A kind of gel dressing carrying probiotics and preparation method thereof

Abstract

The present invention belongs to the field of bioengineering medicine technology, and specifically relates to a gel dressing carrying probiotics and a preparation method thereof. The composition of the gel dressing includes the following components: a probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk protein. The present invention constructs a probiotic composition through Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies long and Lactococcus lactis, exerts antibacterial and immunomodulatory functions, inhibits the growth of pathogens and promotes tissue repair, and the addition of chitosan-hyaluronic acid cross-linked network and cellulose nanocrystals enhances the mechanical strength, water absorption capacity and antibacterial properties of the material; the probiotic layer is combined with the honey layer, and the microecological balance is optimized by adjusting pH, providing nutrition and maintaining a moist environment, and accelerating wound healing; the mechanical stability of the highly water-absorbent composite layer and the controlled release ability of hyaluronic acid enhance the activity and function of probiotics.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering medicine, and in particular relates to a probiotic-carrying gel dressing and a preparation method thereof. Background Art

[0002] As an important medical product, gel dressing plays an indispensable role in wound care and skin repair. This type of dressing is mainly composed of gelling substances and purified water, usually with a water content of more than 50%, which gives the gel dressing good moisturizing and softness, can fit the wound surface closely, and provide a comfortable use experience. The gelling substances usually use highly absorbent polymers such as sodium polyacrylate, polyvinyl alcohol, and carbomer. These materials not only have excellent moisture absorption and water retention properties, but also can form a stable three-dimensional network structure, giving the gel dressing appropriate viscoelasticity and mechanical strength. In contrast, traditional dressings such as gauze and bandages have relatively single functions and may cause secondary damage to the wound surface during the dressing change process. Although modern new wound dressings such as foam dressings, hydrogels, and tissue-engineered skin have improved these problems to a certain extent, they are still insufficient in maintaining the skin microbial community. For example, in some cases, gel dressings may need to be fixed with an additional second layer of dressing or tape, which increases the complexity and cost of use. When facing wounds with more exudate, if the dressing is not changed in time, it may cause maceration of the surrounding skin, thereby affecting the healing process. In addition, traditional gel dressings are not suitable for eschar or dry wounds; although traditional antibacterial hydrogels can effectively inhibit pathogens, they also have a negative impact on the beneficial bacteria on the skin, disrupting the natural balance of the skin microbiome. This imbalance is not only not conducive to wound healing, but may also cause other complications.

[0003] Probiotics are used as functional ingredients in gel dressings to maintain the ecological balance of microorganisms during wound healing, effectively control inflammatory responses, and promote tissue regeneration. Probiotics can survive and function in gel dressings, prevent pathogens from attaching to the surface of host cells through competitive exclusion mechanisms, and use their own metabolites to inhibit the growth of harmful bacteria, thereby maintaining or restoring the normal microbial community structure on the skin surface. In addition, the presence of probiotics helps regulate local immune responses and reduce the occurrence of excessive inflammation. Therefore, based on the above problems, it is extremely necessary to develop a gel dressing with high safety, excellent biocompatibility and carrying probiotics. Summary of the invention

[0004] In view of the defects of the prior art, the object of the present invention is to provide a gel dressing carrying probiotics and a preparation method thereof.

[0005] The technical effect described in the present invention is achieved through the following technical scheme: a gel dressing carrying probiotics, which comprises the following components: a probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk fibroin.

[0006] Preferably, the probiotic composition is composed of Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis in a mass ratio of 0.1:0.4:0.3:0.2.

[0007] Preferably, the Akkermansia metabolite is obtained by culturing Akkermansia ATCC BAA-835 in LB medium at 37° C. for 24 hours, centrifuging to remove the bacterial bodies, and then freeze-drying.

[0008] Preferably, the antioxidant is any one of vitamin C, vitamin E, tea polyphenols and rosemary extract.

[0009] Preferably, the specific preparation steps of the functionalized cellulose nanocrystals are as follows:

[0010] S1: adding cellulose nanocrystals to deionized water, stirring and dispersing them uniformly, to obtain a 1-2 wt% cellulose nanocrystal dispersion; adding hexadecyltrimethylammonium bromide to deionized water, stirring and dissolving them uniformly, to obtain a 0.2-0.5 wt% quaternary ammonium salt solution;

[0011] S2: slowly adding the quaternary ammonium salt solution prepared in step S1 to the cellulose nanocrystal dispersion, adjusting the pH to 8.5-10, stirring at 200-400 rpm for 2-4 hours, centrifuging, filtering, washing with anhydrous ethanol for 3 times, and drying at 40° C. to constant weight to obtain functionalized cellulose nanocrystals;

[0012] Preferably, in step S2, the volume ratio of the quaternary ammonium salt solution to the cellulose nanocrystal dispersion is 1:2-3.

[0013] Preferably, another aspect of the present invention is to provide a method for preparing a gel dressing carrying probiotics, and the specific preparation steps are as follows:

[0014] S101: dissolving chitosan in a 1wt% acetic acid solution, stirring and dissolving uniformly, to obtain a 1-2wt% chitosan solution; dissolving hyaluronic acid in deionized water, stirring and dissolving uniformly, adjusting the pH to 5-6, to obtain a 0.2-0.5wt% hyaluronic acid solution; slowly adding the hyaluronic acid solution to the chitosan solution, stirring and mixing uniformly, to obtain a chitosan-hyaluronic acid solution;

[0015] S102: dissolving polyvinyl alcohol in deionized water, raising the temperature to 80-90° C., stirring and dissolving uniformly to obtain a 5-10 wt % polyvinyl alcohol solution; cooling the polyvinyl alcohol solution to 40-50° C., adding functionalized cellulose nanocrystals, and ultrasonically treating at 60-100 W for 10-20 min to obtain a mixed solution;

[0016] S103: slowly adding the 2wt% silk fibroin solution and the mixed solution prepared in step S102 to the chitosan-hyaluronic acid solution prepared in step S101, and then slowly dropping 8wt% EDC solution and 3wt% NHS solution, controlling the pH to 4.5-6, stirring and reacting for 2-4h, to obtain a composite solution A;

[0017] S104: coating the composite solution A prepared in step S103 to a thickness of 1 to 2 mm, performing a double freeze-thaw treatment, then slowly adding the resuscitated probiotic composition, and adding sucrose, maintaining the temperature at 4°C, adjusting the pH to 4.5 to 5.5, stirring and mixing at a speed of 80 rpm for 5 to 10 min, then stirring at 200 rpm for 10 to 15 min, standing for 1 to 2 h, freezing at -80°C for 60 min, and then slowly thawing at room temperature for 12 h to obtain a composite solution B;

[0018] S105: adding honey to deionized water and adding an antioxidant, stirring and mixing evenly to obtain a honey solution; evenly coating the composite solution B prepared in step S104 to control the thickness to 1-2 mm, then freezing at -40°C for 6 hours, then slowly and evenly dripping the honey solution to control the thickness to 0.5-1 mm, then adding 1wt% sucrose, and performing double freeze-thaw treatment to obtain a gel dressing;

[0019] Preferably, in step S101, the volume ratio of the hyaluronic acid solution to the chitosan solution is 1:1;

[0020] Preferably, in step S102, the ratio of the amount of the polyvinyl alcohol solution to the amount of the functionalized cellulose nanocrystals is 1 mL: 0.01-0.02 g;

[0021] Preferably, in step S103, the silk fibroin solution is prepared from silk fibroin and deionized water, the EDC solution is prepared from 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride and deionized water; the NHS solution is prepared from N-hydroxysuccinimide and deionized water;

[0022] Preferably, in step S103, the volume ratio of the silk fibroin solution, the mixed solution, the chitosan-hyaluronic acid solution, the EDC solution and the NHS solution is 1:1-2:0.5-1:0.5:0.2-0.3;

[0023] Preferably, in step S104, the activity of Lactobacillus rhamnosus in the probiotic composition is 1×10 8 CFU / g; the activity of the long bifidobacterium subspecies longum is 5×10 7 CFU / g; the activity of Lactococcus lactis is 5×10 7 CFU / g; the ratio of the composite solution A, the probiotic composition and sucrose is 100mL: 1-1.5g: 1-2g;

[0024] Preferably, in step S104, the specific operation of the double freeze-thaw treatment is freezing at -20°C for 12 hours, then slowly thawing at room temperature for 4 hours, freezing at -20°C for 12 hours again, and slowly thawing at room temperature for 4 hours;

[0025] Preferably, in step S105, the ratio of the amount of honey, deionized water and antioxidant is 3g:2mL:0.015g; the specific operation of the double freeze-thaw treatment is freezing at -20°C for 12 to 16 hours, then slowly thawing at room temperature for 4 to 6 hours, freezing at -20°C again for 12 to 16 hours, and slowly thawing at room temperature for 4 hours.

[0026] The beneficial effects of the present invention are as follows:

[0027] The present invention uses Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis to construct a probiotic composition, wherein Lactobacillus rhamnosus can secrete metabolites such as lactic acid, and inhibit pathogenic microorganisms by reducing the local pH value, competitive exclusion, etc.; when the lactic acid of Lactobacillus rhamnosus cooperates with the bacteriocin of Lactococcus lactis, a wider spectrum of pathogen inhibition can be covered, and the overall antibacterial efficiency can be improved. In addition, short-chain fatty acids such as acetic acid and lactic acid produced by the metabolites of Bifidobacterium longum and Akkermansia can further reduce the local pH value and enhance the activity of antibacterial substances. The multi-strain combination can also deeply inhibit the growth of harmful colonies by occupying "adhesion sites", secreting hydrogen peroxide and a variety of bacteriocins, etc. Lactobacillus rhamnosus and Lactococcus lactis can both produce lactic acid, and Bifidobacterium longum can grow better in this slightly acidic environment, and in turn provide a stable nutritional environment for other probiotics through its own polysaccharides or other metabolites, forming a mutually beneficial symbiotic relationship and further inhibiting the invasion of pathogens. Lactobacillus rhamnosus plays a significant role in immune regulation, helping to balance the Th1 / Th2 immune response and inhibit the excessive release of inflammatory cytokines (such as IL-6 and TNF-α); the combined action of Lactococcus lactis and Bifidobacterium can more effectively regulate immune cell activity, relieve inflammation and promote tissue repair. Short-chain fatty acids not only further maintain a slightly acidic environment and inhibit pathogens, but also provide energy for local cell proliferation and repair, promote the proliferation and migration of fibroblasts, keratinocytes, etc., and accelerate the wound healing process.

[0028] The present invention utilizes chitosan cross-linking and hyaluronic acid (HA) covalent cross-linking to form a stable network, reduce excessive swelling of the material after water absorption, and retain the excellent moisturizing properties of hyaluronic acid; cellulose nanocrystals (CNCs) are functionalized with surface quaternary ammonium salts to avoid their agglomeration, and can rely on positive charges to form a uniformly dispersed nano-support skeleton in matrices such as chitosan, hyaluronic acid and polyvinyl alcohol, while synergistically improving the overall water absorption capacity and network stability of the composite layer; in addition, the addition of cellulose nanocrystals gives the material high mechanical strength and toughness. Chitosan-hyaluronic acid cross-linking and polyvinyl alcohol copolymerization modification form a dense interpenetrating network inside the material, which still has high deformation stability after absorbing exudate. Chitosan retains its natural antibacterial properties and inhibits the reproduction of pathogens to a certain extent; hyaluronic acid helps cells adhere and migrate, and assists tissue regeneration; polyvinyl alcohol-g-chitosan reduces irritation to the skin and enhances mild antibacterial effects. The modified network structure allows hyaluronic acid and other active substances to be released in a controlled manner, enhances synergy with probiotic metabolites, and accelerates wound healing. The highly absorbent composite layer quickly absorbs and disperses the exudate, without drying up or being overly wet, thus prolonging the survival time of probiotics and enhancing their functions; quaternized cellulose nanocrystals complement chitosan, polyvinyl alcohol, etc. at the molecular level, forming more hydrogen bonds and electrostatic cross-linking points, so that the material still maintains a stable three-dimensional structure after swelling with water absorption, and is not easy to collapse or break. The viscoelasticity and hydrophilicity of hyaluronic acid promote the transfer of probiotic nutrients between the composite layer and the wound surface, optimizing the balance of the microecology. Chitosan forms a primary antibacterial barrier in the composite layer, while probiotics form secondary inhibition through competitive exclusion and bacteriocin secretion, superimposed with the anti-inflammatory and cell repair-promoting properties of hyaluronic acid, jointly improving the wound microenvironment, accelerating regeneration and healing, and reducing the risk of excessive washing of probiotic active substances. The excellent mechanical properties and water absorption capacity of the modified highly absorbent composite layer can also reduce the frequency of dressing changes and reduce interference with the probiotic layer; the probiotics are combined with the honey layer to further kill or inhibit pathogens through high osmotic pressure, hydrogen peroxide and acidic pH, and provide sugar and trace nutrients for probiotics to promote proliferation. The honey layer and the bottom water-absorbing composite layer achieve two-way moisturizing - the upper viscous film reduces water loss, and the lower layer locks in excess exudate, so that the middle probiotic layer is moderately moist and has nutritional supplements; in addition, the upper and lower layers form a reasonable pH gradient, maintaining a slightly acidic state at the middle probiotic layer, which is conducive to the reproduction and function of probiotics. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings in the following description are only for the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0030] Figure 1 is a graph showing the biocompatibility test results of the gel dressings prepared in Examples 1 to 3 of the present invention;

[0031] Figure 2 1 is a graph showing the antibacterial test results of the gel dressings prepared in Example 2 of the present invention and Comparative Examples 2, 3, and 5;

[0032] Figure 3 1 is a graph showing the anti-inflammatory test results of the gel dressings prepared in Example 2 of the present invention and Comparative Examples 1, 3, and 5;

[0033] Figure 4 is a SEM scanning electron microscope image of the gel dressing prepared in Example 2 of the present invention;

[0034] Figure 5 1 is a graph showing the water vapor permeability test results of the gel dressings prepared in Example 2 of the present invention and Comparative Examples 2, 4, and 5. DETAILED DESCRIPTION

[0035] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them; based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. It should be noted that, unless otherwise specified, the raw materials involved in the present invention are purchased through conventional commercial channels.

[0036] Example 1: A probiotic-carrying gel dressing, comprising the following components: a probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk fibroin.

[0037] The specific preparation steps of functionalized cellulose nanocrystals are as follows:

[0038] S1: Add 2 g of cellulose nanocrystals to 200 mL of deionized water, stir and disperse evenly to obtain a 1 wt% cellulose nanocrystal dispersion; add 0.2 g of hexadecyltrimethylammonium bromide to 100 mL of deionized water, stir and dissolve evenly to obtain a 0.2 wt% quaternary ammonium salt solution;

[0039] S2: Slowly add 100 mL of the quaternary ammonium salt solution prepared in step S1 into 200 mL of the cellulose nanocrystal dispersion, adjust the pH to 8.5, stir at 200 rpm for 4 h, centrifuge, filter, wash with anhydrous ethanol for 3 times, and dry at 40°C to constant weight to obtain functionalized cellulose nanocrystals;

[0040] The specific preparation steps of the probiotic gel dressing are as follows:

[0041] S101: dissolving 1 g of chitosan into 100 mL of 1 wt% acetic acid solution, stirring and dissolving uniformly to obtain a 1 wt% chitosan solution; dissolving 0.2 g of hyaluronic acid into 100 mL of deionized water, stirring and dissolving uniformly, adjusting the pH to 6, and obtaining a 0.2 wt% hyaluronic acid solution; slowly adding 100 mL of the hyaluronic acid solution into 100 mL of the chitosan solution, stirring and mixing uniformly, and obtaining a chitosan-hyaluronic acid solution;

[0042] S102: dissolving 10 g of polyvinyl alcohol in 200 mL of deionized water, raising the temperature to 80° C., stirring and dissolving uniformly to obtain a 5 wt % polyvinyl alcohol solution; cooling the polyvinyl alcohol solution to 40° C., adding 4 g of functionalized cellulose nanocrystals, and ultrasonically treating at 60 W for 20 min to obtain a mixed solution;

[0043] S103: 200 mL of a 2 wt% silk fibroin solution prepared in deionized water and 200 mL of the mixed solution prepared in step S102 were slowly added to 100 mL of the chitosan-hyaluronic acid solution prepared in step S101, and then 100 mL of an 8 wt% EDC solution prepared in deionized water and 40 mL of a 3 wt% NHS solution were slowly added dropwise, the pH was controlled to 6, and the reaction was stirred for 2 h to obtain a composite solution A;

[0044] S104: 500 mL of the composite solution A prepared in step S103 is coated to control the thickness to 1 mm, and double freeze-thaw treatment is performed, the parameters are freezing at -20°C for 12 hours, then slowly thawing at room temperature for 4 hours, freezing at -20°C for 12 hours again, slowly thawing at room temperature for 4 hours, and then slowly adding 5 g of the resuscitated probiotic composition of Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis, and adding 5 g of sucrose, maintaining the temperature at 4°C, adjusting the pH to 5.5, stirring and mixing at a speed of 80 rpm for 5 minutes, then stirring at 200 rpm for 10 minutes, standing for 1 hour, freezing at -80°C for 60 minutes, and then slowly thawing at room temperature for 12 hours to obtain a composite solution B;

[0045] S105: Add 60g honey to 40mL deionized water, and add 0.3g vitamin C, stir and mix evenly to obtain a honey solution; evenly apply 500mL of the composite solution B prepared in step S104, control the thickness to 1mm, and then freeze it at -20℃ for 6h, then slowly and evenly add the honey solution, control the thickness to 0.5mm, and then add 5g sucrose, and perform double freeze-thaw treatment, the parameters are freezing at -20℃ for 12h, then slowly thawing at room temperature for 4h, freezing at -40℃ again for 12h, and slowly thawing at room temperature for 4h, to obtain a gel dressing.

[0046] Example 2: A probiotic-carrying gel dressing, comprising the following components: a probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk fibroin.

[0047] The specific preparation steps of functionalized cellulose nanocrystals are as follows:

[0048] S1: Add 4.5 g of cellulose nanocrystals to 300 mL of deionized water, stir and disperse evenly to obtain a 1.5 wt% cellulose nanocrystal dispersion; add 0.4 g of hexadecyltrimethylammonium bromide to 100 mL of deionized water, stir and dissolve evenly to obtain a 0.4 wt% quaternary ammonium salt solution;

[0049] S2: Slowly add 100 mL of the quaternary ammonium salt solution prepared in step S1 into 260 mL of the cellulose nanocrystal dispersion, adjust the pH to 9.5, stir at 300 rpm for 3 h, centrifuge, filter, wash with anhydrous ethanol for 3 times, and dry at 40°C to constant weight to obtain functionalized cellulose nanocrystals;

[0050] The specific preparation steps of the probiotic gel dressing are as follows:

[0051] S101: dissolving 1.5 g of chitosan into 100 mL of 1 wt% acetic acid solution, stirring and dissolving uniformly, obtaining a 1.5 wt% chitosan solution; dissolving 0.4 g of hyaluronic acid into 100 mL of deionized water, stirring and dissolving uniformly, adjusting the pH to 5.5, obtaining a 0.4 wt% hyaluronic acid solution; slowly adding the hyaluronic acid solution into the chitosan solution, stirring and mixing uniformly, obtaining a chitosan-hyaluronic acid solution;

[0052] S102: dissolving 24 g of polyvinyl alcohol into 300 mL of deionized water, raising the temperature to 85° C., stirring and dissolving uniformly to obtain an 8 wt % polyvinyl alcohol solution; cooling the polyvinyl alcohol solution to 45° C., adding 3 g of functionalized cellulose nanocrystals, and ultrasonically treating at 80 W for 15 min to obtain a mixed solution;

[0053] S103: 100 mL of 2 wt% silk fibroin solution prepared in deionized water and 300 mL of the mixed solution prepared in step S102 were slowly added to 150 mL of chitosan-hyaluronic acid solution prepared in step S101, and then 100 mL of 8 wt% EDC solution prepared in deionized water and 50 mL of 3 wt% NHS solution were slowly added dropwise, the pH was controlled to 5.5, and the mixture was stirred for 3 h to obtain a composite solution A;

[0054] S104: 500 mL of the composite solution A prepared in step S103 is coated to control the thickness to 1.5 mm, and double freeze-thaw treatment is performed, the parameters are freezing at -20°C for 12 hours, then slowly thawing at room temperature for 4 hours, freezing at -20°C for 12 hours again, slowly thawing at room temperature for 4 hours, and then slowly adding 6.5 g of the resuscitated probiotic composition of Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis, and adding 8 g of sucrose, maintaining the temperature at 4°C, adjusting the pH to 5, stirring and mixing at a speed of 80 rpm for 8 minutes, then stirring at 200 rpm for 12 minutes, standing for 1.5 hours, freezing at -80°C for 60 minutes, and then slowly thawing at room temperature for 12 hours to obtain a composite solution B;

[0055] S105: Add 60g honey to 40mL deionized water, and add 0.3g tea polyphenols, stir and mix evenly to obtain a honey solution; evenly apply the composite solution B prepared in step S104, control the thickness to 1.5mm, then freeze it at -20℃ for 6h, then slowly and evenly drip the honey solution, control the thickness to 0.8mm, then add 5g sucrose, and perform double freeze-thaw treatment, the parameters are freezing at -20℃ for 15h, then slowly thawing at room temperature for 5h, freezing at -40℃ again for 15h, and slowly thawing at room temperature for 4h, to obtain a gel dressing.

[0056] Example 3: A probiotic-carrying gel dressing, comprising the following components: a probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk fibroin.

[0057] The specific preparation steps of functionalized cellulose nanocrystals are as follows:

[0058] S1: Add 6 g of cellulose nanocrystals to 300 mL of deionized water, stir and disperse evenly to obtain a 2 wt % cellulose nanocrystal dispersion; add 0.5 g of hexadecyltrimethylammonium bromide to 100 mL of deionized water, stir and dissolve evenly to obtain a 0.5 wt % quaternary ammonium salt solution;

[0059] S2: Slowly add 100 mL of the quaternary ammonium salt solution prepared in step S1 into 300 mL of the cellulose nanocrystal dispersion, adjust the pH to 10, stir at 400 rpm for 2 h, centrifuge, filter, wash with anhydrous ethanol for 3 times, and dry at 40°C to constant weight to obtain functionalized cellulose nanocrystals;

[0060] The specific preparation steps of the probiotic gel dressing are as follows:

[0061] S101: dissolving 2 g of chitosan into 100 mL of 1 wt% acetic acid solution, stirring and dissolving uniformly, to obtain a 2 wt% chitosan solution; dissolving 0.5 g of hyaluronic acid into 100 mL of deionized water, stirring and dissolving uniformly, adjusting the pH to 5, to obtain a 0.5 wt% hyaluronic acid solution; slowly adding the hyaluronic acid solution into the chitosan solution, stirring and mixing uniformly, to obtain a chitosan-hyaluronic acid solution;

[0062] S102: dissolving 40 g of polyvinyl alcohol in 400 mL of deionized water, raising the temperature to 90° C., stirring and dissolving uniformly to obtain a 10 wt % polyvinyl alcohol solution; cooling the polyvinyl alcohol solution to 50° C., adding 4 g of functionalized cellulose nanocrystals, and ultrasonically treating at 100 W for 10 min to obtain a mixed solution;

[0063] S103: 100 mL of 2 wt% silk fibroin solution prepared with deionized water and 400 mL of the mixed solution prepared in step S102 are slowly added to 200 mL of the chitosan-hyaluronic acid solution prepared in step S101, and then 100 mL of 8 wt% EDC solution prepared with deionized water and 60 mL of 3 wt% NHS solution are slowly added dropwise, the pH is controlled to 4.5, and the reaction is stirred for 4 hours to obtain a composite solution A;

[0064] S104: 500 mL of the composite solution A prepared in step S103 is coated to control the thickness to 2 mm, and double freeze-thaw treatment is performed, the parameters are freezing at -20°C for 12 hours, then slowly thawing at room temperature for 4 hours, freezing at -20°C for 12 hours again, slowly thawing at room temperature for 4 hours, and then slowly adding 7.5 g of the probiotic composition Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis after recovery, and adding 10 g of sucrose, maintaining the temperature at 4°C, adjusting the pH to 4.5, stirring and mixing at a speed of 80 rpm for 10 minutes, then stirring at 200 rpm for 15 minutes, standing for 2 hours, freezing at -80°C for 60 minutes, and then slowly thawing at room temperature for 12 hours to obtain a composite solution B;

[0065] S105: Add 60g of honey to 40mL of deionized water, and add rosemary extract, stir and mix evenly to obtain a honey solution; evenly apply 500mL of the composite solution B prepared in step S104, control the thickness to 2mm, then freeze at -20℃ for 6h, then slowly and evenly add the honey solution, control the thickness to 1mm, then add 5g of sucrose, and perform double freeze-thaw treatment, the parameters are freezing at -20℃ for 16h, then slowly thawing at room temperature for 6h, freezing at -40℃ again for 16h, and slowly thawing at room temperature for 4h, to obtain a gel dressing.

[0066] Comparative Example 1: The operations of Comparative Example 1 and Example 2 are basically the same, except that no silk fibroin is added in Comparative Example 1.

[0067] Comparative Example 2: The operation of Comparative Example 2 is basically the same as that of Example 2, except that in Comparative Example 2, cellulose nanocrystals are used to replace quaternary ammonium salt functionalized cellulose nanocrystals.

[0068] Comparative Example 3: The operation of Comparative Example 3 is basically the same as that of Example 2, except that Lactobacillus rhamnosus is not added to the probiotic composition in Comparative Example 3.

[0069] Comparative Example 4: The operations of Comparative Example 4 and Example 2 are basically the same, except that in Comparative Example 4, steps S104 and S105 are not subjected to double freeze-thaw treatment, and the operations are modified to directly freeze at -20°C for 24 hours and then thaw.

[0070] Comparative Example 5: The operation of Comparative Example 5 is substantially the same as that of Example 2, except that no honey layer is added in Comparative Example 5.

[0071] Performance Testing:

[0072] Biocompatibility test: The gel dressing samples prepared in Examples 1 to 3 were placed at the bottom of the culture plate. Eight wells were set for each group of samples as repeated experiments. 100 μL of serum-free DMEM medium was added to each well, and then Fibroblasts were diluted to 1×10 4 / mL, and inoculate 100 μL of the diluted cell suspension in each well, and use 8 wells without sample as negative control group, and 8 wells with Fibroblasts fibroblast culture medium as positive control group, set the temperature to 37°C, 5% CO2, add 10 μL of 5 mg / mL MTT solution after 24h and 72h of incubation, respectively, and continue to incubate for 4h, then aspirate the supernatant in the well, and then add 100 μL of dimethyl sulfoxide to the well, use a microplate reader to measure the absorbance (OD value) at 570nm, and measure the activity percentage [cell survival rate (%) = (OD value of the embodiment - OD value of the negative control group) / (OD value of the positive control group - OD value of the negative control group) × 100%], the cell survival rate results are as follows Figure 1 shown.

[0073] Depend on Figure 1 The results show that the gel dressing prepared by the present invention has a promoting effect on cell activity, has excellent biocompatibility, and can be safely and effectively applied to the skin as a gel dressing.

[0074] Antibacterial test: Prepare fresh bacterial suspensions of Staphylococcus aureus, Escherichia coli and Candida albicans at a concentration of 1×10 8CFU / mL, the gel dressing samples prepared in Example 2 of the present invention and Comparative Examples 2, 3, and 5 (1 mL of bacterial solution + 9 mL of deionized water + 0.1 g of gel dressing sample) were added respectively, and the mixed bacterial solution was placed in a constant temperature oscillator at 37°C for 24 h. After the culturing, 100 μL of the mixed bacterial solution was taken and gradiently diluted (1:10 dilution), and then spread on a culture medium plate. After culturing at 37°C for 24 h, the total number of colonies in the bacterial solution was recorded by the plate colony counting method, and the bacterial solution without the sample was used as a control (the strains used in this experiment were all purchased from the market), and the antibacterial rate was calculated [inhibition rate (%) = (1-the number of bacteria in the test sample / the number of bacteria in the control sample) × 100%], and the results are as follows Figure 2 shown.

[0075] Depend on Figure 2 As a result, the antibacterial rate of the gel dressing prepared by the present invention for bacteria can reach more than 90%, especially the antibacterial rate for Staphylococcus aureus can reach more than 98%, showing an excellent antibacterial effect; from the results of Comparative Example 2 and Example 2, it can be seen that the quaternary ammonium salt functionalized cellulose nanocrystals can interfere with the bacterial membrane through the action of positive charge, and the absence of quaternary ammonium salt functionalized cellulose nanocrystals has a greater impact on the antibacterial rate of Gram-negative bacteria, resulting in a more obvious decrease in the antibacterial property against Escherichia coli; from the results of Comparative Example 3 and Example 2, it can be seen that the synergistic effect of Lactobacillus rhamnosus is missing, the effects of short-chain fatty acids and lactic acid are weakened, and the antibacterial efficiency is significantly reduced; from the results of Comparative Example 5 and Example 2, it can be seen that the antibacterial mechanism of hydrogen peroxide provided by honey is missing, which has a certain degree of influence on the antibacterial rate.

[0076] Anti-inflammatory performance test: RAW264.7 (mouse macrophages) were inoculated in a sterile culture medium containing DMEM + 10% fetal bovine serum at 37°C and 5% CO2. After 24 h of culture, the cells were diluted to a concentration of 1×10 5The cell solution of cells / mL was divided into 15 groups (1 mL per group). The gel dressings prepared in Example 2 and Comparative Examples 1, 3, and 5 were taken as test samples and saline was taken as control group samples. Three groups were set for each sample, and each sample was 0.1 mL. The above samples were added to the 15 groups of cell solutions in sequence, gently mixed and continued to be cultured for 24 hours, and then 100 μL of sample from each group was added to the ELISA plate pre-coated with TNF-α antibody and incubated at room temperature for 2 hours. After the incubation, the plates were washed three times with PBS buffer, and 100 μL of 1000 diluted samples were added. The cells were incubated at room temperature for 1 h with a secondary antibody solution labeled with horseradish peroxidase (HRP). After the incubation, the cells were washed three times with PBS buffer and added with 100 μL of 0.1 mg / mL TMB solution. The cells were incubated for 15 min in a dark environment. 100 μL of 1 mol / L hydrochloric acid was added and the absorbance was read at 450 nm using an enzyme reader. The anti-inflammatory activity was calculated [anti-inflammatory activity (%) = (average TNF-α concentration in the control group - average TNF-α concentration in the example) / average TNF-α concentration in the control group × 100%]. The results are shown in Table 1. Figure 3 shown.

[0077] Depend on Figure 3 The results show that the synergistic effect of the probiotics, silk fibroin and honey in the gel dressing prepared by the present invention significantly reduces the inflammation level, and the anti-inflammatory property reaches an excellent level; from the results of Comparative Example 1 and Example 2, it can be seen that the lack of silk fibroin will reduce the cell adhesion ability and the ability to regulate the inflammatory response, and the anti-inflammatory performance is slightly reduced; from the results of Comparative Example 3 and Example 2, it can be seen that the lack of Lactobacillus rhamnosus significantly affects the anti-inflammatory ability of probiotics, and the local microecological balance may be destroyed; from the results of Comparative Example 5 and Example 2, it can be seen that the lack of the honey layer may lead to a decrease in the activity of the probiotics, and at the same time the moisture regulation ability becomes worse, resulting in a significant impact on the anti-inflammatory performance.

[0078] Spectrum test: The SEM spectrum of the inner layer of the gel dressing prepared in Example 2 was observed using a scanning electron microscope. The results are as follows: Figure 4 shown.

[0079] Depend on Figure 4 The results show that the pores have an irregular honeycomb morphology, there is an obvious interconnected network between the pores, the pore walls are relatively smooth, and show a good cross-linked structure. This structure is the result of the combined action of freeze-thaw cycles and chemical cross-linking, which helps to improve the air permeability and moisture retention capacity of the material; in addition, there are fine granular features on the pore walls, which are related to the effect of nano-scale additives.

[0080] Water vapor transmission rate test: According to "YY / T0471.2-2004 Test Method for Contact Wound Dressing Part 2", the water vapor transmission rate of the gel dressings prepared in Example 2 and Comparative Examples 2, 4, and 5 was tested at a test temperature of 23°C and a relative humidity of 50%. The test results are as follows: Figure 5 shown.

[0081] Depend on Figure 5 The results show that the gel dressing prepared by the present invention has excellent water vapor permeability. From the results of Comparative Example 1 and Example 2, it can be seen that the density of the gel network is slightly reduced due to the lack of silk fibroin, and water vapor is more likely to pass through, resulting in a slight increase in air permeability; from the results of Comparative Example 4 and Example 2, it can be seen that the double freeze-thaw treatment is not performed but a single freeze and then thaw is performed, and the internal pore structure of the gel becomes uneven, which leads to a significant decrease in air permeability; from the results of Comparative Example 5 and Example 2, it can be seen that the lack of the honey layer loses the moisture locking function, water vapor is more likely to dissipate, and the air permeability is significantly improved, which may lead to ineffective moisturizing.

[0082] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A gel dressing carrying probiotics, characterized in that: The composition includes the following components: probiotic composition, chitosan, functionalized cellulose nanocrystals, polyvinyl alcohol, hyaluronic acid, honey, antioxidants and silk fibroin; The probiotic composition is composed of Akkermansia metabolites, Lactobacillus rhamnosus, Bifidobacterium longum subspecies longum and Lactococcus lactis in a mass ratio of 0.1:0.4:0.3:0.2; The specific preparation steps of the probiotic-carrying gel dressing are as follows: S101: dissolving chitosan in a 1 wt % acetic acid solution, stirring and dissolving uniformly to obtain a chitosan solution; dissolving hyaluronic acid in deionized water, stirring and dissolving uniformly, and adjusting the pH to obtain a hyaluronic acid solution; Slowly adding the hyaluronic acid solution into the chitosan solution, stirring and mixing evenly to obtain a chitosan-hyaluronic acid solution; S102: dissolving polyvinyl alcohol in deionized water, increasing the temperature, stirring and dissolving uniformly to obtain a polyvinyl alcohol solution; cooling the polyvinyl alcohol solution, adding functionalized cellulose nanocrystals, and ultrasonically treating to obtain a mixed solution; S103: slowly adding the silk fibroin solution and the mixed solution prepared in step S102 to the chitosan-hyaluronic acid solution prepared in step S101, and then slowly dropping the EDC solution and the NHS solution, controlling the pH to 4.5-6, stirring the reaction, and obtaining a composite solution A; S104: coating the composite solution A prepared in step S103 to a thickness of 1 to 2 mm, performing a double freeze-thaw treatment, then slowly adding the resuscitated probiotic composition, and adding sucrose, maintaining the temperature at 4°C, adjusting the pH, stirring and mixing, and then increasing the speed of stirring, standing, freezing, and then slowly thawing at room temperature to obtain a composite solution B; S105: adding honey to deionized water and adding an antioxidant, stirring and mixing evenly to obtain a honey solution; evenly coating the composite solution B prepared in step S104 to control the thickness to 1-2 mm, then freezing, then slowly and evenly dripping the honey solution to control the thickness to 0.5-1 mm, then adding sucrose, performing a double freeze-thaw treatment, and obtaining a gel dressing; In step S101, the volume ratio of the hyaluronic acid solution to the chitosan solution is 1:1; in step S102, the volume ratio of the polyvinyl alcohol solution to the functionalized cellulose nanocrystals is 1 mL: 0.01-0.02 g; In step S103, the volume ratio of the silk fibroin solution, the mixed solution, the chitosan-hyaluronic acid solution, the EDC solution and the NHS solution is 1:1-2:0.5-1:0.5:0.2-0.3; In step S104, the activity of Lactobacillus rhamnosus in the probiotic composition is 1×10 8 CFU / g; the activity of the long bifidobacterium subspecies longum is 5×10 7 CFU / g; the activity of Lactococcus lactis is 5×10 7 CFU / g; the ratio of the composite solution A, the probiotic composition and sucrose is 100mL: 1-1.5g: 1-2g; In step S104, the specific operation of the double freeze-thaw treatment is freezing at -20°C for 12 hours, then slowly thawing at room temperature for 4 hours, freezing at -20°C for 12 hours again, and slowly thawing at room temperature for 4 hours; In step S105, the ratio of the amount of honey, deionized water and antioxidant is 3g:2mL:0.015g; the specific operation of the double freeze-thaw treatment is freezing at -20°C for 12 to 16 hours, then slowly thawing at room temperature for 4 to 6 hours, freezing at -20°C again for 12 to 16 hours, and slowly thawing at room temperature for 4 hours.

2. The probiotic-carrying gel dressing according to claim 1, characterized in that: The antioxidant is any one of vitamin C, vitamin E, tea polyphenols and rosemary extract.

3. The probiotic-carrying gel dressing according to claim 2, characterized in that: The specific preparation steps of the functionalized cellulose nanocrystals are as follows: S1: adding cellulose nanocrystals to deionized water, stirring and dispersing them uniformly to obtain a cellulose nanocrystal dispersion; adding hexadecyltrimethylammonium bromide to deionized water, stirring and dissolving them uniformly to obtain a quaternary ammonium salt solution; S2: slowly adding the quaternary ammonium salt solution prepared in step S1 to the cellulose nanocrystal dispersion, adjusting the pH, stirring the reaction, centrifuging, filtering, repeatedly washing with anhydrous ethanol, and drying to a constant weight to obtain functionalized cellulose nanocrystals; In step S2, the volume ratio of the quaternary ammonium salt solution to the cellulose nanocrystal dispersion is 1:2-3.

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