Method for preparing medical hydrogels
High-pressure steam sterilization of aldonic acid-bonded chitosan derivatives simplifies the preparation of medical hydrogels, addressing sterilization and biotoxicity issues, resulting in biocompatible wound dressings with enhanced mechanical strength.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Patents
- Current Assignee / Owner
- KAGOSHIMA UNIV
- Filing Date
- 2023-02-27
- Publication Date
- 2026-07-01
AI Technical Summary
Existing methods for preparing chitosan-based hydrogels for medical applications require sterilization steps like freezing and thawing, which are cumbersome, and the use of cross-linking agents like glutaraldehyde introduces biotoxicity concerns.
A method involving high-pressure steam sterilization of an aqueous solution containing a chitosan derivative with bonded aldonic acid, such as gluconic acid, to form a medical hydrogel without harmful additives, ensuring biocompatibility and mechanical strength.
The method allows for simple, effective sterilization and gelling of medical hydrogels, maintaining biocompatibility and mechanical strength, suitable for wound dressings that promote healing by providing a moist environment.
Smart Images
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Abstract
Description
Technical Field
[0001] The present invention relates to a method for preparing a medical hydrogel. In the law It relates thereto.
Background Art
[0002] A wound dressing is required to have properties that promote wound treatment. A hydrogel-like wound dressing that can provide a moist environment at the wound site has been reported to promote granulation tissue formation and re-epithelialization at the wound site and thus promote healing.
[0003] Chitosan is a natural polysaccharide having antibacterial properties and a wound healing effect. Due to its properties, chitosan has been widely used as a wound dressing material. However, since chitosan is poorly water-soluble at physiological pH, it is difficult to prepare a hydrogel-like chitosan wound dressing. On the other hand, although chitosan derivatives that are water-soluble at physiological pH have been reported, in order to prepare a hydrogel from this chitosan derivative, a cross-linking agent such as glutaraldehyde must be used, and the biotoxicity of the cross-linking agent remaining in the gel is a problem.
[0004] In view of such a situation, a method for preparing a chitosan-derived hydrogel without using additives harmful to the living body has been proposed (Patent Document 1). In Patent Document 1, an aqueous solution containing aldonic acid-modified chitosan is frozen and thawed to prepare a hydrogel having no biotoxicity.
Prior Art Documents
Patent Documents
[0005]
Patent Document 1
Summary of the Invention
Problems to be Solved by the Invention
[0006] Patent Document 1 states that when hydrogels are used for medical purposes such as wound dressings, it is necessary to sterilize them after preparation, which involves the steps of freezing, thawing, and sterilization.
[0007] The present invention has been made in view of the above matters, and its purpose is to provide a method for preparing a medical hydrogel that can be sterilized and gelled by a simple method. Law The purpose is to provide. [Means for solving the problem]
[0008] Honpatsu Clearly The method for preparing the medical hydrogel is as follows: A method for preparing a medical hydrogel derived from a chitosan derivative in which an aldonic acid is bonded to the amino group of the glucosamine unit of chitosan by dehydration condensation, The process includes a step of sterilizing an aqueous solution containing the chitosan derivative, placed in a mold of a predetermined shape, by high-pressure steam sterilization to obtain a sterilized medical hydrogel of the predetermined shape. It is characterized by the following:
[0009] Furthermore, it is preferable to perform the high-pressure steam sterilization treatment at 121°C or higher for 15 minutes to 2 hours.
[0010] Furthermore, it is preferable to use the chitosan derivative having a deacetylation degree of 70-90% and an aldonic acid introduction rate of 5-60%.
[0011] Furthermore, it is preferable that the aldonic acid is gluconic acid.
[0012] Furthermore, it is preferable that the obtained medical hydrogel has a fibrous structure. 。 [Effects of the Invention]
[0013] According to the present invention, a method for preparing a hydrogel that can be sterilized and gelled by a simple method. Law It can be provided. [Brief explanation of the drawing]
[0014] [Figure 1] It is a diagram showing a synthesis scheme of a chitosan derivative. [Figure 2] It is a graph explaining the introduction rate of gluconic acid in the chitosan derivative. [Figure 3] It is a photograph of the medical hydrogel prepared in the examples. [Figure 4] It is a graph showing the compressive strength of the medical hydrogel prepared in the examples. [Figure 5] It is a photograph showing a SEM image of the GC54 hydrogel. [Figure 6] It is a photograph showing a SEM image of the GC54FM hydrogel.
Mode for Carrying Out the Invention
[0015] A method for preparing a medical hydrogel according to this embodiment will be described. The medical hydrogel obtained by the preparation method according to this embodiment is used as a wound dressing material that maintains a moist environment of a wound surface such as an incision, laceration, contusion, burn, or bedsore and exhibits a wound treatment effect by promoting cell growth.
[0016] The method for preparing a medical hydrogel according to this embodiment is a method for preparing a medical hydrogel derived from a chitosan derivative in which an aldonic acid is bonded to an amino group of a glucosamine unit of chitosan by dehydration condensation, and includes a step of subjecting an aqueous solution containing the chitosan derivative placed in a mold of a predetermined shape to high-pressure steam sterilization treatment to obtain a medical hydrogel of a predetermined shape that has been sterilized.
[0017] In another embodiment of this implementation, the chitosan used to synthesize the chitosan derivative is a deacetylated product of chitin. As is well known, the conversion of chitin to chitosan (deacetylation reaction) does not proceed completely, and a part of N-acetylglucosamine is contained on the sugar chain. In commercially available chitosan, the degree of deacetylation is usually in the range of 50 to 100%, and particularly many are about 70 to 90%.
[0018] Unless otherwise specified, the chitosan used in relation to this embodiment refers to chitosan with a degree of deacetylation of 50-100%, and therefore also includes chitosan with a degree of deacetylation of less than 100%.
[0019] The aldonic acid used to synthesize the chitosan derivative used in this embodiment is a general term for carboxylic acids obtained by oxidizing monosaccharides, in which the formyl group at position 1 of the aldose is replaced with a carboxyl group, and can be represented, for example, by the following formula (I). In formula (I), n is an integer from 2 to 4.
[0020] [ka]
[0021] In this embodiment, gluconic acid is a preferred aldonic acid for the synthesis of the chitosan derivative, and threonic acid and xylonic acid can also be suitably used. Furthermore, any carboxylic acid known as an aldonic acid, such as galactonic acid, mannonic acid, lyxonic acid, erythronic acid, ribonic acid, arabinonic acid, aronic acid, altronic acid, guronic acid, idonic acid, and talonic acid, can be used.
[0022] As can be understood from the above explanation, the chitosan derivative used in this embodiment can generally be represented as having a repeating unit consisting of a site where an aldonic acid is bonded to the amino group of a glucosamine unit and a site where an acetylglucosamine unit remains, as shown in the following formula (II). In formula (II), n is an integer from 2 to 4.
[0023] [ka]
[0024] Therefore, when aldonic acid is gluconic acid, the chitosan derivative used in this embodiment can generally be represented as having repeating units shown by the following formula (III).
[0025] [ka]
[0026] The chitosan derivative used in this embodiment can be synthesized by condensing and dehydrating aldonic acid with chitosan, thereby attaching (introducing) the aldonic acid to the amino group at position 2 of the glucosamine unit of chitosan. The aldonic acid is generally reacted as a suitable salt (e.g., a sodium salt). Figure 1 shows the synthesis scheme when gluconic acid is used as the aldonic acid.
[0027] This condensation dehydration reaction can be carried out at room temperature under acidic conditions using a condensing agent (dehydration condensing agent). Suitable condensing agents in this condensation dehydration reaction between chitosan and aldonic acid include, but are not limited to, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), which activates the carboxyl group of aldonic acid, and N-hydroxysuccinimide (NHS), which suppresses side reactions.
[0028] The rate of aldonic acid, such as gluconic acid, being introduced into chitosan can be adjusted by changing the relative amount of aldonic acid used relative to chitosan and the amount of condensing agent used. The introduction rate can be increased by increasing the relative amount of aldonic acid and the amount of condensing agent used. The introduction rate of aldonic acid in the chitosan derivative used in this embodiment is, for example, 5 to 60%.
[0029] Here, the rate of introduction of aldonic acids such as gluconic acid (into chitosan) is expressed by the following formula (A). Aldonic acid introduction rate (%) = [Y / (X+Y)] × 100 (A) In equation (A), X is the amount of substance (in moles) of glucosamine units contained in the chitosan derivative, and Y is the amount of substance (in moles) of aldonic acid-modified glucosamine units contained in the chitosan derivative.
[0030] This introduction rate can be calculated by measuring the change in conductivity when a sodium hydroxide solution of an equivalent molar concentration is added to an aqueous solution prepared by dissolving a chitosan derivative (aldonic acid-modified chitosan) in an aqueous hydrochloric acid solution of appropriate concentration.
[0031] This measurement method, using gluconic acid as the aldonic acid to obtain gluconic acid-modified chitosan (hereinafter sometimes abbreviated as GC), is described in detail with reference to Figure 2 as follows: When 0.1 M sodium hydroxide is added to a 0.1 M hydrochloric acid aqueous solution in which gluconic acid-modified chitosan is dissolved, a neutralization reaction occurs and H in the solution + The amount of OH decreases, and consequently the conductivity of the aqueous solution decreases (Figure 2A-B). Once the neutralization reaction is complete, deprotonation of the protonated amino group in the glucosamine unit begins, and the conductivity does not change until this deprotonation is complete (Figure 2B-C). Once deprotonation is complete, the amount of OH in the aqueous solution decreases. - As the amount increases, the conductivity also increases (Figure 2C-D). Here, the amount of sodium hydroxide added to the aqueous solution in BC in Figure 2 corresponds to the amount of protonated amino groups in the glucosamine units. Furthermore, considering that most of the amino groups in the glucosamine units are protonated in this hydrochloric acid aqueous solution, the amount of sodium hydroxide added to the aqueous solution in BC in Figure 2 corresponds to the amount of glucosamine units that have not been modified with gluconic acid. Therefore, by calculating the amount of glucosamine units in gluconic acid-modified and unmodified chitosan using this measurement method and comparing them, the gluconic acid introduction rate can be determined.
[0032] In this embodiment, an aqueous solution containing the chitosan derivative synthesized as described above is placed in a mold of a predetermined shape and subjected to high-pressure steam sterilization. This causes the aqueous solution containing the chitosan derivative to gel and is also sterilized, forming a biocompatible medical hydrogel of a predetermined shape.
[0033] High-pressure steam sterilization can be performed using an autoclave. The temperature and time for this process should be set to a temperature and time that allows for sterilization, and as with general autoclave sterilization, it is sufficient to sterilize at 2 atmospheres and 121°C or higher for 15 minutes or more, although 20 minutes to 2 hours is also acceptable.
[0034] An aqueous solution containing a chitosan derivative can be obtained by dissolving the dried powder of the chitosan derivative mentioned above in an acidic aqueous solution such as hydrochloric acid, and then adding an alkali such as sodium hydroxide to neutralize it to a pH of around 7. The content of the chitosan derivative in the aqueous solution does not need to be high; for example, 1-5% (by weight / volume) is sufficient as long as the chitosan derivative is sufficiently dissolved in the aqueous solution.
[0035] The medical hydrogel formed as described above is an elastic hydrogel. Depending on the shape of the mold used, medical hydrogels can be obtained in various shapes, such as sheets or spheres, depending on the application. Furthermore, as mentioned above, since no crosslinking agents are used and no additives harmful to living organisms are used in obtaining the medical hydrogel, it has excellent biocompatibility.
[0036] Furthermore, by using a chitosan derivative with a high aldonic acid introduction rate and extending the autoclaving time, a medical hydrogel with higher mechanical strength can be obtained. The higher the mechanical strength of the medical hydrogel, the easier it is to process it to the size of the wound and to attach it to an outer covering material to hold it on the wound. Therefore, it is possible to obtain a medical hydrogel with mechanical strength appropriate for the application.
[0037] Furthermore, in this embodiment, a medical hydrogel is manufactured without freezing and thawing the aqueous solution containing the chitosan derivative. As a result, the internal structure of the obtained medical hydrogel is fibrous and dense. The chitosan contained in the medical hydrogel has the property of being broken down by enzymes in bodily fluids that seep out from the wound, but in the case of a medical hydrogel with a fibrous and dense structure, enzymatic breakdown takes time. Therefore, the medical hydrogel according to this embodiment can remain on the wound surface for a long period of time, which can accelerate wound healing.
[0038] As described above, in this embodiment, a medical hydrogel can be obtained in a single step of autoclaving an aqueous solution containing a chitosan derivative. Therefore, it has the advantage of being able to prepare a medical hydrogel in a very simple manner, and even medical institutions equipped with autoclaves can easily prepare medical hydrogels according to their intended use. [Examples]
[0039] <Synthesis of gluconate-modified chitosan> 3.0 g of chitosan (product name "Chitosan LL", degree of deacetylation 80%, manufactured by Yaizu Suisan Kagaku Kogyo Co., Ltd.) was dissolved in 300 ml of distilled water (pH 4.0) in which 2-morpholinoethanesulfonic acid was dissolved at a concentration of 23.5 mM, and the pH was adjusted to 4.0 by adding a 1 M hydrochloric acid aqueous solution. As shown in Table 1, sodium gluconate (hereinafter abbreviated as GA), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter abbreviated as EDC), and N-hydroxysuccinimide (hereinafter abbreviated as NHS) were dissolved in this aqueous solution, and the mixture was stirred at room temperature for 24 hours to introduce gluconic acid to the amino group at position 2 of the glucosamine unit of chitosan (see Figure 2).
[0040] Next, the pH of the reaction solution was adjusted to 8.0 by adding a 1M sodium hydroxide aqueous solution. Then, 99.5% ethanol was added to precipitate the gluconate-modified chitosan and unreacted sodium gluconate, and the precipitate was collected by centrifugation. After that, the precipitate was sealed in a dialysis membrane and immersed in distilled water for one week to remove the sodium gluconate. During this time, the distilled water was changed twice a day. Subsequently, the precipitate and aqueous solution in the dialysis membrane were collected, and 99.5% ethanol was added to precipitate the gluconate-modified chitosan, and the precipitate was collected. After that, a dried powder of gluconate-modified chitosan was obtained by freeze-drying and stored.
[0041] Three types of dried gluconate-modified chitosan powders with different raw material ratios were prepared. These gluconate-modified chitosans are denoted as GC8, GC26, and GC54, respectively. The gluconate introduction rate was then calculated for GC8, GC26, and GC54. As previously described, the gluconate introduction rate was calculated by dissolving 0.2 g of dried gluconate-modified chitosan powder in 40 ml of 0.1 M hydrochloric acid aqueous solution and measuring the change in conductivity when 0.1 M sodium hydroxide aqueous solution was added to the solution. Table 1 shows the preparation conditions and gluconate introduction rates for each gluconate-modified chitosan.
[0042] [Table 1]
[0043] <Sterilization by autoclave and preparation of hydrogel> The dried powders of each gluconic acid-modified chitosan synthesized as described above were added to distilled water (2.0% [weight / volume]). Then, the powders were completely dissolved by adding a 0.1 M hydrochloric acid aqueous solution. Subsequently, the pH was adjusted to 7.0 by adding a 0.1 M or 1.0 M sodium hydroxide aqueous solution.
[0044] This gluconate-modified chitosan aqueous solution was placed in a cylindrical glass container with an inner diameter of 15 mm and sealed. This was then placed in an autoclave and sterilized by high-pressure steam (121°C, 2 atm). The sterilization was performed for 20 minutes, 60 minutes, and 120 minutes, respectively.
[0045] As a result, regardless of which gluconate-modified chitosan aqueous solution was used or the processing time, the gluconate-modified chitosan aqueous solution gelled, yielding medical-grade hydrogels (hereinafter referred to as GC8 hydrogel, GC26 hydrogel, and GC54 hydrogel, respectively). A representative photograph of the GC54 hydrogel is shown in Figure 3.
[0046] <Compression Test> The compressive strength of each medical hydrogel obtained as described above was measured. A compression test was performed on each medical hydrogel in a glass container by lowering a 10 mm diameter indenter from above at a rate of 5 mm / min. The compressive strength was then measured when the strain reached 10%.
[0047] The results are shown in Figure 4. For GC8 hydrogel, there was almost no change in compressive strength with increasing processing time. On the other hand, GC26 hydrogel and GC54 hydrogel showed a tendency for compressive strength to increase with increasing processing time. It was found that medical-grade hydroxygels with higher mechanical strength can be obtained as the gluconic acid introduction rate increases and the autoclaving processing time increases.
[0048] <Structural comparison with hydrogels prepared by freezing and thawing> An aqueous solution of gluconic acid-modified chitosan using the aforementioned GC54 dried powder was placed in a metal mold and frozen at -20°C for 12 hours. It was then dissolved by standing at room temperature to produce a medical-grade hydrogel (hereinafter referred to as GC54FM hydrogel).
[0049] SEM (Scanning Electron Microscope) images of GC54 hydrogel and GC54FM hydrogel were taken. The SEM image of GC54 hydrogel is shown in Figure 5. The SEM image of GC54FM hydrogel is shown in Figure 6.
[0050] GC54FM hydrogel is manufactured using a freezing and thawing process, resulting in numerous pores derived from ice crystals formed during freezing. On the other hand, GC54 hydrogel is manufactured without freezing and thawing, and therefore lacks the pores found in GC54FM hydrogel, exhibiting a fibrous and dense structure.
[0051] This invention allows for various embodiments and modifications without departing from the broad spirit and scope of the invention. Furthermore, the embodiments described above are for illustrative purposes only and do not limit the scope of the invention. In other words, the scope of the invention is indicated not by the embodiments, but by the claims. Various modifications made within the scope of the claims and the equivalent scope of the meaning of the invention are considered to be within the scope of this invention.
[0052] This application is based on Japanese Patent Application No. 2022-30659, filed on March 1, 2022. The entire specification, claims, and drawings of Japanese Patent Application No. 2022-30659 are incorporated herein by reference. [Industrial applicability]
[0053] It can be used as a wound dressing for treating wounds such as cuts, lacerations, bruises, burns, and pressure ulcers.
Claims
1. A method for preparing a medical hydrogel derived from a chitosan derivative in which an aldonic acid is bonded to the amino group of the glucosamine unit of chitosan by dehydration condensation, The process includes a step of sterilizing an aqueous solution containing the chitosan derivative, placed in a mold of a predetermined shape, by high-pressure steam sterilization to obtain a sterilized medical hydrogel of the predetermined shape. A method for preparing a medical hydrogel characterized by the following:
2. The aforementioned high-pressure steam sterilization treatment is performed at 121°C or higher for 15 minutes to 2 hours. A method for preparing a medical hydrogel according to feature 1.
3. Using the chitosan having a degree of deacetylation of 70-90%, the chitosan derivative is used in which the introduction rate of the aldonic acid is 5-60%. A method for preparing a medical hydrogel according to feature 1.
4. The aforementioned aldonic acid is gluconic acid. A method for preparing a medical hydrogel according to feature 1.
5. The obtained medical hydrogel has a fibrous structure. A method for preparing a medical hydrogel according to feature 1.