Artificial skin with antibacterial and inflammation regulating functions and preparation method thereof

Abstract

The invention particularly relates to artificial skin with antibacterial and inflammation regulating functions and a preparation method thereof, and belongs to the technical field of artificial skin. Collagen is used as a carrier material, a corium layer is formed by doping sulfadiazine silver and simvastatin-loaded polylactic acid microspheres, and the obtained corium layer is combined with a silica gel epidermal layer to obtain the artificial skin. 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 period of skin regeneration, macrophages are promoted to be converted into M2 type, the macrophages participate in the anti-inflammatory and repairing process, and skin regeneration is accelerated. The silica gel epidermal layer can prevent body fluid loss and bacteria invasion.

Description

Technical Field

[0001] The invention belongs to the technical field of artificial skin, and particularly relates to an artificial skin with antibacterial and inflammation regulating functions and a preparation method thereof. Background Art

[0002] Skin is the body's natural protective layer that isolates the body from direct contact with the outside world and protects the body from harmful substances. Since the skin is exposed to the external environment over a large area, it may be damaged at any time. Common skin injuries in daily life include trauma, burns, bacterial infection, etc. At present, people use biomaterials for skin reconstruction and have successfully developed various types of artificial skin. They have good cytoplasmic proliferation activity, certain mechanical strength, low price and other characteristics, but there are still safety risks such as bacterial infection and inflammatory response.

[0003] As the first line of defense of the human body, when the skin is damaged, its barrier function is destroyed, making it easier for bacteria to invade the body, increasing the risk of infection. Bacterial infection can hinder skin regeneration, and in severe cases it can even be life-threatening. The most common methods for treating bacterial infections are oral or intravenous medications, which have the disadvantages of low absorption and utilization efficiency and unnecessary systemic side effects, respectively. Topical medications are simple to operate and have fewer systemic side effects, making them a very promising option.

[0004] There are four main processes for skin regeneration after damage: the onset of inflammatory response, wound contraction, granulation tissue proliferation and scar formation, and regeneration of the epidermis and other tissues. The role of inflammation is two-sided. On the one hand, the inflammatory response is conducive to the recruitment of macrophages to phagocytose tissue fragments, which is an important part of the skin regeneration process. On the other hand, long-term and repeated inflammatory responses may lead to the formation of non-healing chronic wounds. Macrophages play an important role in the regulation of inflammation. Macrophages have two phenotypes, namely pro-inflammatory (M1) and anti-inflammatory (M2). Under different pathophysiological conditions, macrophages can switch between M1 (pro-inflammatory) and M2 (anti-inflammatory) phenotypes. M1 macrophages can release IL-1β, IL-6, etc. to promote the inflammatory process, and M2 macrophages can release anti-inflammatory factors such as IL-10 and TGF-β to promote wound recovery. Summary of the invention

[0005] The purpose of the present application is to provide an artificial skin with antibacterial and inflammation regulating functions and a preparation method thereof, so as to solve the problems of bacterial infection and inflammatory response after the current use of artificial skin for treatment.

[0006] The artificial skin with antibacterial and inflammation regulating functions provided by the embodiment of the present invention uses collagen as a carrier material, forms a dermis layer by doping silver sulfadiazine and polylactic acid microspheres loaded with simvastatin, and combines the obtained dermis layer with a silicone epidermis layer to obtain the artificial skin.

[0007] The dermis layer is obtained by the following method:

[0008] (1) Polylactic acid and simvastatin are dissolved in dichloromethane, and then polyvinyl alcohol solution is slowly added. The solution is stirred for a period of time and then centrifuged. The supernatant is poured out, and the remaining precipitate is taken and dried to obtain polylactic acid microspheres loaded with simvastatin.

[0009] (2) Dissolve collagen in a dilute acetic acid solution, add silver sulfadiazine powder and polylactic acid microspheres loaded with simvastatin, stir evenly, then add 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC / NHS), continue stirring evenly, inject into a mold, and freeze-dry to obtain a dermis layer.

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

[0011] Optionally, the molecular weight of the polylactic acid is 5 to 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 hours.

[0015] Optionally, the concentration of the dilute acetic acid is 0-1%, and the concentration of the 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 freezing temperature of freeze drying is -50 to -40°C, and the time is 2 hours; the sublimation temperature is -50 to -40°C, and the time is 24 hours; and the re-drying temperature is 30 to 35°C, and the time is 24 hours.

[0019] Optionally, the thickness of the silica gel surface layer is 100 to 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) The porous structure obtained by freeze drying is similar to the dermis structure, which is conducive to cell adhesion and proliferation. The dermis can also be used to load drugs for in situ release, improve drug utilization, and reduce 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 late stage of skin regeneration to control the conversion of macrophages from M1 to M2, and transition from the inflammatory stage to the proliferation 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 only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, it can be implemented according to the contents of the specification. In order to make the above and other purposes, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are listed below. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0026] Figure 1 It is a flow chart of the method provided by the embodiment of the present invention. DETAILED DESCRIPTION

[0027] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0028] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

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

[0030] The technical solution of the embodiment of the present application is to solve the above technical problems, and the overall idea is as follows:

[0031] During the invention process, the applicant discovered that current artificial skin products are often accompanied by the risk of bacterial infection during treatment, and because artificial skin materials cover wounds for a long time, there may also be a risk of inflammatory response.

[0032] The combined use of silver sulfadiazine and simvastatin can effectively avoid bacterial infection and regulate verification reactions.

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

[0034] Silver sulfadiazine loaded in the collagen matrix can effectively prevent bacterial infection. 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 the polylactic acid is 20-60 kDa.

[0036] Controlling the molecular weight of polylactic acid is to control the degradation rate of polylactic acid microspheres. If the molecular weight of polylactic acid is too small, the microspheres will degrade too quickly, which will lead to the rapid release of simvastatin, resulting in the inhibition of the inflammatory response (phagocytic cell fragments, invading microorganisms) necessary for tissue regeneration, which is not conducive to wound regeneration. If the molecular weight is too large, the drug release will be too slow, prolonging the inflammatory response.

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

[0038] The addition of acetic acid will increase the amount of collagen that is dissolved, but too high an acetic acid content will cause solvent residue after freeze-drying; too low a collagen content will cause the mechanical properties of the dermis after freeze-drying to be too poor, 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] The cross-linking of EDC / NHS will change the microstructure of collagen from a disordered state to an ordered network structure, which will improve the mechanical properties and anti-degradation properties of the dermis and reduce the swelling properties. However, high concentrations of EDC / NHS may be cytotoxic and make the mechanical properties and anti-degradation properties of the dermis too strong.

[0041] The dermis layer, artificial skin and preparation method thereof of the present application will be described in detail below in combination with embodiments, control examples and experimental data.

[0042] Example 1

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

[0044] (1) 2 g of polylactic acid (5 kDa) and 0.1 g of simvastatin were dissolved in 20 mL of dichloromethane, and then 80 mL of 1% polyvinyl alcohol solution was slowly added. The solution was stirred at 1000 rpm for 3 h and then centrifuged. The supernatant was poured out and the remaining precipitate was taken and dried to obtain polylactic acid microspheres loaded with simvastatin.

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

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

[0047] Example 2

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

[0049] (1) 2 g of polylactic acid (20 kDa) and 0.1 g of simvastatin were dissolved in 20 mL of dichloromethane, and then 80 mL of 1% polyvinyl alcohol solution was slowly added. The solution was stirred at 1000 rpm for 3 h and then centrifuged. The supernatant was poured out and the remaining precipitate was taken and dried to obtain polylactic acid microspheres loaded with simvastatin.

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

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

[0052] Example 3

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

[0054] (1) 2 g of polylactic acid (60 kDa) and 0.1 g of simvastatin were dissolved in 20 mL of dichloromethane, and then 80 mL of 1% polyvinyl alcohol solution was slowly added. The solution was stirred at 1000 rpm for 3 h and then centrifuged. The supernatant was poured out and the remaining precipitate was taken and dried to obtain polylactic acid microspheres loaded with simvastatin.

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

[0056] (3) The obtained dermis layer and the silicone epidermis layer are combined with a 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 a lower dermis layer 1, an upper dermis layer 2, an absorbable epidermis layer 3 and a non-absorbable epidermis layer 4 which are arranged in sequence from bottom to top; the lower dermis layer 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 layer 2 is a three-layer porous acellular matrix membrane adsorbed with active substances, the thickness of a single-layer porous acellular matrix membrane is 0.1 mm, and the porous acellular matrix membrane is provided with a plurality of through holes along the thickness direction, the pore size of the through holes is 0.1 mm, and the density is 500 / cm 2 , the active substances include epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF); the absorbable epidermis layer 3 is a layer of acellular matrix membrane coated with nano-mesoporous bioactive glass on the surface, the thickness of the single-layer acellular matrix membrane is 0.05 mm, the mass ratio of the nano-mesoporous bioactive glass to the acellular matrix membrane is 1:9, the particle size of the nano-mesoporous bioactive glass is 40 μm, and the pore size is 10 nm; the non-absorbable epidermis layer 4 is a silicone rubber film with a thickness of 0.15 mm.

[0060] Comparative Example 2

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

[0062] Step 1: Using 1% acetic acid solution as solvent, taking a certain amount of type I collagen, preparing a collagen solution with a higher concentration, a solution with a concentration of 0.6wt%, and placing it in a dialysis bag for 7 days to form solution 1;

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

[0064] Step 3: Solution II was injected into a mold with a depth of 2 mm, frozen at -80°C, and then freeze-dried into a sponge using a freeze dryer 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, washed with purified water after cross-linking to remove residual formaldehyde, and then freeze-dried again to obtain a cross-linked sponge layer;

[0066] Step 5: Inject solution I into a mold with a depth of 5 mm, and dry it at 55°C to form a film: Step 6: Inject solution I with a thickness of 5 mm on the film obtained in step 5. After leveling for a period of time, directly attach the sponge layer obtained in step 4 to solution I, and then freeze-dry it at -80°C to form an artificial skin with a dermis layer containing a gradient porosity structure and an epidermis layer containing 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) The porous structure obtained by freeze drying is similar to the dermis structure, which is conducive to cell adhesion and proliferation. The dermis can also be used to load drugs for in situ release, improve drug utilization, and reduce 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 late stage of skin regeneration to control the conversion of macrophages from M1 to M2, and transition from the inflammatory stage to the proliferation 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 "comprises," "includes," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements that are inherent to such process, method, article, or apparatus.

[0072] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0073] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for preparing an artificial skin having antibacterial and inflammation regulating functions, characterized in that: The following steps are involved: (1) dissolving polylactic acid and simvastatin in dichloromethane, then slowly adding polyvinyl alcohol solution, stirring the solution for a period of time and then centrifuging, pouring out the supernatant, taking the remaining precipitate, and drying to obtain polylactic acid microspheres loaded with simvastatin; (2) Dissolving collagen in a dilute acetic acid solution, adding silver sulfadiazine powder and polylactic acid microspheres loaded with simvastatin, stirring evenly, adding 1-ethyl-(3-dimethylaminopropyl) carbodiimide and N-hydroxysuccinimide (EDC / NHS), continuing to stir evenly, injecting into a mold, and freeze-drying to obtain a dermis layer; (3) The obtained dermis layer and the silicone epidermis layer are combined with a medical adhesive to obtain artificial skin.

2. The method for preparing an artificial skin having antibacterial and inflammation regulating functions according to claim 1, characterized in that: In the step (1), the concentration of polyvinyl alcohol is 1-3%, the stirring speed is 500-1500 rpm, and the stirring time is 1-5 hours.

3. The method for preparing an artificial skin having antibacterial and inflammation regulating functions according to claim 1, characterized in that: In the polylactic acid microspheres loaded with simvastatin in step (2), the molecular weight of the polylactic acid is 5 to 60 kDa, and the mass ratio of simvastatin to polylactic acid is 0.1 to 1:

10.

4. The method for preparing an artificial skin having antibacterial and inflammation regulating functions according to claim 1, characterized in that: The concentration of the dilute acetic acid is 0-1%, and the concentration of the collagen solution is 5-20 mg / mL; the mass ratio of sulfadiazine silver powder, polylactic acid microspheres loaded with simvastatin and collagen is 0.1-0.5:0.5-1:10; 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; the freezing temperature of freeze drying is -50--40°C, and the time is 2h; the sublimation temperature is -50--40°C, and the time is 24h; the re-drying temperature is 30-35°C, and the time is 24h.

5. The method for preparing an artificial skin having antibacterial and inflammation regulating functions according to claim 1, characterized in that: The thickness of the silica gel skin layer is 100 to 300 μm.

6. An artificial skin with antibacterial and inflammation regulating functions obtained according to the preparation method described in any one of claims 1 to 5.

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