KR101853432B1 - Transparent hydrogel membrane containing hyaluronic acid and silk protein and method for preparing thereof - Google Patents

Abstract

The present invention relates to a transparent hydrogel film copolymerized with a hyaluronic acid and a silk protein, specifically silk fibroin by a linker, and a method for producing the same, and has excellent transparency, flexibility, A transparent hydrogel film is provided.

Description

TECHNICAL FIELD [0001] The present invention relates to a transparent hydrogel film containing hyaluronic acid and a silk protein, and a process for producing the same. [0002] The present invention relates to a transparent hydrogel film containing hyaluronic acid and a silk protein,

The present invention relates to a transparent hydrogel membrane in which hyaluronic acid and silk proteins, specifically silk fibroin, are copolymerized by a linker, and a method for producing the same.

Hyaluronic acid (HA) is a polymer composed of two sugars, glucuronic acid and N-acetylglucosamine. It is a natural mucopolysaccharide widely distributed in the epilepsy in the tissue. It exists in a large amount in human joint fluid, cartilage, skin, eyeball, etc., plays a three-dimensional cross-linking role of intercellular molecules and protects cartilage between cartilage . It contains a large amount of a carboxyl group which is a hydrophilic group and has a characteristic that the mechanical strength is weakened upon contact with moisture.

In general, hyaluronic acid is used as a viscous supplement, a medicine, and an adjunct to surgery, which plays a positive role in cell culture, promotes the regeneration of corneal epithelial cells, and is used as a main ingredient of artificial tears for treating ocular surface diseases in ophthalmic fields have. Long-term clinical application of hyaluronic acid has already secured eye safety. Also, lubricants administered by intra-articular injection have been extensively tested in viscous supplements to alleviate joint pain caused by osteoarthritis of different nature.

Silk protein extracted from silkworm cocoon is composed of sericin of hydrophilic structure surrounding fibrous structure and fibroin of hydrophobic structure with inner gloss and is one of traditional medical polymer materials which has been used for a long time as a folk remedy. After the possibility of being used as a medical material since 2000, many researchers have been studied as various types of medical materials such as membranes, sponges, hydrogels, and fibers. Among them, silk used as fiber is sericin-free fibroin protein, which is a natural protein natural polymer material that protects the skin from ultraviolet rays and static electricity, which is excellent in humidity control, excellent in antibacterial effect and harmful to human body.

Many studies have been reported on the effect of fibroin protein on skin health, especially fibroin protein promotes the production of collagen to maintain skin elasticity and helps to regenerate wounded skin cells. Furthermore, it has excellent biocompatibility, biodegradability, air permeability and mechanical properties and is used for tissue engineering supports and medical applications.

Hydrogels have a three-dimensional network structure, can contain a large amount of water, are highly hydrophilic and have biocompatibility. Particularly, hydrogels composed of natural polymers are inexpensive because they are very abundant in nature, are excellent in biocompatibility and biodegradability, and are widely used as drug delivery vehicles. It has high hygroscopicity, stability and non-toxicity and shows excellent biocompatibility when contacted with blood, body fluid and living body. Hydrogels consist of a natural polymer or derivative thereof, a synthetic polymer or a combination of a natural polymer and a synthetic polymer, whose molecules interacting by electrostatic or chemical bonding form a hydrophilic cross-linked polymer and have a dry weight of 10 Lt; RTI ID = 0.0 > 20% < / RTI > In particular, hydrogels are ideal candidates for the production of tissue engineering substrates intended to heal or reconstitute damaged, diseased or degenerated human tissues or organs.

Despite these excellent possibilities, hydrogel membranes containing hyaluronic acid and fibroin have been difficult to apply to ophthalmology due to their opacity.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent No. 10-1154327

The present invention relates to a transparent hydrogel film in which hyaluronic acid and a silk protein, specifically silk fibroin, are copolymerized by a linker, and a process for producing the same, and provides a transparent hydrogel film exhibiting excellent transparency, flexibility and drug releasing property.

One aspect of the present invention provides a transparent hydrogel film wherein hyaluronic acid and a silk protein are copolymerized by a linker.

According to one embodiment of the present invention, the silk protein is contained in an amount of 1.25 to 2 parts by weight based on 1 part by weight of hyaluronic acid.

According to an embodiment of the present invention, the transparent hydrogel film may form a net structure by the linker.

According to one embodiment of the present invention, the linker is selected from the group consisting of butanediol diglycidyl ether (BDDE), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), diepoxy octane, Bis- (2,3-epoxypropyl) -2,3-ethylene, and combinations thereof.

According to an embodiment of the present invention, the molecular weight of the hyaluronic acid may be 800,000 to 1,200,000 daltons.

According to one embodiment of the present invention, the silk protein may be silk fibroin.

According to an embodiment of the present invention, the transparent hydrogel may be an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory agent, an antiseptic agent, an antihistamine agent, a growth promoter, an antiseptic agent, an antimicrobial agent, An anti-inflammatory agent, an antipyretic agent, an immunostimulant, a dermatological agent, an anticancer agent, and a combination thereof.

According to one embodiment of the present invention, the transparent hydrogel can be used for prevention or treatment of ophthalmic diseases.

The present invention provides a hydrogel film containing hyaluronic acid and a silk protein, specifically silk fibroin, which is improved in transparency and flexibility, is excellent in biocompatibility, is effective in the treatment of wound healing and inflammation, It can be applied as an amniotic membrane replacement agent for the treatment of ocular surface disease.

In addition, other drugs carried on the hydrogel membrane can be slowly released below a certain rate, and additional effects such as inflammation inhibition, pain control, and epithelial regeneration promotion can be exerted through the additionally supported drug.

Fig. 1 shows a transparent hydrogel film containing hyaluronic acid and silk fibroin according to the present invention (Fig. 1 (a)) and its transparency (Fig. 1 (b)).
2 shows SEM analysis results of the hyaluronic acid hydrogel (a) and the transparent hydrogel film (b) according to the present invention.
3 shows an evaluation of toxicity of hyaluronic acid hydrogel according to linker concentration.
Fig. 4 shows an evaluation of toxicity of hyaluronic acid hydrogel according to the mixing ratio of hyaluronic acid and silk fibroin.

One aspect of the present invention provides a transparent hydrogel film in which hyaluronic acid and a silk protein are copolymerized by a linker, wherein the silk protein is contained in an amount of 1.25 to 2 parts by weight based on 1 part by weight of hyaluronic acid.

As used herein, the term "hyaluronic acid" is present in the vitreous or umbilical cord of the eye in the human body, and is highly viscous and plays an important role in preventing invasion of bacteria or penetration of toxins. In the present invention, hyaluronic acid is preferably low molecular weight hyaluronic acid, more preferably 800,000 to 1,200,000 daltons.

The term " silk protein " as used herein includes sericin and silk fibroin, and silk fibroin is preferably used in the present invention. Silk fibroin can be obtained from silkworm by removing sericin through a refining process known in the art.

In the transparent hydrogel film of the present invention, the silk protein is preferably contained in an amount of 1.25 to 2 parts by weight, preferably 1.25 to 1.7 parts by weight, more preferably 1.4 to 1.7 parts by weight, more preferably 1.4 to 1.7 parts by weight based on 1 part by weight of hyaluronic acid, If the amount of the protein is more than 2 parts by weight, the formation of the film can not be performed well. If the amount of the protein is less than 1.25 parts by weight, it may become opaque and air bubbles may be generated in the hydrogel film.

By adjusting the content ratio of hyaluronic acid and silk protein in the transparent hydrogel film of the present invention, swelling degree can be adjusted as needed.

As used herein, the term " linker " serves to link and copolymerize the hyaluronic acid monomer with the silk protein, whereby the internal structure of the transparent hydrogel film can form a net structure. The linker can generally be a different type of double- or multi-functional crosslinking agent and is selected from the group consisting of, for example, divinyl sulfone, double or multifunctional epoxides, carbodiimides and formaldehyde But is not limited thereto. The linker is preferably a preparation selected from the group of double- or multi-functional epoxides, more preferably 1,4-butanediol diglycidyl ether (BDDE) Ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), diepoxyoctane or 1,2-bis- 1,3-epoxypropyl) -2,3-ethylene), more preferably 1,4-butanediol diglycidyl ether or 1,2-bis (2,3- Ethyl-3- (3-dimethylaminopropyl) carbodiimide. The 1,4-butanediol diglycidyl ether has a molecular weight of 202.25 and cross-links hyaluronic acid having a short half-life thereof to form a gel, which complements the disadvantage that the degradation proceeds quickly. The concentration of the linker may be 0.1% or less, preferably 0.01% or less.

The above-mentioned transparent hydrogel can be used as an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory agent, an antioxidant, an antiseptic, an antihistaminic agent, But is not limited to, one or more drugs selected from the group consisting of a drug, an agent, an anticancer agent, and a combination thereof. Such a drug, which is additionally contained in the transparent hydrogel film, may assist in healing the wound or increase the effect thereof.

The transparent hydrogel may be used in the treatment of ophthalmic diseases such as dry eye syndrome, keratitis, glaucoma, uveitis, eye inflammation, allergies, eye infections, carcinoma, corneal neovascularization, retinal edema, macular edema, diabetic retinopathy, But are not limited to, for the prevention or treatment of various ocular conditions such as retinopathy, degenerative diseases of the retina (macular degeneration, retinopathy), retinal diseases associated with proliferation, and the like.

(A) dissolving hyaluronic acid in an aqueous solution to prepare a hyaluronic acid solution; (b) dissolving the silk protein in distilled water to prepare a silk protein solution; (c) mixing the hyaluronic acid solution and the silk protein solution into any one selected from the group consisting of an organic salt solution, an inorganic salt solution, a fluorinated solvent, an ionizing solution and a strong acid; And (d) mixing the linker with the mixed solution, and pouring the gel into a mold, wherein the silk protein is contained in an amount of 0.6 to 1 part by weight based on 1 part by weight of hyaluronic acid And a method for producing the transparent hydrogel film.

As used herein, the term "basic aqueous solution" may be an aqueous solution of a hydroxide of an alkali metal or hydroxide of an alkaline earth metal such as lithium hydroxide, sodium hydroxide, potassium hydroxide, magnesium hydroxide, beryllium hydroxide, calcium hydroxide, But is not limited thereto. Aqueous solution of sodium hydroxide can be facilitated in terms of pH control and concentration control.

Hereinafter, the present invention will be described in more detail with reference to one or more embodiments. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Manufacturing example  1. Hyaluronic acid Hydrogel  Production of film

0.75 g of the non-crosslinked hyaluronic acid raw material was dissolved in 10 ml of distilled water and 0.01% by weight of BDDE was added to prepare a uniform hyaluronic acid solution of 7.5% by weight. The hyaluronic acid solution was poured into a polyethylene (PE) mold and gelated at room temperature for 24 hours. The hyaluronic acid film of a predetermined size prepared by gelling was washed with distilled water every 24 hours for a total of three times, and after the washing process for 72 hours, a hyaluronic acid hydrogel film was obtained.

The size of the hyaluronic acid hydrogel film was 3 cm in diameter and 0.5 cm in thickness, and the hyaluronic acid solution corresponding to the above was 1000 쨉 l.

Example  One. silk  Extraction of fibroin

5 g of cocoon was added to 8% sodium carbonate solution and heated for 30 minutes to remove sericin. The treated cocoons were spread out thinly in a clean hood, and then dried at room temperature for 24 hours.

The dried cocoons were placed in a 9.3 M LiBr solution and dissolved for 4 to 48 hours, then the solution was filtered through a filter paper. Impurities or precipitates were selectively filtered, dialyzed with a dialysis cassette of 3 to 12 ml capacity and washed with distilled water for a total of 6 times for 48 hours to obtain silk fibroin.

Example  2. Hyaluronic acid and silk  Preparation of fibroin membrane (1:10 Hydrogel  membrane)

First, 5 g of the silk fibroin extracted in Example 1 was mixed with distilled water to prepare a 8 wt% silk fibroin solution.

1 ml of a 7.5% by weight hyaluronic acid solution and 1 ml of an 8% by weight silk fibroin solution were mixed, stirred for 6 hours or more, and then dried.

Example  3. Hyaluronic acid and silk  Fabrication of fibroin membrane

A hydrogel film was prepared in the same manner as in Example 2, except that the 7.5 wt% hyaluronic acid solution and the 8 wt% silk fibroin solution were mixed in the volume ratio shown in Table 1 below.

Experimental Example  One. Hydrogel  Observation of film surface

The surface of the membrane was observed with a scanning electron microscope (JSM-7800F, JEOL) in order to obtain information on the surface of the sample that was not influenced by the size and thickness of the hydrogel film.

As a result of observation, it was confirmed that a regular net structure was formed, and thus a smooth surface was formed so as not to cause microscopic scratches when used in an eyeball (see FIG. 2).

Experimental Example  3. Hyaluronic acid Hydrogel  Toxicity assessment

3.1. Cell culture

Human corneal epithelial cells (HCEC) (Cat. No. PCS-700-010) purchased from the American Type Culture Collection (ATCC, Manassas, Va.) Were inoculated into a proliferation kit And resuspended in cell-based medium. The cells were inoculated into a 75 cm ² tissue culture flask and maintained at 37 ° C, 5% CO ₂ and 95% humidity air air. The culture medium was changed every 3 days, and the cells were treated with 0.05% trypsin-EDTA (Gibco BRL, Calif., USA). In this experiment, cells having a subculture number of 5 or less were used.

3.2. BDD  Processing and Cell survival rate  analysis

For toxicity assessment of BDDE treatment, cell viability analysis was performed according to the manufacturer's protocol using Cell Count Kit (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, to process BDDE (Sigma Aldrich) 24 hours, HCEC were cultured in 96-well plates at a concentration of 1? 10 ⁴ / well.

After the cells were adhered, the BDDE solution was diluted to 0.01, 0.1, 1, 10% with the culture medium and treated for 6, 24, 48 hours. After incubation, 10 CC of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured 2 hours after incubation of the HCEC with the reagent.

As a result, there was no significant difference in cell viability after 6 hours of treatment. However, in case of 24 hours after BDDE treatment, in case of 10% solution, in case of 48 hours after treatment, more than 1% The cell survival rate was significantly reduced in the treated group (see Fig. 3).

3.3. silk Hydrogel  Processing and Cell survival rate  analysis

For evaluation of toxicity in the silk hydrogel according to the present invention, cell viability analysis was performed according to the manufacturer's protocol using a cell counting kit (CCK-8) reagent (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). Briefly, 24 hours before treatment with the effluent from the silk hydrogel, HCEC was incubated at a concentration of 1 10 ⁴ cells / well in a 96-well plate.

Among the hydrogel membranes prepared in Example 3, hydrogel membranes (HA: silk fibroin in a ratio of 1: 2, 1: 1.67, 1: 1.43 and 1: 1.25 in different ratios of hyaluronic acid and silk fibroin, Is not included in silk fibroin), and each silk hydrogel was incubated at 37 DEG C for 2 hours in 1 mL of the culture medium. The exudate was treated with HCEC for 6, 24, 48 and 72 hours. After incubation, 10 CC of CCK-8 solution was added to each well, and the absorbance at 450 nm was measured 2 hours after incubation of the HCEC with the reagent.

As a result, it was confirmed that regardless of the presence or the ratio of silk fibroin, the cell viability was maintained at a high level without any difference (see FIG. 4).

Experimental Example  4. Hydrogel  Characterization of membranes

The hydrogel membranes prepared in Example 3 were evaluated for bubble generation, transparency, viscousity, degree of decomposition and usability of handling, and the evaluation results are shown in Table 2 below.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than the foregoing description, and all changes or modifications derived from the meaning and scope of the claims and equivalents thereof are included in the scope of the present invention. .

Claims (7)

In a transparent hydrogel membrane in which a hyaluronic acid and a silk protein are copolymerized by a linker,
Wherein the silk protein is contained in an amount of 1.25 to 2 parts by weight based on 1 part by weight of hyaluronic acid,
The linker is butanediol diglycidyl ether (BDDE)
Wherein the silk protein is silk fibroin. delete delete The method according to claim 1,
Wherein the hyaluronic acid has a molecular weight of 800,000 to 1,200,000 daltons. delete The method according to claim 1,
The transparent hydrogel can be used as an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory agent, an anti-inflammatory agent, an antioxidant, an antiseptic, an antihistamine, An anticancer agent, and a combination thereof. ≪ RTI ID = 0.0 > 11. < / RTI > The method according to claim 1,
Wherein the transparent hydrogel is for preventing or treating ophthalmic diseases.

Images