)carbodiimide and N-hydroxysuccinimide (EDC / NHS) were added, and after stirring evenly, the mixture was injected into a mold and freeze-dried to obtain the dermis.

[0010] (3) The obtained dermis layer and silicone epidermis layer are combined with medical adhesive to obtain artificial skin.

[0011] Optionally, the molecular weight of polylactic acid is 5–60 kDa.

[0012] Optionally, the mass ratio of simvastatin to polylactic acid is 0.1 to 1:10.

[0013] Optionally, the concentration of polyvinyl alcohol is 1-3%.

[0014] Optionally, the stirring speed is 500–1500 rpm and the stirring time is 1–5 h.

[0015] Optionally, the concentration of dilute acetic acid is 0-1%, and the concentration of collagen solution is 5-20 mg / mL.

[0016] Optionally, the mass ratio of silver sulfadiazine powder, polylactic acid microspheres loaded with simvastatin, and collagen is 0.1–0.5:0.5–1:10.

[0017] Optionally, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is 50–150 mmol / L, and the concentration of N-hydroxysuccinimide (NHS) is 25–75 mmol / L.

[0018] Optionally, the freeze-drying temperature is -50 to -40℃ for 2 hours; the sublimation temperature is -50 to -40℃ for 24 hours; and the re-drying temperature is 30 to 35℃ for 24 hours.

[0019] Optionally, the thickness of the silicone epidermis is 100–300 μm.

[0020] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0021] (1) Freeze-drying yields a porous structure similar to that of the dermis, which is beneficial for cell adhesion and proliferation. Furthermore, the dermis can be used to load drugs for in-situ release, improving drug utilization and reducing systemic side effects.

[0022] (2) The combined use of the two drugs can avoid the risk of infection and accelerate the skin regeneration process. The loaded silver sulfadiazine can prevent bacterial infection, and the drug-loaded polylactic acid microspheres can release simvastatin in the later stage of skin regeneration to control the conversion of macrophages from M1 to M2 type, and transition from the inflammatory stage to the proliferative stage of wound repair.

[0023] (3) The raw materials are widely available, the production process is simple, and it is suitable for mass production.

[0024] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, and in order to make the above and other objects, features and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described below. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a flowchart of the method provided in an embodiment of the present invention. Detailed Implementation

[0027] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0028] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In the event of any conflict, this specification shall prevail.

[0029] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0030] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0031] During the invention process, the applicant discovered that current artificial skin products often carry the risk of bacterial infection during treatment, and that the artificial skin material may also pose a risk of inflammatory reaction due to prolonged coverage of the wound.

[0032] The combined use of silver sulfadiazine and simvastatin can effectively prevent bacterial infection and regulate the validation reaction.

[0033] According to a typical embodiment of the present invention, an artificial skin is provided, the raw materials of which include: collagen, silver sulfadiazine and polylactic acid microspheres loaded with simvastatin.

[0034] Silver sulfadiazine loaded in the collagen matrix can effectively prevent bacterial infection, and the sustained-release function of polylactic acid microspheres can release simvastatin in the later stage of skin regeneration, promote the transformation of macrophages to M2 type, participate in the anti-inflammatory and repair process, and accelerate skin regeneration.

[0035] In some embodiments, the molecular weight of polylactic acid is 20–60 kDa.

[0036] Controlling the molecular weight of polylactic acid (PLA) is crucial for controlling the degradation rate of PLA microspheres. If the PLA molecular weight is too small, the microspheres degrade too quickly, leading to a rapid release of simvastatin and inhibiting the essential inflammatory response (phagocytic cell debris, invading microorganisms) necessary for tissue regeneration, thus hindering wound healing. Conversely, a molecular weight that is too large results in slow drug release, prolonging the inflammatory response.

[0037] In some embodiments, the concentration of dilute acetic acid is 0-1%, and the concentration of collagen solution is 5-20 mg / mL.

[0038] The addition of acetic acid increases the amount of collagen that can be dissolved, but too high an acetic acid content will result in solvent residue after freeze-drying; too low a collagen content will lead to poor mechanical properties of the dermis after freeze-drying, making it easy to break and unable to meet the requirements for use.

[0039] In some embodiments, the concentration of 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) is 50–150 mmol / L, and the concentration of N-hydroxysuccinimide (NHS) is 25–75 mmol / L.

[0040] Cross-linking of EDC / NHS transforms the disordered microstructure of collagen into an ordered network structure, which enhances the mechanical properties and anti-degradation properties of the dermis and reduces swelling. However, high concentrations of EDC / NHS may exhibit cytotoxicity and cause excessively strong mechanical properties and anti-degradation properties in the dermis.

[0041] The dermis, artificial skin, and their preparation methods of this application will be described in detail below with reference to embodiments, comparative examples, and experimental data.

[0042] Example 1

[0043] A method for preparing artificial skin, the method comprising:

[0044] (1) Dissolve 2g polylactic acid (5kDa) and 0.1g simvastatin in 20mL dichloromethane, then slowly add 80mL of 1% polyvinyl alcohol solution, stir the solution at 1000rpm for 3h, centrifuge, pour off the supernatant, take the remaining precipitate, dry it to obtain polylactic acid microspheres loaded with simvastatin.

[0045] (2) Dissolve 0.5g of collagen in 100mL of 1% dilute acetic acid solution, add 0.01g of silver sulfadiazine powder and 0.05g of polylactic acid microspheres loaded with simvastatin, stir evenly, add 0.776g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.288g of N-hydroxysuccinimide, continue stirring evenly, inject into a mold, freeze dry to obtain the dermis.

[0046] (3) The obtained dermis layer and silicone epidermis layer are combined with medical adhesive to obtain artificial skin.

[0047] Example 2

[0048] A method for preparing artificial skin, the method comprising:

[0049] (1) Dissolve 2g polylactic acid (20kDa) and 0.1g simvastatin in 20mL dichloromethane, then slowly add 80mL of 1% polyvinyl alcohol solution, stir the solution at 1000rpm for 3h, centrifuge, pour off the supernatant, take the remaining precipitate, dry it to obtain polylactic acid microspheres loaded with simvastatin.

[0050] (2) Dissolve 1g of collagen in 100mL of 1% dilute acetic acid solution, add 0.01g of silver sulfadiazine powder and 0.05g of polylactic acid microspheres loaded with simvastatin, stir evenly, add 0.776g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.288g of N-hydroxysuccinimide, continue stirring evenly, inject into a mold, freeze dry to obtain the dermis.

[0051] (3) The obtained dermis layer and silicone epidermis layer are combined with medical adhesive to obtain artificial skin.

[0052] Example 3

[0053] A method for preparing artificial skin, the method comprising:

[0054] (1) Dissolve 2g polylactic acid (60kDa) and 0.1g simvastatin in 20mL dichloromethane, then slowly add 80mL of 1% polyvinyl alcohol solution, stir the solution at 1000rpm for 3h, centrifuge, pour off the supernatant, take the remaining precipitate, dry it to obtain polylactic acid microspheres loaded with simvastatin.

[0055] (2) Dissolve 2g of collagen in 100mL of 1% dilute acetic acid solution, add 0.01g of silver sulfadiazine powder and 0.05g of polylactic acid microspheres loaded with simvastatin, stir evenly, add 0.776g of 1-ethyl-(3-dimethylaminopropyl)carbodiimide and 0.288g of N-hydroxysuccinimide, continue stirring evenly, inject into a mold, freeze dry to obtain the dermis.

[0056] (3) The obtained dermis layer and silicone epidermis layer are combined with medical adhesive to obtain artificial skin.

[0057] Comparative Example 1

[0058] An antibacterial artificial skin was prepared by referring to the method in Example 1 of Chinese Patent CN 115887785B.

[0059] An antibacterial artificial skin comprises, from bottom to top, a lower dermis 1, an upper dermis 2, an absorbable epidermis 3, and a non-absorbable epidermis 4. The lower dermis 1 is a sponge-like porous collagen layer with a thickness of 2.5 mm, a pore size of 100 μm, and a porosity of 93%. The upper dermis 2 is a three-layer porous decellularized matrix membrane adsorbed with active substances. Each single-layer porous decellularized matrix membrane has a thickness of 0.1 mm and multiple pores with a pore size of 0.1 mm and a density of 500 pores / cm² along its thickness. 2 The active substances include epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermal layer 3 is a decellularized matrix membrane with a surface coated with nanoporous bioactive glass. The thickness of a single layer of decellularized matrix membrane is 0.05 mm, the mass ratio of nanoporous bioactive glass to decellularized matrix membrane is 1:9, the particle size of nanoporous bioactive glass is 40 μm, and the pore size is 10 nm; the non-absorbable epidermal layer 4 is a silicone rubber film with a thickness of 0.15 mm.

[0060] Comparative Example 2

[0061] A tissue-engineered artificial skin was prepared by referring to the method in Example 1 of Chinese Patent CN104984407B.

[0062] Step 1: Using a 1% acetic acid solution as a solvent, take a certain amount of type 3 collagen and prepare a high-concentration collagen solution with a concentration of 0.6 wt%. Place the solution in a dialysis bag and dialyze for 7 days to form solution 1.

[0063] Step 2: Using a 1% acetic acid solution as solvent, take a certain amount of type 1 collagen to prepare a collagen solution with a lower concentration of 0.3 wt%, forming solution II;

[0064] Step 3: Inject Solution II into a mold with a depth of 2mm, freeze at -80℃, and then freeze-dry it into a sponge using a freeze dryer to serve as the lower layer of the dermis;

[0065] Step 4: The sponge obtained in Step 3 is cross-linked with a 0.01% formaldehyde ethanol solution for 24 hours. After cross-linking, it is washed with purified water to remove formaldehyde residue, and then freeze-dried again to obtain the cross-linked sponge layer.

[0066] Step 5: Inject Solution I into a mold with a depth of 5 mm and dry it in a ventilated environment at 55°C to form a thin film. Step 6: Inject Solution I with a thickness of 5 mm onto the film obtained in Step 5. After leveling for a period of time, directly attach the sponge layer obtained in Step 4 onto the solution mold, and then freeze-dry it at -80°C to form artificial skin with a dermal layer with a gradient porosity structure and an epidermal layer with a microporous structure.

[0067] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0068] (1) Freeze-drying yields a porous structure similar to that of the dermis, which is beneficial for cell adhesion and proliferation. Furthermore, the dermis can be used to load drugs for in-situ release, improving drug utilization and reducing systemic side effects.

[0069] (2) The combined use of the two drugs can avoid the risk of infection and accelerate the skin regeneration process. The loaded silver sulfadiazine can prevent bacterial infection, and the drug-loaded polylactic acid microspheres can release simvastatin in the later stage of skin regeneration to control the conversion of macrophages from M1 to M2 type, and transition from the inflammatory stage to the proliferative stage of wound repair.

[0070] (3) The raw materials are widely available, the production process is simple, and it is suitable for mass production.

[0071] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0072] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for producing an artificial skin having antibacterial and inflammation-regulating functions, characterized by, The method comprises the following steps: (1) dissolving polylactic acid and simvastatin in dichloromethane, then slowly adding polyvinyl alcohol solution, stirring the solution for a period of time, centrifuging, pouring out the supernatant, taking the remaining precipitate, and drying to obtain simvastatin-loaded polylactic acid microspheres; the molecular weight of the polylactic acid is 20-60 kDa; (2) dissolving collagen in dilute acetic acid solution, adding silver sulfadiazine powder and simvastatin-loaded polylactic acid microspheres, stirring uniformly, then adding 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide and N-hydroxysuccinimide, continuing to stir uniformly, then pouring into a mold, and freeze-drying to obtain a dermal layer; (3) combining the obtained dermal layer with a silicone epidermal layer by a medical adhesive to obtain artificial skin; wherein: the concentration of the dilute acetic acid is 0-1%, the concentration of the collagen solution is 5-20 mg / mL; the mass ratio of silver sulfadiazine powder, simvastatin-loaded polylactic acid microspheres and collagen is 0.1-0.5:0.5-1:10; the concentration of 1-ethyl-(3-dimethylaminopropyl) carbonyl diimide is 50-150 mmol / L, and the concentration of N-hydroxysuccinimide is 25-75 mmol / L; the freezing temperature of freeze-drying is-50--40℃, and the time is 2 h; the sublimation temperature is-50--40℃, and the time is 24 h; the re-drying temperature is 30-35℃, and the time is 24 h.

2. The method for preparing artificial skin with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, In step (1), the concentration of polyvinyl alcohol is 1-3%, the stirring speed is 500-1500 rpm, and the stirring time is 1-5 h.

3. The method for preparing artificial skin with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, In the simvastatin-loaded polylactic acid microspheres of step (2), the mass ratio of simvastatin to polylactic acid is 0.1-1:

10.

4. The method for preparing artificial skin with antibacterial and anti-inflammatory functions according to claim 1, characterized in that, The thickness of the silicone epidermal layer is 100-300 μm.

5. Artificial skin with antibacterial and inflammation regulating functions prepared by the method according to any one of claims 1-4.

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