KR102083002B1 - Probiotics-loaded double-layered wound dressing and method for preparing the same - Google Patents

Abstract

The present invention relates to a hydrogel / hydrocolloid bilayer wet dressing agent containing probiotics and a method of manufacturing the same, and more particularly, to a hydrogel layer and a hydrogel layer comprising probiotics for wound healing such as wounds and burns, in which stability of probiotics is ensured. The present invention relates to a probiotic-containing bilayer hydrogel / hydrocolloid wet dressing agent consisting of a colloidal layer and a method of manufacturing the same.
The probiotic-containing hydrogel / hydrocolloid double layer dressing agent according to the present invention ensures the stability of probiotics during the manufacturing process, and has excellent physical properties of the dressing agent and prevents infection and promotes epithelial tissue treatment. Excellent effect and excellent effect on skin regeneration treatment.

Description

Probiotics-loaded double-layered wound dressing and method for preparing the same}

The present invention relates to a hydrogel / hydrocolloid double layer wound healing dressing containing probiotics and excellent in swelling and releasing ability, and a method of manufacturing the same.

Materials used in the treatment of wounds should reduce the duration of wound healing due to epithelialization, avoid discomfort due to bleeding or pain during replacement, and minimize scar formation after epithelialization. In addition, the secondary infection rate (secondary infection rate) is low, and easy to manufacture and store should be available. On the basis of this, various wet dressing agents in the form of hydrogel, hydrocolloid, and foam applied with wet wound healing are used to promote wound healing, and the effect of assisting epithelial regeneration can be seen.

Hydrocolloid formulations react with exudate when adhering to wounds to provide a wet environment in the form of gels to promote epithelialization of wounds, and have a higher storage stability than hydrogel formulations and self-adhesion, requiring a second dressing agent unlike other formulations. It does not have the advantage. Hydrocolloid formulations are composed of hydrophilic polymers having hydrophilic functional groups, which absorb water and are insoluble in water, and hydrophobic polymers having elasticity while giving adhesion to skin.

Korean Patent No. 1,651,574 discloses a drug delivery of hydrocolloids as a dressing for use and hydrophilic polymers and hydrophobic polymers. However, the manufacturing method of the dressing makes the active drug exposed to high temperature, It is difficult to apply to unstable active drugs.

In addition, the hydrogel formulation is a formulation consisting of a water-soluble polymer and water and is an excellent biocompatible material and is safe for the human body, and is widely used as an oral drug carrier.

Probiotics are microorganisms that have beneficial effects on the human body, assisting the body's immune system and reducing inflammatory reactions (Zahedi et al. Journal of Cell and Animal Biology. 5 (12): 265-270. 2011; Heydari Nasrabadi et al. African Jounal of Pharmacy and Pharmacology. 5 (21): 2395-2401. 2011), some probiotics have an excellent effect on wound healing, have excellent therapeutic capabilities in the treatment of burns, and Resistance has been reported to have excellent antibacterial activity against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Enterococci and Klebsiella (Brachkova et al. Journal of Hospital Infection 79: 375-377. 2011). In addition, probiotics reduce the risk of tissue infections during the regeneration of wounds and contribute to angiogenic capacity (El-Fawal et al. AAPS PharmSciTech. 18: 1605-1616. 2016).

However, probiotics have a property of being rapidly decomposed when exposed to heat or an organic solvent, there is a problem that it is difficult to formulate into a hydrocolloid type dressing agent using heat or an organic solvent while maintaining the excellent physical properties of the probiotics.

In the case of conventional polyurethane foam dressing and hydrocolloid dressing formulation, when the active agent is unstable in heat and organic solvent, there is a problem that the stability of the active drug is reduced by exposure to heat and organic solvent during the manufacturing process. In addition, polyurethane foam formulations, hydrogel formulations do not have a self-adhesive secondary dressing is required, hydrogel formulations are difficult to exhibit excellent physical properties, there is a problem that the application to the active drug is limited.

As a technology related to wound healing containing probiotics, Korean Laid-Open Patent Publication No. 2008-0049797 discloses a wound dressing including a hydrogel and a hydrocolloid containing Lactobacillus bacteria, but does not disclose a dressing of a bilayer structure. Korean Patent Publication No. 2013-0111849 discloses a wound dressing having a double layer structure including a polyvinyl alcohol and a hydrogel layer containing a water-soluble polymer, but does not describe probiotics and hydrocolloid layers. There is no disclosure of wound repair dressing formulations having excellent physical properties while maintaining the activity of unstable probiotics.

Therefore, there is a need for the development of dressing formulations having excellent physical properties, including active agents that are unstable in heat and organic solvents, probiotics having excellent wound repair ability.

It is an object of the present invention to solve the above problems, bilayer wound healing containing probiotics containing probiotics that do not occur degeneration, hydrogels having wound healing effects and hydrocolloids possessing optimal physical properties, and probiotics The dressing agent and its manufacturing method are provided.

The present invention also provides a hydrogel / hydrocolloid double layer wound healing dressing containing probiotics, and having excellent swelling and releasing ability, and a method of manufacturing the same.

The present invention relates to a bilayer dressing agent composition containing a probiotic, which is an active drug, and a hydrogel and a hydrocolloid.

In order to solve the problems shown in the prior art, a dressing agent containing an active drug 1) ensures the stability of active drugs such as probiotics during the manufacturing process; 2) has good physical properties; 3) the ability to release the active drug is good; 4) It has to be capable of absorbing enough exudate to maintain an effective wetting environment of the drug at the wound site; 5) It should show excellent wound recovery ability through the above effects.

The present invention was able to remarkably improve the stability of probiotics by containing probiotics in the hydrogel layer, and in particular, by using a freeze-thaw method to produce a hydrogel containing probiotics to maximize the above effects, hydrocolloid layer and hydrogel layer The bilayer dressing agent was prepared to prepare a dressing agent having excellent physical properties, exudate absorption ability, and active drug release ability.

Accordingly, the hydrogel / hydrocolloid bilayer wet dressing agent according to the present invention contains probiotics as an active ingredient, a hydrogel layer containing probiotics and a water-soluble polymer included in the hydrocolloid upper layer and hydrocolloid upper layer having excellent physical properties. It includes. That is, the wet dressing agent according to the present invention includes a hydrogel layer on the hydrocolloid layer and the hydrocolloid layer, and includes probiotics as an active ingredient in the hydrogel layer. For example, the hydrocolloid layer includes a pressure sensitive adhesive material, a hydrophobic polymer, a hydrophilic polymer, an oil, and an antioxidant, and the hydrogel layer includes polyvinyl alcohol and a water soluble polymer.

The hydrogel / hydrocolloid bilayer wet dressing agent containing probiotics as the active ingredient according to the present invention absorbs wound exudates and creates a moist environment, and can be used for wound healing applications such as wounds and burns.

In addition, the dressing agent according to the present invention further includes a pharmaceutically acceptable additive to provide a dressing agent that promotes stability and wound recovery.

In addition, the present invention comprises the steps of (a) forming a hydrocolloid layer of a rubbery elastomer matrix; (b) preparing a hydrogel layer comprising a water-soluble polymer and a probiotic as an active ingredient, on top of the hydrocolloid layer, and (c) freezing and thawing the bilayer. A method for producing a double layer dressing agent.

Specifically, the method for preparing a hydrogel / hydrocolloid bilayer wet dressing containing probiotics according to the present invention comprises the following steps:

 (a) a hydrophobic polymer comprising a pressure-sensitive adhesive, a thermoplastic elastomer, a tackifier and a hydrophilic polymer, and optionally a mixture containing one or more additives selected from the group consisting of oils and antioxidants, uniformly mixed at high temperature Preparing a rubbery elastomer matrix and forming a hydrocolloid layer;

(b) dissolving the probiotics and the water-soluble polymer in a solvent to prepare an aqueous solution, and then adding the aqueous solution to the upper portion of the hydrocolloid layer of (a); And

(c) thawing after freezing to prepare a hydrogel / hydrocolloid bilayer wet dressing agent.

Hereinafter, the present invention will be described in more detail.

The present invention is characterized in providing a hydrogel / hydrocolloid bilayer dressing agent containing a probiotic comprising a hydrogel layer containing the probiotics having improved stability of the probiotics and a hydrocolloid layer having excellent physical properties.

The active drug according to the present invention is a drug that can be expected to treat wounds such as antibiotics, disinfectants, wound healing agents, including probiotics, drugs that are unstable in heat and organic solvents, such as fusidic acid, centella asiatica, mupirosine, Neomycin, bacitracin, gentamycin, rincomycin, erythromycin, heparin, benzalkonium chloride, bentrinium, achilinol, triclosan, triclocarban, cetylpyridinium, donepyne bromide, cephayeonzoex and allantoin, Granulation promoters and probiotics such as tyrotricin, fibroblast growth factor (FGF), hepatic fibroblast growth factor (HGF), and marker cell proliferation factor (EGF), and the like, and are preferably selected from the group consisting of probiotics. Can be.

Probiotics, which is a feature of the present invention, is included to enhance the wound healing effect of the pharmaceutical composition comprising the mixture and serves to enhance the wound healing effect of the wet dressing agent. Probiotics that can enhance wound healing are L. acidophilus, L. casei and L. delbrueckii of the genus Lactobacillus . ssp bulgaricus, L. helveticus, L. fermentum , L. reuteri, L. rhamnosus, L. salvarius, L.brevis, L. plantarum, L. paracasei, Lactococcus genus Lc. Lactis , E. faecium of Enterococcus , E. faecalis , S. thermophiles of Streptococcus and B. bifidum of Bifidobacterium , B. breve , B. longum , B. animalis ssp Various probiotic genera including lactis may all be used, and may be one or two or more selected from probiotics, but is not limited thereto. Preferably, one or more selected from the group consisting of L.brevis, and L. plantarum groups can be used.

The amount of probiotics included in the hydrogel layer may be appropriately adjusted as necessary, but specifically 1 x 10 ⁶ To 1 x 10 ¹³ , preferably 1 x 10 ⁸ , to 1 x 10 ¹² , for example, 1 x 10 ¹⁰ CFU.

The solvent used in the preparation of the bilayer dressing agent containing the probiotic of the present invention is not particularly limited in its kind, but water, purified water, or phosphate buffered saline (PBS) may be used, and preferably the stability of the probiotic is improved. In order to ensure the phosphate buffered saline (PBS) in the volume ratio of 0.1 to 1 times the hydrogel may be used in the preparation of the double-layer dressing agent.

In addition, the cryopreservative may be included in order to secure the stability of probiotics in the freezing and thawing of the bacteria and freeze-thaw of the hydrogel, and the cryopreservative is not particularly limited to the type, but DMSO (dimethyl sulfoxide, glycerol, 1, One or more of 2-propan-diol, sufrose, methanol, glucose, proline, galactose, lactose, glycine betanine, fructose, and skim milk can be used. Preferably, glycerol, skim milk, etc. Can be used.

The cryopreservative may comprise 0.1% to 5% by weight, preferably 0.3% to 3% by weight, most preferably 0.5% to 2% by weight, based on 100% by weight of the hydrogel layer.

In the present invention, a pharmaceutically acceptable water-soluble polymer that absorbs wound exudates and creates a wet environment may include polyacrylic acid, polyvinylpyrrolidone, hydroxyalkyl cellulose, carbomer, Polyvinyl alcohol, chitosan, alginates, carboxymethylcellulose, polyhexamethylene biguanide, carrageenan, polyethylene oxide, polysaccharides, collagen, gelatin, hyaluronic acid, xanthan rubber, acacia rubber, guar rubber, arginic acid, agar, Gelatin, gum arabic, tragacanth, karaya gum, pectin, textan and starch or salts thereof, and may be one or two or more selected from the group consisting of, but not limited to. Preferably, 1 or 2 or more from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, carbomer, alginates, carboxymethyl cellulose, dextran, pectin, xanthan rubber and hydroxyalkyl cellulose Can be selected. In one embodiment of the invention, the water-soluble polymer of the hydrogel layer is polyvinyl alcohol alone, or polyvinylpyrrolidone, sodium alginate, copovidone, carbomer, dextran, carboxymethylcellulose or salts thereof, hydroxypropyl It may be one containing at least one selected from the group consisting of cellulose, and hydroxypropyl methyl cellulose.

The water-soluble polymer included in the hydrogel layer may include 1 wt% to 50 wt%, preferably 3 wt% to 30 wt%, and most preferably 5 wt% to 20 wt%, based on 100 wt% of the hydrogel layer. Can be.

In the present invention, the hydrocolloid layer includes a hydrophobic polymer and a hydrophilic polymer, and may optionally include one or more additives selected from the group consisting of oils and antioxidants. In addition, the hydrophobic polymer used in the hydrocolloid layer in the present invention plays a role of imparting the adhesiveness and physical properties of the hydrocolloid formulation, and includes one or two or more from the group consisting of a pressure-sensitive adhesive, a thermoplastic elastomer, and a tackifier. can do.

Pressure-sensitive adhesive materials suitable for the present invention include 1 selected from the group consisting of various natural or synthetic viscous or elastomeric materials such as natural rubber, silicone rubber, acrylonitrile rubber, polyurethane rubber, polyisobutylene, polyisoprene and the like. Or two or more, but is not limited thereto. Preferably polyisoprene and polyisobutylene may be selected.

The thermoplastic elastomers are polyisoprene, polyisobutylene, isobutylene rubber, polyethylene-propylene rubber, polyethylene-propylene diene-modified rubber, styrene-butadiene-styrene, styrene-isoprene-styrene, styrene-ethylene-propylene-styrene and It may be one or two or more selected from the group consisting of styrene-ethylene-butylene-styrene copolymer polymer, but is not limited thereto. More preferably styrene-butadiene-styrene copolymer and styrene-isoprene-styrene copolymer may be selected.

In addition, the tackifier according to the present invention is a thermoplastic material having a molecular weight of 300 ~ 3,000, the petroleum resin system is mainly used aliphatic C ₅ , aromatic C ₉ , C ₅ / C ₉ and hydrogenated DCPD (Dicyclopentadiene) resin, preferably Hydrogenated DCPC resin can be selected.

The hydrophobic polymer included in the hydrocolloid layer may include 20% to 80%, preferably 30% to 70%, and most preferably 40% to 60% based on 100% by weight of the hydrocolloid layer.

In the present invention, a pharmaceutically acceptable hydrophilic polymer for preparing a hydrocolloid layer may use a hydrophilic polymer generally used in a hydrocolloid dressing agent, and the hydrogel polymer used in the hydrocolloid layer according to the present invention may be used as the hydrogel. It may be one or two or more selected from the group consisting of water-soluble polymers used in the layer, but is not limited thereto. For example, pharmaceutically acceptable hydrophilic polymers include polyacrylic acid, polyvinylpyrrolidone, hydroxyalkyl cellulose, carbomer, polyvinyl alcohol, chitosan, alginates, carboxymethylcellulose, polyhexamethylene biguanide, Carrageenan, polyethylene oxide, polysaccharide, collagen, gelatin, hyaluronic acid, xanthan rubber, acacia rubber, guar rubber, arginic acid, agar, gelatin, gum arabic, tragacanth, karaya rubber, pectin, textan and starch or It may be one or two or more selected from the group consisting of salts, but is not limited thereto. Preferably, 1 or 2 or more selected from the group consisting of polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, carbomer, alginates, carboxymethyl cellulose, dextran, pectin, xanthan rubber and hydroxyalkyl cellulose Can be. Preferably, polyvinylpyrrolidone, alginates or sodium carboxymethylcellulose can be selected.

The hydrophilic polymer included in the hydrocolloid layer may be contained in 10 to 50% by weight, preferably 20 to 40% by weight based on 100% by weight of the total hydrocolloid layer.

The hydrocolloid layer according to the present invention may further include one or more selected from oils, antioxidants and the like commonly used in hydrocolloid formulations. The oil plays a role in imparting flexibility and processability in the hydrocolloid layer. Paraffinic oils and naphthenic oils are mainly used, preferably paraffinic oils may be selected.

The antioxidant plays a role of preventing adhesive oxidation at high temperature, and may be a type of BHT (Butylated hydroxytoluene), Vulcanox BKF, LANXESS, IGRANOX, Ciba Specialty Chemical and the like.

According to one embodiment of the present invention, the hydrogel / hydrocolloid bilayer dressing agent containing the probiotic of the present invention is fixed to a probe using a Young's modulus evaluation method and applied a constant force to a texture analyzer (Micro systems) As a result of measuring the elastic modulus in Equation 1, it has an elastic force of 0.1 N / mm ² to 1.0 N / mm ² , preferably 0.2 N / mm ² to 0.8 N / mm ² , most preferably 0.493 ± 0.032 N / mm Has ² .

[Equation 1]

E = Tensile strength / Elongation at break

According to one embodiment of the present invention, the hydrogel / hydrocolloid bilayer dressing agent containing the probiotics of the present invention has a water absorption of 300% to 2,000% as a result of measuring the water absorption by Equation 2 based on the swelling test. Preferably 500% to 1,500%, most preferably 600 to 900%, for example 837.0 ± 21.8%.

[Equation 2]

Absorption (%) = (We / Wo) x 100

According to one embodiment of the present invention, the hydrogel / hydrocolloid bilayer dressing agent containing the probiotics of the present invention was measured by the drug release test while maintaining the conditions of 32 ℃, 100 rpm by the Paddle over disc method, 120 It has a release of 45% to 80% after minutes, preferably 50% to 75%, most preferably 55% to 70%.

In the hydrocolloid layer according to the present invention, a hydrophobic polymer, an oil, an antioxidant, and the like are uniformly mixed at a high temperature in a stirrer to prepare a rubbery elastomer matrix, and then a hydrophilic polymer is added to the uniform rubbery elastomer matrix. It can be prepared by mixing and uniformly mixing.

As a result of measuring the content of probiotics contained in the finally prepared hydrogel / hydrocolloid bilayer wet dressing agent of the present invention by the following Equation 3, 50% to 100%, preferably 60% to 100%, most preferably May have a content of 80% to 100%.

 [Equation 3]

Content (%) = {quantity of probiotics in medium (CFU) / amount of probiotics initially injected during manufacture (CFU)} × 100

Another embodiment of the present invention comprises the steps of (a) uniformly mixing a mixture of hydrophobic polymer, hydrophilic polymer, oil, and antioxidant at high temperature to prepare a rubbery elastomer matrix to form a hydrocolloid layer;

(b) dissolving the probiotics and the water-soluble polymer in a solvent to prepare an aqueous solution, and then adding the aqueous solution to the upper portion of the hydrocolloid layer of (a); And

(c) thawing after freezing to prepare a hydrogel / hydrocolloid bilayer wet dressing agent, the method of producing a hydrogel / hydrocolloid bilayer wet dressing containing probiotics.

The hydrophobic polymer used in the hydrocolloid layer may include one or two or more from the group consisting of a pressure-sensitive adhesive, a thermoplastic elastomer and a tackifier. The hydrocolloid layer may further include at least one selected from the group consisting of oils and antioxidants, in addition to hydrophobic polymers and hydrophilic polymers. The hydrophobic polymers, hydrophilic polymers, pressure-sensitive adhesives, thermoplastic elastomers, tackifiers, oils, and antioxidants are as described in the above description of the double layer dressing agent.

The hydrogel layer includes a water soluble polymer and probiotics, and may further include a cryopreservative to increase the stability of the probiotics. The water soluble polymers, probiotics, solvents, and cryopreservatives are as described in the above description of double layer dressing agents.

The freezing process is 12 hours to 24 hours, preferably 14 hours to 20 hours at a temperature of -50 ° C to -10 ° C, preferably -40 ° C to -15 ° C, more preferably -30 ° C to -20 ° C. More preferably 16 to 19 hours.

The thawing process is 2 hours to 10 hours, preferably 3 hours to 8 hours, more preferably 4 hours at 0 ° C. to 50 ° C., preferably 10 ° C. to 40 ° C., more preferably 15 ° C. to 30 ° C. It is appropriate to carry out for 7 to 7 hours.

The freezing and thawing process is suitably repeated 1 to 5 times, preferably 2 to 4 times.

If the freezing, thawing temperature and the reaction time is out of the range, the activity of the probiotics may be reduced, the safety may be deteriorated, and the physical properties of the dressing agent may change, so it is appropriate to perform the freeze thawing process in the above range.

Hydrogel / hydrocolloid bilayer dressings containing probiotics effective for wound healing such as wounds and burns according to the present invention can be prepared in pharmaceutical forms comprising conventional excipients including conventional gel properties and release controlling agents.

The present invention is excellent in the manufacturing method of the hydrogel / hydrocolloid double-pack wetting dressing agent containing the probiotics is not destroyed in the physical environment and the efficacy is maintained, and the physical properties produced by the above method, and improve the ease of wound application It is possible to provide a dressing agent having an improved wound healing effect.

1 is a shape photograph of a probiotic-containing hydrogel / hydrocolloid bilayer dressing agent according to the present invention, FIG. 1A is a hydrocolloid monolayer, FIG. 1B is a hydrogel layer dressing that does not contain probiotics, and FIG. 1C contains probiotics. The shape photograph of the hydrogel layer dressing is shown.
Figure 2 is a graph comparing the release pattern according to the hydrophilic polymer of the probiotic-containing hydrogel / hydrocolloid bilayer dressing agent according to the present invention.
FIG. 3 shows: 1. control (gaze treated group), 2. conventional product (Duoderm ™), 3. control 5, and 4. probiotic-containing hydrogel / hydrocolloid bilayer dressing agent (Example 13) 1), after 1 day, after 3 days, after 7 days, after 10 days, after 13 days, the wound portion photograph is shown.
Figure 4 shows a wound site infected with Staphylococcus aureus: 1. control (gaze treatment group), 2. conventional product (DuodermTM), 3. control 5, and 4. probiotics containing hydrogel / hydrocolloid bilayer dressing of the present invention After processing the (Example 13), the wound part photograph is shown after 1 day, after 3 days, after 7 days, after 10 days, and after 13 days.
Figure 5 shows the wound site infected with Pseudomonas aeruginosa, 1. control (gaze treatment group), 2. conventional product (DuodermTM), 3. control 5, and 4. probiotics containing hydrogel / hydrocolloid bilayer dressing agent of the present invention ( After treating Example 13), the photograph of the wound site after 1 day, 3 days, 7 days, 10 days, and 13 days is shown.
Figure 6 shows the size reduction of the general wound area treated with the control group (gaze treatment group), conventional product (duoderm), Comparative Example 5, and the probiotic-containing hydrogel / hydrocolloid dressing agent (Example 13) It is a graph. * p <0.05: Statistics for control (gaze), #p <0.05: Statistics for control (gaze) and existing product,? p <0.05: Statistics for control (gaze) and existing product and Comparative Example 5
7 is a wound site infected with Staphylococcus aureus treated with a control group (gauze treatment group), a conventional product (Duoderm), Comparative Example 5, and the probiotic-containing hydrogel / hydrocolloid dressing agent (Example 13) Is a graph showing the decrease in size. * p <0.05: Statistics for control (gaze), #p <0.05: Statistics for control (gaze) and existing product,? p <0.05: Statistics for control (gaze) and existing product and Comparative Example 5
Figure 8 is the size of the wound site infected with Pseudomonas aeruginosa treated with a control group (gaze treatment group), conventional products (Duoderm), Comparative Example 5, and the probiotic-containing hydrogel / hydrocolloid dressing agent (Example 13) It is a graph showing the decrease. * p <0.05: Statistics for control (gaze), #p <0.05: Statistics for control (gaze) and existing product,? p <0.05: Statistics for control (gaze) and existing product and Comparative Example 5
9 is 1. control group (gaze treated group), 2. conventional product (duoderm), 3. control example 5, and 4. A photograph of the organization of a general wound treated with a probiotic containing hydrogel / hydrocolloid dressing agent (Example 13) according to the present invention.
Figure 10 is a control group (gaze treatment group), conventional products (duodom), 3. control 5, and 4. Staphylococcus aureus treated with a probiotic-containing hydrogel / hydrocolloid dressing agent (Example 13) according to the present invention This is a picture of the organization of wounds infected with.
11 is infected with Pseudomonas aeruginosa treated with a control group (gauze treatment group), a conventional product (duoderm), 3. control examples, and 4. a probiotic-containing hydrogel / hydrocolloid dressing agent (Example 13). The organized picture of the wound.

Hereinafter, the present invention will be described in detail by Examples and Experimental Examples. However, the following Examples and Experimental Examples are only for illustrating the present invention, and the scope of the present invention is not limited by the Examples and Experimental Examples.

< Example  1> Probiotics  Preparation of the suspension

Among several species of probiotics, L. brevis ( Lactobacillus brevis KCTC3498) was collected in small amounts with a sterile kolle handle, and streaked on a solid medium of MRS agar (DifcoTM Lactobacilli MRS Broth + BactoTM agar, Becton, Dickinson and Company, USA). plate method). One colony was pipetted into 3 ml MRS broth (DifcoTM Lactobacilli MRS Broth, Becton, Dickinson and Company, USA) liquid medium and incubated for 16 hours with stirring at 37 ° C. After that. 50% glycerol was mixed as a cryopreservative in the culture solution and stored in a frozen stock at -80 ° C deep freezer.

The stock stored during the preparation of the probiotic-containing hydrogel / hydrocolloid bilayer dressing was buried in a small amount with a kolle handle and incubated in MRS agar medium by a streak plate method. One colony was pipetted into a liquid medium, stirred at 37 ° C. for 16 hours, and the culture was inoculated with MRS broth broth for 16 hours at 37 ° C. Next, centrifuge 1 L culture solution at 6000 g, 4 ° C for 10 minutes, discard the remaining MRS medium in the supernatant, and vortex with 100 ml of phosphate-buffered saline (PBS). It was. After centrifugation at 6000 g and 4 ° C. for 10 minutes, the supernatant was discarded and this process was repeated again. Finally, 100 ml of PBS was added to vortex, redispersed, and glycerol was added as a cryopreservative, mixed well, and stored at -80 ° C deep freezer. The stored probiotics were used in the preparation of the hydrogel / hydrocolloid bilayer dressing agent, and the concentration of the probiotics in use was measured by Colony Forming Unit (CFU) to prepare a hydrogel / hydrocolloid bilayer dressing agent of the desired concentration.

< Example  2> Hydrocolloid layer  Produce

The hydrocolloid layer was prepared in the following composition: styrene-isoprene-styrene copolymer: polyisobutylene rubber: Hydrogenated hydrocarbons: sodium carboxymethylcellulose: paraffin oil: IGRANOX 1010: 10: 25: It was prepared by mixing at a ratio of 20: 34.5: 10: 0.5% by weight. Specifically, the styrene-isoprene-styrene copolymer was stirred and dissolved in a vessel heated to 180 to 200 ° C, and Irganox was added thereto and stirred. After the temperature was lowered, the mixture was stirred with polyisobutylene rubber, petroleum resin, and paraffin oil at 140 to 160 ° C., and then added with sodium carboxymethyl cellulose at 100 to 120 ° C., stirred, and placed in a hot melt coater. Laminated with. After drying at room temperature it was laminated with a polyurethane film to prepare a general hydrocolloid layer.

< Example  3 to 19> Hydrogel Of Hydrocolloid Double layer  Preparation of Dressing Agent

Next, the components shown in Tables 1 and 2 were dissolved in purified water according to the following composition (based on 100% by weight of the hydrogel layer) to prepare an aqueous solution, and a hydrocolloid layer prepared in Example 2 Probiotic-containing hydrogel / hydrocolloid bilayer dressings were prepared at concentrations of 1 × 10 ¹⁰ CFU.

Specifically, the freeze / thaw method was used, and after freezing at -20 ° C. for 18 hours, thawed at 20 ° C. for 6 hours, the freeze thawing process was repeated three times.

division Example 3 4 5 6 7 8 9 10 11 12 Example 1 Probiotic Suspensions
(Unit: weight%) One One One One One One One One One One Polyvinyl alcohol
(Unit: weight%) 4 5 10 15 20 10 10 10 10 10 Sodium alginate
(Unit: weight%) - - - - - 0.25 0.5 One 1.5 3

division Example 13 14 15 16 17 18 19 Example 1 Probiotic Suspensions
(weight%) One One One One One One One Polyvinyl alcohol
(weight%) 10 10 10 10 10 10 10 Polyvinylpyrrolidone
(weight%) 0.5 - - - - - - Copovidone (% by weight) - 0.5 - - - - - Carbomer (wt%) - - 0.5 - - - - Carboxymethylcellulose Sodium (% by weight) - - - 0.5 - - - Dextran (% by weight) - - - - 0.5 - - Hydroxypropyl cellulose (% by weight) - - - - - 0.5 - Hydroxypropylmethylcellulose (% by weight) - - - - - - 0.5

< Comparative example  1> Hydrogel Single layer  Preparation of Dressing Agent

Without the hydrocolloid layer prepared in Example 2, the probiotics containing hydrogel monolayer dressing agent at a concentration of 1 × 10 ¹⁰ CFU was directly mixed in the composition of Example 13 at freezing using a freeze thaw method at -20 ° C. 18 hour thawing was prepared by repeating freeze thawing three times at 20 ° C. for 6 hours.

< Comparative example  2> L. plantarum contain Hydrogel Of Hydrocolloid Double layer  Preparation of Dressing Agent

L. plantarum , another kind of probiotics A hydrogel / hydrocolloid bilayer dressing agent was prepared in the same manner as in Example 13, including ( Lactobacillus plantarum subsp. Plantarum KCTC3105).

Comparative Examples 3 to 5 Preparation of Hydrogel / Hydrocolloid Bilayer Dressing Agent

As shown in Table 3 below, the probiotics prepared in Example 1 were directly mixed with the composition of Comparative Example 3-5 (based on 100 wt% of the hydrogel layer), and then attached to the hydrocolloid layer prepared in Example 2. Hydrogel / hydrocolloid bilayer dressings with or without probiotics were prepared. Specifically, freeze thawing was repeated three times at 20 ° C. for 6 hours at -20 ° C. and freeze thawing three times at 20 ° C. for freezing using the freeze thawing method. It was prepared by.

division Comparative example 3 4 5 Probiotic Suspension (% by weight) One One - Polyvinyl Alcohol (Unit: wt%) 10 21 10 Sodium alginate (unit: weight%) 3.1 - Polyvinylpyrrolidone (Unit: wt%) - 0.5

< Experimental Example  1> Evaluation of elastic force characteristics

In order to evaluate whether the hydrogel / hydrocolloid bilayer dressing agent containing the active ingredient such as the probiotic of the present invention can be applied well to curved wound sites as a formulation, the physical properties of the bilayer dressing agent of the present invention were evaluated.

Comparative Examples 2 to 19, which are hydrogel / hydrocolloid bilayer dressing agents containing active ingredients such as probiotics according to the present invention, and Comparative Examples 2 to 4, which are hydrocolloid monolayer dressing agents, and Example 2 and hydrogel monolayer dressing agents Physical property evaluation was performed including Example 1. Physical properties of hydrogel / hydrocolloid bilayer dressings containing active ingredients such as probiotics were evaluated by Young's modulus, which is commonly used in the art, and was determined by Equation 1.

Young's modulus evaluation was performed with a texture analyzer (Micro systems) by immobilizing the test agent on the probe and applying a constant force.

[Equation 1]

E = Tensile strength / Elongation at break

division Young's modulus
(Unit: N / mm ² ) division Young's modulus
(Unit: N / mm ² ) Example 2 0.213 ± 0.022 Example 14 0.523 ± 0.043 Example 3 Not measurable Example 15 0.702 ± 0.104 Example 4 0.553 ± 0.073 Example 16 0.712 ± 0.120 Example 5 0.584 ± 0.086 Example 17 0.702 ± 0.104 Example 6 0.694 ± 0.095 Example 18 0.660 ± 0.050 Example 7 0.887 ± 0.105 Example 19 0.539 ± 0.096 Example 8 0.753 ± 0.069 Comparative Example 1 0.941 ± 0.157 Example 9 0.706 ± 0.078 Comparative Example 2 0.481 ± 0.042 Example 10 0.883 ± 0.092 Comparative Example 3 Not measurable Example 11 1.106 ± 0.122 Comparative Example 4 Not measurable Example 12 1.306 ± 0.153 Comparative Example 5 0.249 ± 0.042 Example 13 0.493 ± 0.032

As can be seen in Table 4, Examples 3 to 19 of the hydrogel / hydrocolloid double-layer dressing agent according to the present invention showed a higher elasticity index expressed as Young's modulus compared to the single-layer hydrocolloid containing no active drug. And, it can be seen that the elastic force falls.

On the other hand, Comparative Examples 3 and 4 did not form a hydrogel. In the case of Comparative Example 3, if the amount of alginate is 3 or more, it is determined that hydrogel is not formed due to the freeze thawing process of polyvinyl alcohol. In addition, even when the polyvinyl alcohol of Comparative Example 4 contains 20 or more, it was impossible to manufacture due to excessive viscosity. In the case of using the polyvinylpyrrolidone of Example 13 it was confirmed to exhibit the best elastic force.

In addition, looking at the elastic force of Comparative Example 2 in which the probiotics were changed, it was confirmed that there is almost no change according to the types of probiotics. In addition, it was confirmed that the elastic force is lowered due to probiotics when compared with Comparative Example 5 without probiotics and Example 13 with probiotics.

In the above experimental results, it was confirmed that the present invention can control the elastic force in a variety of components depending on the components used when the active drug is prepared in the dressing agent containing a hydrogel layer.

< Experimental Example  2> Absorption Rate Evaluation

This experiment is to evaluate the effluent absorption of dressing agent, which is important for maintaining the wet environment. Comparative Examples 2 to 19, which are hydrogel / hydrocolloid bilayer dressing agents containing active ingredients such as probiotics according to the present invention, and Comparative Examples 2 to 4, which are hydrocolloid monolayer dressing agents, and Example 2 and hydrogel monolayer dressing agents Absorption rate evaluation was performed including Example 1. Absorption rate evaluation is shown in Table 5 evaluated by the swelling test commonly used in the art.

The dried dressing agent was taken in a size of 2 cm X 2 cm and immersed in 37 ° C. PBS (Phospahte buffered saline) for 24 hours to measure the weight (We), and dried in an oven at 60 ° C. for 8 hours. Measured. Finally, it was calculated using Equation 2.

[Equation 2]

Absorption (%) = (We / Wo) x 100

division Absorption rate (%) division Absorption rate (%) Example 2 835.3 ± 27.4 Example 14 829.3 ± 27.6 Example 3 Not measurable Example 15 677.9 ± 29.1 Example 4 803.5 ± 38.8 Example 16 728.8 ± 31.9 Example 5 852.1 ± 10.1 Example 17 778.6 ± 35.9 Example 6 655.8 ± 25.5 Example 18 748.3 ± 29.5 Example 7 617.8 ± 60.3 Example 19 749.3 ± 20.7 Example 8 852.1 ± 10.1 Comparative Example 1 187.6 ± 11.8 Example 9 886.2 ± 24.9 Comparative Example 2 842.5 ± 30.9 Example 10 892.3 ± 11.3 Comparative Example 3 Not measurable Example 11 854.5 ± 18.2 Comparative Example 4 Not measurable Example 12 848.1 ± 24.2 Comparative Example 5 911.3 ± 30.7 Example 13 837.0 ± 21.8

As can be seen in Table 5, Examples 3 to 19, which is a hydrogel / hydrocolloid bilayer dressing agent according to the present invention, is a single layer hydrocolloid having an absorption rate, which is an indicator of absorbing exudates in a wound, containing no active drug. It was confirmed that the level is similar to.

In addition, in the case of Example 3 in which the amount of polyvinyl alcohol is less than 5 among the components constituting the hydrogel layer in the same manner as the evaluation of the elastic force, measurement was not possible because no hydrogel was formed, and Comparative Examples 3 and 4 also did not form a hydrogel. Did.

When the alginate, polyvinylpyrrolidone and copovidone of Examples 8 to 14 were used, it was confirmed to exhibit the same or more absorbent power as a single layer hydrocolloid.

In addition, the absorption power of Comparative Example 2, in which the probiotics were changed, confirmed that there was almost no difference due to the change in the probiotics, and the absorption power was lowered due to the probiotics compared with the Comparative Example 5 without the probiotics and the Example 13 containing the probiotics. Confirmed.

As a result of the experiment, when the active agent of the present invention contains a hydrogel layer to prepare a dressing agent, it was confirmed that the absorption power can be adjusted in various ways depending on the components used, among them alginate, polyvinylpyrrolidone and nose It was confirmed that the best when using povidone, in particular, the case of Example 13 using polyvinylpyrrolidone was confirmed to be the best.

< Experimental Example  3> shape evaluation

This experiment is an experiment for confirming the shape of a hydrogel / hydrocolloid bilayer dressing agent containing an active ingredient such as probiotics according to the present invention.

A hydrogeloid, a hydrogel / hydrocolloid bilayer dressing agent containing no probiotics, and a hydrogel / hydrocolloid bilayer dressing agent containing probiotics with Example 2, Example 13 and Comparative Example 5 were prepared by scanning electron microscopy. , SEM; S-4800; Hitachi, Tokyo, Japan) to observe the surface morphology is shown in Figure 1a to 1c.

As shown in Figure 1a, the hydrocolloid layer prepared in Example 2 was found to be intricately intertwined hydrocolloids according to the network of hydrophobic polymers.

As shown in Figure 1b, it was confirmed that the hydrogel / hydrocolloid bilayer dressing agent containing no probiotics prepared in Comparative Example 5 is very transparent and clean surface of the hydrogel.

As shown in FIG. 1C, the probiotics contained in the hydrogel layer were confirmed in the form of the hydrogel / hydrocolloid bilayer dressing agent containing the probiotics prepared in Example 13.

< Experimental Example  4> Probiotics  Content evaluation

This experiment is to evaluate the content of probiotics, the active drug of hydrogel / hydrocolloid dressing agent.

The hydrogel / hydrocolloid bilayer dressing agent according to the present invention was chopped finely, put into 200 ml of PBS, stirred with a stirrer, sampled with 1 ml, diluted appropriately with PBS, and cultured at 37 ° C. by spreading 100 ul into MRS agar medium. The number of colonies was directly counted to determine the number of viable cells, and the content was calculated as follows.

[Equation 3]

Content (%) = {quantity of probiotics in medium (CFU) / amount of probiotics initially injected during manufacture (CFU)} × 100

The content showed at least 80% content in all examples.

< Experimental Example  5> Probiotics  Emission rating

This experiment is to evaluate the change of release of probiotics, the active drug of hydrogel / hydrocolloid bilayer dressing.

The probiotic release characteristics of Examples 5 to 9 and 13 to 19 and Comparative Examples 1 and 2 of the hydrogel / hydrocolloid bilayer dressing agent according to the present invention were evaluated.

The hydrogel / hydrocolloid bilayer dressing formulation containing the active drug was subjected to a drug release test while maintaining the conditions at 32 ° C. and 100 rpm using the Paddle over disc method, the fifth method of transdermal drug dissolution evaluation of USP. 1 ml of each sample was taken at regular time intervals, and the same amount of the discharged solution was added after sampling to maintain the sink condition of the discharged liquid. The concentration of the collected sample was shown in the graph of FIG. 2 as the release amount (%) relative to the total amount contained in the dressing formulation measured according to Experiment 4 by actually culturing the sample solution and measuring the number of viable cells.

As can be seen in Figure 2, the hydrogel / hydrocolloid bilayer dressing formulation according to the present invention showed a sustained release of probiotics and it was confirmed that the initial and final release rate can be properly adjusted according to the polymer used.

In particular, the hydrogel / hydrocolloid bilayer dressing formulation using the polyvinylpyrrolidone of Example 13 showed the highest release rate, and similar release rate was confirmed when compared with Example 13 and Comparative Examples 1 and 2, The hydrogel / hydrocolloid bilayer dressing formulation according to the present invention was confirmed that there is no difference between the monolayer hydrogel and the probiotic type.

< Experimental Example  6> Wound Recovery Evaluation

Wound recovery was evaluated as follows for Example 13 and Comparative Example 5 and the commercially available DuodermTM (Convatec Co.) and the control group as a general gauze treatment.

SD rats with an average age of 6-8 weeks and a weight of 250-300 g were used for 1 week. The breeding environment was reared in the general breeding room (temperature 23 ± 2 degrees, humidity 55 ~ 60%, lighting from 7 am to 7 pm) until the end of the experiment, and the feed was supplied with the general feed, and the water was used with the normal purified water. . Wound induction is anesthetized with Ketamine and Xylazine in rats without any abnormality during the acclimatization period. After hair removal and disinfection, the skin is cut with scissors to cause 10 mm x 10 mm wounds or the skin is cut with scissors 10 mm x 10 The wound that caused the wound of mm was infected with Pseudomonas aeruginosa and Staphylococcus aureus. After the dressing was carried out by measuring the area by using the grid paper according to the time and the wound size was measured and the recovery rate was evaluated by the evaluation of the recovery and visual observation by Equation 4 below, the results of the wound healing is measured in Figures 3, 4 and 5 is shown.

[Equation 4]

Wound Recovery Rate = (First scratch size-Later wound size) / First scratch size * 100

As can be seen in Figures 3, 4 and 5, Example 13, a hydrogel / hydrocolloid bilayer dressing agent containing the probiotics according to the present invention was confirmed that there is a very excellent wound recovery effect.

In addition, as can be seen in the graph of the wound recovery rate in FIGS. 6, 7 and 8, Example 13, a hydrogel / hydrocolloid bilayer dressing containing probiotics similarly to visual observation, showed the best wound recovery rate. It was. In particular, the wound recovery rate was faster in wounds infected with Staphylococcus aureus and Pseudomonas aeruginosa than in general wounds.

Commercial formulations showed less effective wound healing than no treatment, but showed lower wound healing effects than Example 13 according to the present invention. From this, it can be confirmed that the hydrogel hydrocolloid preparation containing probiotics is a composition containing probiotics which is superior to commercially available preparations and helps in wound recovery.

< Experimental Example  7> Histopathology Evaluation

Final wounds after the wound recovery evaluation of the wounds infected with the common wounds, yellow grapes, and Pseudomonas aeruginosa in Example 13 and Comparative Example 5 and commercially available DuodermTM (Convatec Co., Ltd.) and the control group according to the present invention. Histopathological evaluation was performed on the site, and the results are shown in FIGS. 9, 10, and 11.

Compared with the gauze treatment control group, a significant decrease in the wound epithelial defect site was recognized in Comparative Example 5 and Example 13, Duoderm ™ treatment groups, and as a result, a significant increase in epithelial regeneration rate was recognized, respectively. Example 13 treatment group compared with Comparative Example 5 treatment group and DuodermTM similarly to the wound recovery evaluation experiment of Experiment 13 with a significant effect of promoting wound healing, that is, better granulation tissue loss, epithelial regeneration and collagen fiber proliferation Inhibitory effect on inflammatory cell and mast cell invasion and angiogenesis. These experimental results indicate that there is an effect of promoting wound healing due to probiotics.

Claims (6)

A hydrocolloid layer, and a hydrogel layer,
The hydrogel layer is a probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent, comprising a probiotic, a water-soluble polymer and a solvent,
The water-soluble polymer of the hydrogel layer is polyvinyl alcohol alone, or
At least one selected from the group consisting of polyvinylpyrrolidone, sodium alginate, copovidone, carbomer, dextran, carboxymethylcellulose or salts thereof, hydroxypropylcellulose, and hydroxypropylmethylcellulose together with polyvinyl alcohol Including,
The content of the polyvinyl alcohol is 5 to 20% by weight based on 100% by weight of the hydrogel layer, probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent.
The probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent according to claim 1, wherein the probiotics are selected from the group consisting of lactic acid bacteria strains.
The probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent according to claim 1, wherein the content of sodium alginate is 0.25 to 3% by weight based on 100% by weight of the hydrogel layer.
The probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent of claim 1, wherein the water soluble polymer of the hydrogel layer comprises polyvinyl alcohol and polyvinylpyrrolidone.
The probiotic-containing hydrogel / hydrocolloid bilayer wet dressing agent according to any one of claims 1 to 4, wherein the bilayer wet dressing agent is for treating wounds infected with Staphylococcus aureus or Pseudomonas aeruginosa.
(a) uniformly mixing a mixture of hydrophobic polymer, hydrophilic polymer, oil, and antioxidant at high temperature to prepare a rubbery elastomer matrix to form a hydrocolloid layer;
(b) dissolving the probiotics and the water-soluble polymer in a solvent to prepare an aqueous solution, and then adding the aqueous solution to the upper portion of the hydrocolloid layer of (a); And
(c) thawing after freezing to prepare a hydrogel / hydrocolloid bilayer wet dressing agent, the method of producing a hydrogel / hydrocolloid bilayer wet dressing agent containing probiotics, comprising:
In the step (b), the water-soluble polymer is polyvinyl alcohol alone or polyvinylpyrrolidone, sodium alginate, copovidone, carbomer, dextran, carboxymethylcellulose or salts thereof, hydroxypropylcellulose, and hydroxy At least one selected from the group consisting of propylmethylcellulose together with polyvinyl alcohol,
The polyvinyl alcohol content is 5 to 20% by weight based on 100% by weight of an aqueous solution prepared by dissolving probiotics and water-soluble polymers in a solvent, a method for producing a hydrogel / hydrocolloid double layer wet dressing agent.

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