JP2014005211A - Method for producing gelatin gel - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a gelatin gel which contains no chemical cross-linking agent and enables a controlled-release drug to be delivered in vivo and a method for producing the same.SOLUTION: The method is a method for producing a gelatin gel, including the steps of: (i) dissolving, in an organic solvent, a gelatin derivative obtained by introducing a hydrophobic group into gelatin; (ii) freeze-drying the organic solvent containing the gelatin derivative dissolved therein, obtained in the step (i) to obtain a dry matter; and (iii) adding an aqueous solvent to the dried matter obtained in the step (ii) to swell and gelate the dried matter and obtain a gelatin gel.

Description

  The present invention relates to a gelatin gel that does not contain a chemical cross-linking agent and enables sustained-release drug delivery in vivo and a method for producing the same.

  In recent years, a drug delivery system using gelatin gel as a carrier has been researched and developed in order to deliver drugs, nutrients, and the like to a lesion site at an effective concentration over a long period of time (Patent Documents 1-3, Non-patent document 1). The drug delivery system using gelatin gel is based on gelatin with excellent biocompatibility and bioabsorbability, and is fixed in the gel by the degradation of gelatin by the action of various enzymes in vivo. Drugs and nutrients are gradually released.

  A gelatin gel can be produced by dissolving gelatin in a water-soluble solvent and cooling it. However, since the gelatin gel dissolves under body temperature (about 37 ° C.) conditions, It is impossible to maintain the gel state only by attaching (that is, irrespective of the action of the enzyme). Therefore, conventionally, in the production of gelatin gels used for sustained-release drug delivery, chemical cross-linking agents are added to cross-link gelatin so that the gel state can be maintained even under body temperature conditions. ing. As the chemical crosslinking agent, glutaraldehyde, water-soluble carbodiimide, propylene oxide, diepoxy compound and the like are used (Patent Document 3).

  However, since the chemical cross-linking agent generally shows strong toxicity to living tissues and cells, the presence of the chemical cross-linking agent contained in the gelatin gel has been a problem for administration to a living body. In this field, there is a need for a gelatin gel that can maintain a gel state under body temperature conditions without using a chemical crosslinking agent, and that can be used as a carrier for sustained-release drug delivery, and a method for producing the gelatin gel. It was.

JP 2004-277348 A JP 2008-137975 A JP 2009-67732 A

Ma J et al. , Journal of Biomaterials Science: Polymer Edition, 13, 67-80, 2002.

  An object of this invention is to provide the gelatin gel which does not contain a chemical crosslinking agent, and enables the sustained release drug delivery in the living body, and its manufacturing method.

  As a result of intensive studies to solve the above-mentioned problems, the present inventors have used a gelatin derivative in which a hydrophobic group is introduced into gelatin as a base material for gel production. Gelatin gel that can maintain a gel state under body temperature conditions and allows sustained-release drug delivery in vivo without using a chemical cross-linking agent since it is cross-linked with each other via a group The inventors have found that it can be manufactured, and have completed the present invention.

That is, the present invention includes the following inventions.
[1] A method for producing a gelatin gel, comprising:
(I) a step of dissolving a gelatin derivative having a hydrophobic group introduced into gelatin in an organic solvent;
(Ii) a step of lyophilizing the organic solvent obtained by dissolving the gelatin derivative obtained in step (i) to obtain a dried product; and (iii) adding an aqueous solvent to the dried product obtained in step (ii). Swelling and gelling to obtain a gelatin gel,
Including the above method.
[2] The method of [1], wherein the hydrophobic group is an alkyl group having 4 to 20 carbon atoms.
[3] The method of [1] or [2], wherein 1 × 10 ⁻⁵ to 1 × 10 ⁻² mol of hydrophobic groups are introduced into the gelatin derivative per 1 g (dry weight) of the gelatin derivative.
[4] The method according to any one of [1] to [3], wherein in step (i), the gelatin derivative is dissolved in an organic solvent so as to have a concentration of 0.1 to 50% (w / v).
[5] The method according to any one of [1] to [4], wherein the organic solvent is dimethyl sulfoxide.
[6] The method according to any one of [1] to [5], wherein (iv) an aqueous solvent containing a drug is added to the gelatin gel obtained in step (iii).
[7] The method of [6], wherein the drug is a cell growth factor.
[8] The method of [6] or [7], wherein the drug is a basic fibroblast growth factor.
[9] A gelatin gel produced by the method of any one of [1] to [8].
[10] A gelatin gel comprising a gelatin derivative formed by introducing a hydrophobic group into gelatin and bonded to each other by hydrophobic interaction, and does not contain a chemical crosslinking agent.
[11] The gelatin gel according to [10], wherein the hydrophobic group is an alkyl group having 4 to 20 carbon atoms.
[12] The gelatin gel according to [10] or [11], wherein 1 × 10 ⁻⁵ to 1 × 10 ⁻² mol of hydrophobic groups are introduced into the gelatin derivative per 1 g (dry weight) of the gelatin derivative.
[13] The gelatin gel according to any one of [10] to [12], comprising a gelatin derivative in an amount of 0.1 to 50% (w / v).
[14] The gelatin gel according to any one of [10] to [13], further comprising a drug.
[15] The gelatin gel according to [14], wherein the drug is a cell growth factor.
[16] The gelatin gel according to [14] or [15], wherein the drug is a basic fibroblast growth factor.
[17] An angiogenesis promoter comprising the gelatin gel of [16].

  ADVANTAGE OF THE INVENTION According to this invention, the gelatin gel which does not contain a chemical crosslinking agent and enables the sustained release drug delivery in the living body, and its manufacturing method can be provided.

  Since the gelatin gel of the present invention does not contain a chemical cross-linking agent, it has lower cytotoxicity than conventionally known gelatin gels and can be suitably used as a carrier for performing sustained-release drug delivery.

FIG. 1 shows a method for producing a gelatin derivative having an alkyl group introduced therein. FIG. 2 shows a schematic structural diagram of a gelatin gel. In gelatin gel, gelatin derivatives are cross-linked with each other through hydrophobic groups by hydrophobic interaction. FIG. 3 is a photographic diagram showing the results of analyzing the cytotoxicity of gelatin gel. FIG. 4 shows the results of analyzing the in vivo stability of gelatin gel. (A) GD1 gelatin gel; (B) GD3 gelatin gel; (C) GD5 gelatin gel; (D) GD8 gelatin gel. FIG. 5 shows the analysis result of cytochrome C adsorption rate of gelatin gel. FIG. 6 shows the analysis results of the gelatin gel degradation rate and cytochrome C release rate. FIG. 7 shows the results of angiogenesis analysis using a bFGF-containing gelatin gel. (A) Gelatin gel containing bFGF; (B) Gelatin gel not containing bFGF (control). FIG. 8 shows the analysis results of angiogenesis (hemoglobin amount) by bFGF-containing gelatin gel. bFGF (+): gelatin gel with bFGF; gelatin gel without bFGF (−) bFGF (control).

  The gelatin gel of the present invention can be produced using a gelatin derivative in which a hydrophobic group is introduced into gelatin as a base material.

  “Gelatin” can be obtained by subjecting collagen collected from organs / tissues of animals (eg, mammals, birds, fish, etc.) to alkaline hydrolysis, acid hydrolysis, and enzymatic degradation. In addition, gelatin obtained using techniques such as chemical synthesis, fermentation using microorganisms, and gene recombination can also be used. Commercially available gelatin can be used. For example, bovine skin-derived acidic gelatin (manufactured by Sigma) can be used in the present invention.

  The gelatin used in the present invention may be either acidic gelatin or basic gelatin. “Acid gelatin” is gelatin obtained by alkali treatment of collagen, and has an isoelectric point of 2.0 to 7.0, preferably 4.0 to 6.5, more preferably 4.5 to 5. .5 is intended. “Basic gelatin” is gelatin obtained by acid treatment of collagen, and has an isoelectric point of 7.0 to 13.0, preferably 7.5 to 10.0, more preferably 8.5. What is 9.5 is intended. The selection of acidic gelatin and basic gelatin can be appropriately determined according to the use of the gelatin gel and the properties of the drug compounded in the gelatin gel. For example, when a gelatin gel is blended with a drug that is positively charged in the same neutral range (about pH 7) as the living body, such as basic fibroblast growth factor (hereinafter referred to as “bFGF”), It is preferable to use acidic gelatin that is negatively charged in the sex region. On the other hand, when a negatively charged drug such as bone morphogenetic protein (hereinafter referred to as “BMP”) is added to the gelatin gel, a basic gelatin that is positively charged in the neutral region is used. It is preferable.

  Examples of the “hydrophobic group” include an alkyl group, an acyl group, a phenyl group, and a benzyl group, and an alkyl group is preferable. In the present invention, the “alkyl group” refers to a straight chain having 4 to 20 carbon atoms, and particularly preferably an undecyl group (11 carbon atoms).

A “gelatin derivative” can be obtained by bonding a hydrophobic group directly or indirectly through a linker to a functional group of gelatin. Examples of functional groups of gelatin that can be used for the production of gelatin derivatives include amino groups, hydroxy groups, carboxylic acid groups, thiol groups, ester groups, amide groups, ketone groups, and aldehyde groups. is there. The binding of the hydrophobic group to the functional group of gelatin can be performed by a known method. For example, when an alkyl group is used as a hydrophobic group (FIG. 1), gelatin is dissolved in an appropriate solvent (for example, water), and an appropriate linear saturated aliphatic aldehyde is added thereto. The linear saturated aliphatic aldehyde forms a Schiff base by a condensation reaction with the amino group of gelatin. Subsequently, gelatin having an alkyl group bonded thereto can be obtained by reducing the Schiff base. Introduction of hydrophobic groups in the gelatin derivative finally obtained by appropriately changing the amount ratio of gelatin used for the reaction and a substance that imparts hydrophobic groups (in the example of the alkyl group, linear saturated aliphatic aldehyde) The amount can be adjusted. By increasing the introduction amount of the hydrophobic group in the gelatin derivative, the degree of cross-linking of the gelatin gel obtained using the gelatin derivative can be increased, the stability of the gelatin gel can be increased, and the gelatin gel can be hardly decomposed. In the present invention, it is preferable that 1 × 10 ⁻⁵ to 1 × 10 ⁻² mol of hydrophobic groups are introduced per 1 g (dry weight) of the gelatin derivative.

Particularly preferably, in the present invention, “gelatin derivative” refers to a compound in which 1 × 10 ^{−4 to} 9 × 10 ⁻⁴ mol of linear alkyl group having 11 carbon atoms is introduced per 1 g (dry weight) of gelatin derivative. Point to.

  The gelatin gel of the present invention is obtained by dissolving a gelatin derivative in an organic solvent to obtain a gelatin derivative solution, and then freeze-drying the gelatin derivative solution to obtain a dried product, and the dried product is swollen and gelled with an appropriate aqueous solvent. Can be manufactured.

  The organic solvent for dissolving the gelatin derivative is not particularly limited as long as it can dissolve the gelatin derivative. Examples thereof include dimethyl sulfoxide (hereinafter referred to as “DMSO”), ethanol, propanol, and glycerin. DMSO is preferred, and DMSO is preferred.

  The gelatin derivative is dissolved in an organic solvent at a concentration of 0.1 to 50% (w / v), preferably 5 to 20% (w / v). The density of the gelatin derivative in the gelatin gel can be adjusted by the concentration. By increasing the density of the gelatin derivative in the gelatin gel, the degree of cross-linking of the resulting gelatin gel can be increased, the stability of the gelatin gel can be increased, and the degradation can be made difficult.

  The freeze-drying of the gelatin derivative solution can be performed by a known method. That is, the gelatin derivative solution is rapidly frozen at about −196 ° C. to −20 ° C., and dried by sublimating the solvent under vacuum conditions. By using lyophilization, a dried product can be obtained as a porous material in which gelatin derivatives are cross-linked with each other through hydrophobic groups by hydrophobic interaction. The dried product obtained as a result of freeze-drying can be stored at -196 ° C to room temperature until use.

  The swelling and gelation of the dried product can be performed by impregnating the dried product with an aqueous solvent. That is, by impregnating the dried product with an aqueous solvent, water is held inside the dried product which is a porous body (that is, the gap) and swells into a gel. In addition, gelatin derivatives can be kept in a gel state because they are cross-linked with each other through hydrophobic groups by hydrophobic interaction (FIG. 2).

  Examples of the aqueous solvent used for swelling and gelation of the dried product include water and physiological saline, or those added with a buffer (phosphate, citrate, carbonate, acetate, etc.). (But not limited to). The swelling and gelation of the dried product can be performed by dropping an aqueous solvent into the dried product or immersing the dried product in the aqueous solvent. The step of swelling and gelation of the dried product may be performed for a time sufficient for the aqueous solvent to uniformly infiltrate the inside of the dried product. Depending on the size of the dried product and the amount of gelatin derivative contained, 1 minute to 24 hours Just do it. In the swelling and gelation of the dried product, a deaeration treatment can be performed as necessary, whereby the aqueous solvent can be efficiently infiltrated uniformly into the dried product.

  The gelatin gel of the present invention has a water content of 50 to 99.9 w / w%, preferably 75 to 95 w / w% (ie, the gelatin gel of the present invention contains 0.1 to 50% (w / W), preferably in an amount of 5-25 w / w%). The water content is expressed as a weight percent of the aqueous solvent in the gelatin gel. The water content can be adjusted by the introduction amount of the hydrophobic group in the gelatin derivative and / or the density of the gelatin derivative in the gelatin gel (that is, the concentration of the gelatin derivative in the solvent). The moisture content can be increased as the introduction amount of the hydrophobic group in the gelatin derivative is smaller and the density of the gelatin derivative in the gelatin gel is smaller.

  The shape and size of the gelatin gel of the present invention are not particularly limited and can be appropriately selected according to the intended use. Examples of the shape include a columnar shape, a prismatic shape, a sheet shape, a disk shape, a spherical shape, a paste shape, and the like, but if it is a columnar shape, a prismatic shape, a sheet shape, or a disk shape, it can be easily embedded in a living body. Can do. The shape and size of the gelatin gel can be adjusted by pouring the gelatin derivative solution into a mold having a desired shape and size at the time of preparing the gelatin gel, and lyophilizing the gelatin gel. It can also be performed by appropriately cutting and cutting the gelatin gel into a desired shape and size.

  In the gelatin gel of the present invention, a gelatin derivative as a base material is cross-linked with each other through a hydrophobic group by hydrophobic interaction. Unlike a gelatin gel conventionally used, a chemical cross-linking agent (for example, formaldehyde) is used. Or glutaraldehyde). Therefore, the gelatin gel of the present invention can be applied to cells, tissues and organs without showing toxicity or with low toxicity.

  The gelatin gel of the present invention can be used as a sustained-release preparation by including a drug. In sustained-release preparations, as gelatin is degraded by the action of various enzymes in vivo, the drug contained in the gel is gradually released. The drug release rate can be adjusted by the degradation rate of the gelatin gel.

  The degradation rate of the gelatin gel is adjusted by adjusting the degree of crosslinking in the gelatin gel by adjusting the amount of hydrophobic groups introduced into the gelatin, the concentration of the gelatin derivative in the gelatin derivative solution, or a combination thereof. It can be carried out.

  Examples of the “drug” include an antitumor agent, an antibacterial agent, an anti-inflammatory agent, an antiviral agent, an anti-AIDS agent, a hormone, a cell growth factor, a nucleic acid and the like, and has a molecular weight of about 10,000 to 100,000. What it has is preferable. Of these, cell growth factors are preferred. “Cell growth factor” includes fibroblast growth factor (FGF) (preferably bFGF), hepatocyte growth factor (HGF), nerve growth factor (NGF), bone morphogenetic protein (BMP), epidermal growth factor (EGF) , Transforming growth factor (TGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF) and the like (but not limited to). As described in detail in Examples below, the gelatin gel of the present invention containing bFGF can be used as an angiogenesis promoter.

  The drug can be immobilized in the gelatin gel by utilizing physicochemical interaction with the gelatin gel (for example, hydrophobic interaction, hydrogen bond, Coulomb force, etc.) or coordination bond. For example, when the drug is a protein such as a cell growth factor, if the isoelectric point of the drug is 7 or less, the drug is suitably fixed in the gelatin gel by using a gelatin gel prepared from the above basic gelatin. If the isoelectric point of the drug is 7 or more, the drug can be suitably fixed in the gelatin gel by using the gelatin gel prepared from the acidic gelatin.

  After the drug is dissolved in an appropriate solvent, preferably an aqueous solvent, the solution containing the drug is dropped from one side of the gelatin gel, or the gelatin gel is immersed in the solution containing the drug. Can contain drugs. Alternatively, the drug can be included in the gelatin gel by including the drug in an aqueous solvent for swelling and gelling the dried product.

  The amount of drug included in the gelatin gel may vary depending on factors such as the age, weight, severity of disease, size of the gelatin gel to be administered, etc., but is 0.01 μg to 1000 μg, preferably 0.1 μg to An amount appropriately selected from the range of 100 μg can be included.

  In addition to the drug, the gelatin gel can contain a stabilizer, a preservative, a solubilizer, a pH adjuster, a thickener and the like as necessary.

  Gelatin gel containing a drug can be administered to a living body by any method, but local administration is preferred in order to continuously release the drug at a target site. The dosage of the gelatin gel containing the drug can be appropriately selected according to the required dosage of the drug.

EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples.
Example 1: Preparation of gelatin gel (1-1) Synthesis of gelatin derivative Cattle skin-derived acidic gelatin (manufactured by Sigma) was dissolved in 8 mM phosphate buffer (pH 7.4) at a concentration of 3% (w / v). To obtain an aqueous solution. 99.9% ethanol of 0.7 times the amount of the aqueous solution was added to the obtained aqueous solution. 1-dodecanal was added to the obtained aqueous solution, and a Schiff base was formed by a condensation reaction with an amino group of gelatin (25 ° C., 6 hours). To the obtained reaction product, sodium cyanoborohydride was added, and a gelatin derivative into which an alkyl group was introduced was prepared by reductive amination reaction (FIG. 1).

  The amount of alkyl group introduced into the gelatin can be adjusted by the ratio of the gelatin used in the reaction to 1-dodecanal. The total amount of amino groups in the gelatin is defined as 1, 0.1, 0.5, 1 0.0 (, 5.0 (GD8) substance amount of 1-dodecanal was added to the gelatin aqueous solution, and a gelatin derivative was prepared by the above reaction.

As a result, a gelatin derivative into which 1.1 × 10 ⁻⁴ mol of alkyl group was introduced per 1 g (dry weight) of the gelatin derivative (hereinafter referred to as “GD1”), 2.9 × 10 ⁻⁴ mol of alkyl group An introduced gelatin derivative (hereinafter referred to as “GD3”), a gelatin derivative into which 4.8 × 10 ⁻⁴ mol of an alkyl group was introduced (hereinafter referred to as “GD5”), and 8.3 × 10 ⁻⁴ A gelatin derivative having a mol alkyl group introduced therein (hereinafter referred to as “GD8”) was obtained and used in the following experiments.

(1-2) Preparation of gelatin gel 0.125 g of dry powder of each gelatin derivative of GD1, GD3, GD5, and GD8 was added to 10 mL of distilled water or dimethyl sulfoxide (DMSO), and stirred for 15 minutes at 80 ° C., and then dissolved. The amount of gelatin derivative was measured. As a control, the same amount of gelatin without an alkyl group was used.
The results are shown in Table 1 below.

  The gelatin derivative decreased in solubility in distilled water as the number of introduced alkyl groups increased. That is, it was shown that the hydrophobicity of the gelatin derivative improved with the increase of the introduced alkyl group. Hereinafter, DMSO was used as an organic solvent for dissolving the gelatin derivative.

  Dry powders of gelatin derivatives of GD1, GD3, GD5, and GD8 were each dissolved in DMSO at a concentration of 10% (w / v), 1 mL each was placed in a 12-well well plate, and freeze-vacuum drying was performed. While the obtained dried product is immersed in 8 mM phosphate buffer (pH 7.4), it is degassed for about 1 to 10 minutes so that the phosphate buffer solution penetrates into the inside of the dried product, and each gelatin derivative. A gelatin gel consisting of In the following, gelatin gel prepared from GD1 is described as GD1 gelatin gel, gelatin gel prepared from GD3 is described as GD3 gelatin gel, gelatin gel prepared from GD5 is described as GD5 gelatin gel, and gelatin gel prepared from GD8 is described as GD8 gelatin gel. To do.

Example 2: Stability of gelatin gel at in-vivo temperature Gelatin gel (dry weight 0.1 g) prepared from each gelatin derivative obtained in Example 1 above was immersed in 8 mM phosphate buffer (pH 7.4). The mixture was shaken at 37 ° C. and 100 rpm, and the time until the gelatin gel was completely dissolved was measured. As a control, the results of using a gelatin gel prepared in the same manner as described in Example 1 from gelatin into which no alkyl group was introduced are shown in Table 2 below.

  As the alkyl group introduced into the gelatin derivative increased, the stability of the gelatin gel at 37 ° C. improved. The GD8 gelatin gel was hardly dissolved even after 16 days from the start of the experiment.

Example 3: Cytotoxicity of gelatin gel A cell culture plate partially coated with gelatin gel prepared from each gelatin derivative obtained in Example 1 above was prepared, and L929 fibroblasts were seeded on the plate, Culture was performed based on a conventionally known technique.
The results on the fifth day from the start of culture are shown in FIG.

  As is clear from the results in FIG. 3, cell proliferation was confirmed even in a region close to the GD1 gelatin gel or GD8 gelatin gel, indicating that the influence of the gelatin gel on the cells (ie, cytotoxicity) was low. It was done.

Example 4: In vivo stability of gelatin gel A gelatin gel (dry weight 0.025 g) prepared from each gelatin derivative obtained in Example 1 above was implanted subcutaneously in the back of a DDY mouse (6 weeks old). The size of the gelatin gel after one week was confirmed.
The results are shown in FIG.

  As is clear from the results of FIG. 4, GD1 and GD3 were almost decomposed, and GD5 was decomposed by about half. On the other hand, GD8 was hardly decomposed.

  That is, as the alkyl group introduced into the gelatin derivative increased, the stability of the gelatin gel in vivo was improved.

Example 5: Adsorption and release of bFGF model protein (5-1) Adsorption of cytochrome C The molecular weights and isoelectric points of bFGF and cytochrome C are shown in Table 3 below.

  Since cytochrome C has the same molecular weight and isoelectric point as bFGF, cytochrome C was used as a model protein for bFGF.

  The GD3 gelatin gel (dry weight 0.025 g) obtained in Example 1 was added to an aqueous cytochrome C solution (0.15 mg / ml), and the mixture was shaken at 37 ° C. and 400 rpm. A part of the aqueous solution was collected at a predetermined time, the amount of cytochrome C in the aqueous solution was measured, and the amount of cytochrome C adsorbed on the gelatin gel was examined. The results are shown in FIG. The adsorption rate (%) in the figure can be obtained by the following equation.

  As is clear from the results of FIG. 5, cytochrome C was adsorbed to the gelatin gel over time, and all cytochrome C contained in the aqueous solution was adsorbed to the gelatin gel 60 minutes after the start of the experiment.

(5-2) Release of cytochrome C Into an aqueous solution of cytochrome C (0.15 mg / ml), the GD3 gelatin gel (dry weight 0.025 g) obtained in Example 1 was added, and the temperature was 60 ° C. at 37 ° C. and 400 rpm. Shake for a minute to prepare a gelatin gel adsorbed with cytochrome C. Papain, a gelatinolytic enzyme, was dissolved (0.1 mg / ml) in 8 mM phosphate buffer (pH 7.4), and the gelatin gel adsorbed with cytochrome C was added thereto, followed by shaking at 37 ° C. and 200 rpm. A part of the aqueous solution was collected at a predetermined time, the amount of cytochrome C in the aqueous solution was measured, and the amount of cytochrome C released into the aqueous solution was examined. The results are shown in FIG. The cytochrome C release rate (%) in the figure can be obtained by the following equation.

  Further, the weight of the gelatin gel remaining in a predetermined time was measured, and the degradation rate (%) of the gelatin gel was examined.

  As is clear from the results of FIG. 6, there was a correlation between the degradation of the gelatin gel and the amount of cytochrome C in the aqueous solution. This result indicates that the adsorbed cytochrome C is released by the degradation of the gelatin gel.

  From the above results, it is shown that gelatin gel can adsorb bFGF in the same manner as cytochrome C, and that adsorbed bFGF can be released by degradation of gelatin gel.

Example 6: Induction of angiogenesis using gelatin gel 20 μL of an aqueous solution containing 10 μg of bFGF was dropped onto the top surface of the gelatin gel prepared from gelatin induction of GD3 obtained in Example 1 above, and 1 at 37 ° C. After standing for a while, bFGF was adsorbed on the gelatin gel.

  Gelatin gel (dry weight 0.025 g) adsorbed with bFGF was implanted subcutaneously in the back of Wistar rats (male, 6 weeks old). As a control, a gelatin gel prepared in the same manner except that 20 μL of purified water not containing bFGF was added dropwise instead of the bFGF aqueous solution was used.

  A photograph of the tissue around the gelatin gel on the third day after implantation is shown in FIG. As is clear from the results of FIG. 7, when a gelatin gel adsorbed with bFGF was embedded, a large number of blood vessels were formed around the gelatin gel as compared to the case where the control gelatin gel was embedded. .

  Subsequently, the amount of hemoglobin in the tissue around the gelatin gel on the third day after implantation was measured. The results are shown in FIG.

  As is clear from the results of FIG. 8, when a gelatin gel adsorbed with bFGF is embedded, a significantly larger amount of hemoglobin is present around the gelatin gel as compared with the case of embedding the control gelatin gel. It became clear. This result indicates that a large number of blood vessels are formed around the gelatin gel adsorbed with bFGF.

Claims (17)

A method for producing a gelatin gel, comprising:
(I) a step of dissolving a gelatin derivative having a hydrophobic group introduced into gelatin in an organic solvent;
(Ii) a step of lyophilizing the organic solvent obtained by dissolving the gelatin derivative obtained in step (i) to obtain a dried product; and (iii) adding an aqueous solvent to the dried product obtained in step (ii). Swelling and gelling to obtain a gelatin gel,
Including the above method.   The method according to claim 1, wherein the hydrophobic group is an alkyl group having 4 to 20 carbon atoms. The method according to claim 1, wherein 1 × 10 ⁻⁵ to 1 × 10 ⁻² mol of hydrophobic groups are introduced into the gelatin derivative per 1 g (dry weight) of the gelatin derivative.   The method according to any one of claims 1 to 3, wherein in step (i), the gelatin derivative is dissolved in an organic solvent so as to have a concentration of 0.1 to 50% (w / v).   The method according to any one of claims 1 to 4, wherein the organic solvent is dimethyl sulfoxide.   The method according to any one of claims 1 to 5, further comprising (iv) adding an aqueous solvent containing a drug to the gelatin gel obtained in step (iii).   The method of claim 6, wherein the drug is a cell growth factor.   The method according to claim 6 or 7, wherein the drug is a basic fibroblast growth factor.   The gelatin gel manufactured by the method of any one of Claims 1-8.   A gelatin gel comprising a gelatin derivative which is bonded to each other by hydrophobic interaction and in which a hydrophobic group is introduced into gelatin, and does not contain a chemical crosslinking agent.   The gelatin gel according to claim 10, wherein the hydrophobic group is an alkyl group having 4 to 20 carbon atoms. The gelatin gel according to claim 10 or 11, wherein 1 × 10 ⁻⁵ to 1 × 10 ⁻² mol of hydrophobic groups are introduced into the gelatin derivative per 1 g (dry weight) of the gelatin derivative.   The gelatin gel according to any one of claims 10 to 12, comprising a gelatin derivative in an amount of 0.1 to 50% (w / v).   The gelatin gel according to any one of claims 10 to 13, further comprising a drug.   15. Gelatin gel according to claim 14, wherein the drug is a cell growth factor.   The gelatin gel according to claim 14 or 15, wherein the drug is a basic fibroblast growth factor.   An angiogenesis promoter comprising the gelatin gel according to claim 16.

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