CN112156224A

CN112156224A - Composition for preparing keratin liquid dressing and preparation method and application thereof

Info

Publication number: CN112156224A
Application number: CN202011016782.XA
Authority: CN
Inventors: 查国栋; 余钡; 欧珍; 王伯初
Original assignee: Hainan Haimers Medical Biotechnology Co ltd
Current assignee: Hainan Haimers Medical Biotechnology Co ltd
Priority date: 2020-01-20
Filing date: 2020-09-24
Publication date: 2021-01-01

Abstract

The invention relates to a composition for preparing a liquid dressing, a preparation method and related application. The liquid dressing is prepared from keratin, a film-forming agent, a humectant, a material solvent and a volatile solvent. The method for preparing the liquid dressing by using the keratin is simple, and the preparation or generation process is not complicated. The liquid dressing prepared by the invention can effectively stop bleeding, accelerate wound healing, isolate bacterial infection and has no toxic or side effect. And the product can volatilize and absorb, so that the link of retreating the medicine is avoided, only a proper amount of the medicine is needed to be sprayed on the wound in the actual repeated use process, the use is convenient, the wound can not be injured again due to the treatment of the residual medicine, and excessive pain can be avoided. The liquid dressing prepared by the invention has certain waterproofness, air permeability and extensibility after being filmed, and has a wide application range.

Description

Composition for preparing keratin liquid dressing and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to a composition for preparing a keratin liquid dressing, and a preparation method and application thereof.

Background

Keratin, a non-toxic natural biomaterial, has been successfully applied in tissue repair, and can be made into various types of wound dressings due to its abundant cysteine residues, spontaneous assembly and polymerization properties. As a protein source for human, the excellent biocompatibility and good biodegradability further widen the application of the protein in regenerative medicine. Most of the conventional human hair keratin is an extract obtained by a chemical or enzymatic extraction method. Therefore, it has problems of complicated composition and difficulty in quality control. Human hair contains not only keratin but also abundant keratin-related proteins and intermediate silk proteins, etc. Recombinant expression is an important way to improve the purity and quality of keratin.

The liquid dressing has the advantages of maintaining local moist environment around the wound, good air permeability, protecting the wound from microbial infection, preventing the wound from drying, being simple, convenient and comfortable during dressing change and being beneficial to relieving the pain of the wound. The liquid dressing is convenient to carry and simple to use, can achieve the treatment effect by being directly sprayed on an affected part, and reduces the bacterial infection and the pain of a wound. Liquid dressings are also currently in the market favor of many consumers.

There is currently a 3M Cavilon liquid dressing on the market, which is a polymeric solution, forming a thin film for application to the skin, which is dispersed as a non-cytotoxic, non-irritating solution and dries quickly. The product can help intact or damaged skin from the irritations of incontinent urine/feces, digestive juices, wound exudates, mucus and lesions. The main effect is also to protect the skin from contamination and damage. The main components of the emulsion are acrylate copolymer, hexamethyldisiloxane, polyethyl phenyl methyl siloxane and isooctane.

Compared with the liquid dressing, the liquid dressing can achieve the effect of quickly forming a film and also has the effects of protecting and isolating the skin. In addition, the liquid dressing of the invention is added with drug effect components, which can further promote the repair of skin and the growth of granulation and accelerate the healing of wound. Compared with the liquid dressing, the liquid dressing has wider use scenes, for example, the liquid dressing can be used for conveniently treating acute wounds due to the relief of the red buttocks quadrants of infants and old people, and can also be used for promoting the repair of related conditions such as postoperative recovery and healing of minimally invasive surgery or laser surgery to a certain extent.

Disclosure of Invention

One of the objectives of the present invention is to obtain the composition components for preparing film-forming agent, and the composition can be made into film-forming agent with certain medicinal effect.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the composition for preparing the film forming agent comprises a composite film forming agent and keratin, wherein the composite film forming agent comprises polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, sodium carboxymethyl cellulose and acrylic resin;

specifically, the composite film-forming agent consists of polyvinyl alcohol and polyvinylpyrrolidone; of course, the keratin may be replaced by other pharmacologically active ingredients that have pharmacological effects and are absorbed by the skin and mucous membranes.

Further, the weight ratio of the keratin to the composite film-forming agent is 1: 10-40.

Further, the weight ratio of the polyvinyl alcohol to the polyvinylpyrrolidone is 1-3: 2-4.

The other purpose of the invention is to obtain the liquid dressing composition containing the film-forming agent composition, and the liquid dressing composition can be prepared into liquid dressings with certain drug effects.

the liquid dressing composition comprises the following components in parts by weight:

keratin: 1-5 parts by weight;

5-70 parts of polyvinyl alcohol;

5-100 parts of polyvinylpyrrolidone;

humectant: 5-50 parts by weight;

material solvent: 10-100 parts by weight;

a suitable amount of volatile medium.

Further, the humectant is any one that can be dissolved into the whole system, such as glycerin, propylene glycol, silanes;

specifically, the humectant is propylene glycol;

further, the material solvent is one or two of water and ethanol;

specifically, the material solvent is water;

specifically, the volatile medium is one or more of water, ethanol, isopropanol, ethyl acetate and isooctane;

further, the volatile medium is ethanol and ethyl acetate in a volume ratio of 6-1: 3;

further, the liquid dressing composition comprises the following components in parts by weight:

keratin: 2 plus or minus 0.2 weight portions;

10 plus or minus 1 weight part of polyvinyl alcohol;

30 plus or minus 3 portions of polyvinylpyrrolidone;

propylene glycol: 10 plus or minus 1 part by weight;

water: 20-50 parts by weight;

the volume ratio is 1: ethanol and ethyl acetate of 1: 880 and 950 parts by weight.

Specifically, the pH of the liquid dressing is 6-7; the viscosity of the liquid dressing is 1600-.

It is a further object of the present invention to provide a method of making a liquid dressing using the liquid dressing composition.

the method for preparing the liquid dressing by using the liquid dressing composition is characterized in that the components are fully mixed to obtain the liquid dressing.

Specifically, the method comprises the following steps:

a, dissolving the polyvinyl alcohol and the polyvinylpyrrolidone with the formula amount in water, fully swelling and fully mixing to obtain a composite film-forming agent pretreatment material;

b, adding the keratin with the formula amount into the film-forming agent pretreatment material obtained in the step A, and fully mixing to obtain a film-forming agent containing keratin;

and C, adding the humectant in a formula amount into the keratin-containing film-forming agent obtained in the step B, and fully mixing to obtain the liquid dressing.

Further, after the step C, the ethanol and ethyl acetate are added.

The fourth purpose of the invention is to provide the application of the liquid dressing.

animal experiments show that the liquid dressing can promote acute wound healing;

according to the cell scratch test, the liquid dressing can promote the migration of cells.

Thus, the liquid dressing may act as a skin protectant;

in particular, the liquid dressing can be used as a skin isolation agent, a wound healing promoter and a granulation growth promoter.

The invention has the beneficial effects that:

1. the invention provides components of a composition for preparing a keratin film-forming agent, and provides a component selection for preparing the keratin film-forming agent;

2. the invention provides a liquid dressing composition containing a film-forming agent composition, which provides a component selection for preparing a liquid dressing;

3. the invention provides a method for preparing liquid dressing by using the liquid dressing composition, which can be used for preparing medicaments, is simple and convenient to prepare, is beneficial to operation and is convenient to produce;

4. the invention provides a keratin liquid dressing which can be used for hemostasis and antibiosis, promotion of wound healing, prevention of secondary injury of wounds and the like.

Drawings

FIG. 1 is a graph of the effect of a liquid dressing on cell migration;

FIG. 2 is a photographic image of the effect of a liquid dressing on wound closure rate;

fig. 3 is a graph of the effect of a liquid dressing on wound closure rate.

Detailed Description

The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1

Preparation of recombinant human keratin:

constructing recombinant plasmid containing recombinant keratin target gene segment, and transferring the recombinant plasmid into escherichia coli BL21(DE 3). The transformed bacteria were mixed at a ratio of 1: 5000 in proportion was inoculated into a sterile LB-kana (50ug/mL) liquid medium and cultured at 170rpm at 37 ℃ overnight. The temperature was lowered to 25 ℃ when the OD was 0.8-1.0, 170rpm, and induction was carried out using IPTG at a final concentration of 1 mM. After 8h of induction, the strain is collected by centrifugation at 8000rpm for 5min at 4 ℃. The bacterial cells were resuspended in 5 volumes of Buffer A (50mM Tris pH8.0,150mM NaCl,5mM EDTA,20 mM. beta. -mercaptoethanol) based on the weight of the bacteria. The homogenate was broken at 1000bar, centrifuged at 20000rpm at 4 ℃ for 1 hour, and the supernatant was discarded. Resuspend the pellet with Buffer A of 10 times the pellet weight, sonicate for 3min, centrifuge at 10000rpm for 15min, and discard the supernatant. The pellet was resuspended in 10 volumes Buffer B (50mM Tris pH8.0,150mM NaCl,5mM EDTA,20 mM. beta. -mercaptoethanol, 1M Urea, 0.5% Triton X-100) of the pellet weight, sonicated for 3min, centrifuged at 10000rpm for 15min, and the supernatant discarded. Resuspend the pellet with 10 volumes Buffer C (50mM Tris pH8.0,2.5M NaCl,5mM EDTA,20 mM. beta. -mercaptoethanol) of the pellet weight, sonicate for 3min, centrifuge at 10000rpm for 15min, and discard the supernatant. Resuspend the pellet with 10 volumes Buffer A (50mM Tris pH8.0,150mM NaCl,5mM EDTA,20 mM. beta. -mercaptoethanol) of the pellet weight, sonicate for 3min, centrifuge at 10000rpm for 15min, and remove the supernatant. Washing the precipitate with purified water for 2 times, resuspending, standing at-80 deg.C, and freeze drying with freeze dryer to obtain keratin water solution or powder.

Other prior art references may also be made to the preparation of keratin.

Example 2

Selecting auxiliary materials:

1. film-forming agent: the film forming agent is selected from polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, sodium carboxymethyl cellulose, and acrylic resin. Experiments show that one of the materials is selected for single use, and the viscosity is too high or the film cannot be formed, so that the two materials are selected for use; acrylic resins are fat-soluble and are not suitable for raw material systems; the viscosity of the hydroxyethyl cellulose and the sodium carboxymethyl cellulose is too high, and the spraying form is not good, so that the polyvinyl alcohol and the polyvinylpyrrolidone are finally selected to be used in combination; wherein, the polyvinyl alcohol and the polyvinylpyrrolidone can generate precipitation when the concentration is too high, and the film can not be formed when the concentration is too low.

2. Humectant: since the entire dressing contains alcohol, propylene glycol was chosen as the humectant, which could be replaced with any one that could dissolve into the entire system.

(II) selecting a solvent system:

the solvent is selected from water, ethanol, isopropanol, isooctane and ethyl acetate, if water is used as the solvent alone, the film forming time is too slow, and the isooctane is fat-soluble and unsuitable; therefore, considering the irritation of ethanol or isopropanol and the film forming time, a certain proportion of ethanol or isopropanol, ethyl acetate and water are mixed to be used as a solvent system.

Example 3

Preparation of liquid keratin dressing: the liquid dressing was formulated according to the weight parts values of the components of table 1.

TABLE 1 weight parts of the components

	Keratin protein	Polyvinyl alcohol	Polyvinylpyrrolidone	Propylene glycol	Water (W)	Ethanol	Ethyl acetate
								1	1	5	5	50	100	300	500
2	2	10	30	10	20	450	450
								3	2	20	20	5	10	200	100
4	5	70	100	10	50	100	100

The preparation method comprises the following steps of:

a, adding water in the formula amount to dissolve polyvinyl alcohol and polyvinylpyrrolidone in the formula amount, fully swelling, and fully mixing to obtain a composite film-forming agent pretreatment material;

b, adding the keratin with the formula amount into the film-forming agent pretreatment material obtained in the step A, and fully mixing to obtain a film-forming agent containing the keratin;

and C, adding propylene glycol with the formula amount into the keratin-containing film forming agent obtained in the step B, and fully mixing to obtain the liquid dressing.

And C, adding ethanol and ethyl acetate to obtain the liquid dressing.

The liquid dressing obtained according to the composition ratios can form a film within a certain time, and has medicinal properties. The paint can be suitable for different scenes according to different proportions, for example, the formulas 1 and 2 can be selected to be prepared into spray for use, and the

formulas

3 and 4 can be selected to be prepared into direct coating materials for use.

Example 4

The principle of the cell scratch test (round health) is that when cells grow to be fused into a single layer, a blank area is artificially made on the fused single-layer cells, namely a scratch, and the cells at the edge of the scratch gradually enter the blank area to heal the scratch, thereby simulating the process of cell migration in vivo to a certain extent.

The experimental steps are as follows:

after the pancreatin of each experimental group cell in the logarithmic growth phase is digested, the complete culture medium is re-suspended into cell suspension, and counting is carried out; plating cell density (12-well plate, cell plating number 1.4 x 10⁵cell/well) until the next day the cells reached 90% confluence. 37 ℃ and 5% CO₂The culture system is 0.6 ml.

And vertically aligning the 200ul gun heads to the central part of the lower end of the 12-hole plate on the next day, and slightly pushing upwards to form scratches and cross scratches.

Gently rinsed 2-3 times with PBS, serum-free medium (used to prevent cell proliferation from affecting the scoring results) was added, and photographs were taken for 0 h.

Different sample groups were set to treat cells (liquid dressing group, blank group).

37℃、5％CO₂Culturing in an incubator, and taking pictures with a microscope device according to the healing degree and selecting proper time (time gradient is set to be 0h, 24h and 48 h).

Analysis of migration area: and analyzing scratch data.

Migration area-0 h cell area.

And (4) experimental conclusion: the liquid dressing can increase cell migration as shown in figure 1.

Example 5

Establishing an animal model:

injecting chloral hydrate into SD rat to anaesthetize with injector;

after the anesthesia is effective, shaving off the back hair by using a shaver, and establishing two whole-layer skin wounds of 1cm by 1cm on the back by using an ophthalmic scissors; the rats are divided into a blank control group and a liquid dressing group, each group is subjected to corresponding wound treatment, the rats after operation are raised in a single cage, wounds are debrided every 2 days, and corresponding dressings are smeared.

Determination of post-operative healing curves:

after operation, rats are anesthetized by ethyl ether every 2 days, the dressing is removed, the wound surface and the periphery are disinfected, the wound surface healing condition is observed, photographing record is carried out, and then the corresponding dressing is applied again to cover the wound surface. The photographs were analyzed with imagJ and a healing curve was plotted.

And (4) experimental conclusion: the liquid dressing can promote acute wound healing, as shown in figures 2 and 3.

Example 6

The practical application scene of the liquid dressing is as follows:

1. daily wound protection: can be used for treating acute wound such as common scratch, incised wound, and athletic wound.

For example, the product can be carried about or purchased in a nearby pharmacy, and can be ointment, spray and the like. It is convenient to carry, occupies small space and is simple and convenient to use. Different medicine forms are used for different wounds, for example, treatment of sports wounds like sports wounds can be realized, a spray form can be selected, spraying can be directly performed, pain can be reduced when the wound dressing is used, an isolation film can be generated every 30 seconds, the wound dressing not only can stop bleeding and heal for the first time to prevent external environment feeling, but also has the effects of disinfection and sterilization; in addition, the liquid dressing prepared by the invention has certain ductility after being filmed, and has better effect on protecting and isolating joint wounds compared with common medicines.

2. Auxiliary healing: provides a healing environment for the wound surface after laser treatment, inhibits and relieves the conditions of eczema, dermatitis and the like, and relieves the risk of scar formation.

For example, laser treatment operations or minimally invasive operations are performed in beauty parlors or hospitals, the dressing can protect the wound surface after the operation, isolate environmental infection, play a role in protection and promote the wound surface to be repaired and healed, and is transparent in texture and free of influence when being applied to the face.

The dressing has a certain relieving effect on skin symptoms such as eczema, dermatitis and the like, has a certain sterilization effect, has a promoting effect on skin cell growth, has good isolation, and can prevent qualitative repeated infection. But also has certain relieving effect on the formation of scars.

3. Red buttocks: a layer of protective film is formed on the skin of the baby to isolate the protective film from the diaper and urine, and the protective film has the functions of moisturizing and moistening the skin and preventing the generation of red buttocks of the baby;

the red buttocks phenomenon of infants is caused by tender skin, frequent contact of the skin with urine and excrement, frequent cleaning treatment and the like, and the occurrence of the red buttocks phenomenon indicates that the skin of infants is damaged to a certain extent. The dressing of the invention has a good improvement effect on the phenomenon. After the dressing is sprayed, a layer of protective film can be formed, so that the damage caused by direct contact or long-term contact of skin and excrement and urine can be effectively isolated; for cleaning urine and the like of a baby, the film can further protect the skin of the baby, and the skin damage caused by friction on the skin during cleaning is reduced; moreover, the dressing has a certain disinfection effect under the protection condition, so that the situation that bacteria are easily bred due to frequent contact between the periphery of the buttocks of a baby and excrement and urine can be well controlled; the dressing has a promoting effect on skin repair, and can further relieve the red buttocks phenomenon. On the other hand, the skin protection for the elderly can be used in the same manner as described above.

4. And (3) chronic wound nursing: aiming at chronic wounds which are difficult to heal, such as diabetic foot, bedsore and the like, the formation of new granulation and skin is accelerated in the granulation hyperplasia period and the epithelization period, and the healing of the wounds is promoted.

5. Repairing and healing: the method is used for pretreatment and rapid treatment of bleeding-prone medical detection items such as blood drawing and transfusion; for example, the blood is sprayed on the surface of the skin before and after blood drawing, so that the bleeding condition can be effectively reduced. For the skin peeling and cracking conditions caused by skin dryness and the like, the invention can provide a moisturizing environment for wounds, effectively isolate further damage caused by bacterial infection and provide the effect of promoting wound healing

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Claims

1. The composition for preparing the film forming agent is characterized by comprising a composite film forming agent and keratin, wherein the composite film forming agent comprises multiple of polyvinyl alcohol, polyvinylpyrrolidone, hydroxyethyl cellulose, sodium carboxymethyl cellulose and acrylic resin.

2. The composition according to claim 1, wherein the weight ratio of the keratin to the composite film former is 1: 10-40.

3. The composition according to claim 1, wherein the composite film former consists of polyvinyl alcohol and polyvinyl pyrrolidone; the weight ratio of the polyvinyl alcohol to the polyvinylpyrrolidone is 1-3: 2-4.

4. A liquid dressing composition comprising the composition of claim 1, wherein the liquid dressing composition consists of, in parts by weight:

keratin: 1-5 parts by weight;

5-70 parts of polyvinyl alcohol;

5-100 parts of polyvinylpyrrolidone;

humectant: 5-50 parts by weight;

water: 10-100 parts by weight;

ethanol to ethyl acetate medium in a volume ratio of 6-1: 3.

5. The liquid dressing composition of claim 4, wherein the liquid dressing composition consists of the following components in parts by weight:

keratin: 2 plus or minus 0.2 weight portions;

polyvinyl alcohol: 10 plus or minus 1 part by weight;

polyvinylpyrrolidone: 30 plus or minus 3 parts by weight;

propylene glycol: 10 plus or minus 1 part by weight;

water: 20-50 parts by weight;

the volume ratio is 1: 1 ethanol: ethyl acetate: 880 and 950 parts by weight.

6. A method of making a liquid dressing using the liquid dressing composition of claim 4, wherein the components are thoroughly mixed to provide the liquid dressing.

7. The method according to claim 6, characterized in that it comprises in particular the steps of:

a, dissolving the polyvinyl alcohol and the polyvinylpyrrolidone in the formula amount by adding water in the formula amount, fully swelling, and fully mixing to obtain a film-forming agent pretreatment material;

8. The process according to claim 7, characterized in that after step C, the ethanol and ethyl acetate are added.

9. Use of a liquid dressing prepared by the method of claim 6 as a skin protectant.

10. Use according to claim 9, wherein the liquid dressing is used as a skin barrier agent, a wound healing promoter, a granulation growth promoter.