TW593341B - Method for producing water-insoluble polysaccharides - Google Patents

Abstract

This invention is concerned with a method for producing water-insoluble polysaccharides. A method for making the water-insoluble bio-compatible gel includes activating the hydroxyl-containing polysaccharides with the activating agent to form activated polysaccharides, and cross-linking the activated polysaccharides, under moderate conditions producing different shapes of the water-insoluble bio-compatible gel. The water-insoluble bio-compatible gels, films, porosities, powders, sheets, fibers and spheres of this invention may be applied to various medical and cosmetic uses.

Description

593341.. '·'. · Description of the invention: [Technical field to which the invention belongs]. The present invention relates to a method for manufacturing water-insoluble polysaccharide materials, especially a method suitable for medical or Method for non-water-soluble polysaccharide materials of cosmetics. [Previous Technology] Hyaluronic acid (HA) is a mucopolysaccharide that exists in the tissues and fluids of spinal animals. HA contains uronic acid and amino sugar. It is a linear polymer with a molecular weight of tens of thousands to millions. The repeating unit is composed of D-glucuronic acid and DN-acetyl. -glucosamine) is a dimer with a / 3-(1-3) bond structure, and then a linear polymer with a 0-(1-4) bond. It is widely present in connective tissue, mucous tissue and crystalline lens of vertebrae and capsules of some bacteria in nature. 0 HA has the following advantages: it exists naturally in the human body, has no immune response, and can be broken down and absorbed by the human body. It can be obtained in large quantities and has become a biopolymer commonly used in medicine. HA is mainly used for ocular surgery such as cataract and corneal injury. High molecular weight (millions) HA solution is injected into the eye when viscoelastic fluid 'maintains the normal shape and function of the eye 6. In addition, HA can also be used for arthritic agents or joint surgery. Recently HA has also been developed for general wound healing, anti-adhesion of tissues after surgery, and drug release. Since HA has a function of retaining moisture, it can be applied to cosmetics for preventing skin aging. [(· Tomihata et al. (Biomaterials, 1997, vol 18, 1 89-195) uses hyaluronic acid (HA) with a molecular weight of about 1.5 x 10 6 and uses as many diepoxy compounds ( Poly (ethylene glycol) diglycidyl ether was used as the cross-linker of HA. Tomihata et al. Dissolved the HA powder in distilled water and added the diepoxy compound to the HA solution. And adjust the pH of the HA solution to observe the cross-linking effect of the second epoxy compound on HA under different pH conditions. Tomihata et al. Stirred the mixed solution of HA and the second epoxy compound evenly and poured it into a petri dish ( In a Petri dish, it was left at atmospheric pressure and 25 ° C for 5 days. After drying, a cross-linked HA film (Film) with a thickness of 200 μm was formed. According to the experimental results, Tomihata et al. The cross-linking reaction is most suitable when the pH of the HA solution is controlled at pH = 6.1.

Malson in U.S. Patent No. 4,963,666 discloses a manufacturing method for manufacturing polysaccharides containing a COOH functional group. Malson will include polysaccharides containing a -COOH group, for example, hyaluronic acid and bi- or bi- or Polyfunctional epoxy compounds (epoxides) react under alkaline conditions 5933341 to form so-called ePoxy-activation polysaccharides. There will be no gums at this reaction stage ( gel) is formed, and the polysaccharide material is still soluble at this stage, and then the epoxy compounds remaining in the reaction ik are removed [for example, by a dialysis method], and then the polysaccharide solution is poured into A mold is left to dry in the mold. During the drying process, the ring-activated polysaccharide material undergoes cross-linking. The example of Mai son's patent in this patent is as follows: 0.2g of hyaluronic acid (molecular weight is about 3x106) is first mixed with 6ml of 0.5% NaOH, and then 2ml of 1,4-butanediol is used to shrink It is said that glycerol ether (1,4-butanediol diglycidyl ether; BDDE) is mixed uniformly, and the solution is shaken overnight, and then the solution is transferred to a dialysis tube and dialyzed with distilled water for 24 hours. The pH 値 is 5.5. The dialysis solution was poured into a petri dish made of polystyrene material and dried at room temperature for 2 days to form a transparent, water-insoluble hyaluronic acid film.

Sakurai in U.S. Patent No. 4, 7 1 6, 224 discloses a method for producing cross-linked hyaluronic acid. In this patent, the cross-linking agent is a poly-functional epoxy compound, including halomethyl- Halomethyloxirane and bis-epoxy compound cross-linking agents. The cross-linking index of the cross-linking agent after reacting with the parent chain of hyaluronic acid is 100 disaccharide unit molecules. 5 ~ 20 cross-linking reactions, and the transparent negative acid material after the cross-linking is water-soluble (water 1 ub 1 e) and stringy. 8 593341

Burns et al., In U.S. Patent No. 5,017,229, discloses a method for producing a water-insoluble derivative of hyaluronic acid. In this patent, 5 Burns claims to use a transparent solid solution (Solid content) of 0.4% to 2.6%. An acid solution, combined with an activating agent, can be used to produce transparent acid gels. In addition to HA, the polyanionic polysaccharide materials in the scope of this patent application can also be carboxymethlcellulose, carboxylmethylamylose, and chondroitin-6 -Shondroitin-6-sulfate, dermatin sulfate, hepaim and other polymers. One of the embodiments of the patent is described later: 0.4 g of sodium hyaluronate having a molecular weight between 10 and 2 × l06 is dissolved in 10 ml of distilled water, and then the pH of the HA solution is adjusted to pH = 4.75, and then added 0.314 g EDC (1-ethyl-3-C3-dimethylaminopropyl) carbodiimide hydrochloride; (l-ethyl-3-C3-dimethylaminopropyl) carbodiimide hy dr-chloride), reaction mixture Reaction at room temperature for 5 hours to form a water-insoluble hydrogel (Hydrogel).

Burns et al., In US Pat. No. 5,5,27,893, disclose a method for preparing a water-insoluble derivative of polyanion ® polysaccharide. One of the embodiments of this patent is described later: 0.4 g of sodium hyaluronate is dissolved in 10 ml of distilled water, and the pH of the HA solution is adjusted to pH = 4.75, and 0.3 14 g of EDC (\ -ethyl- 3-C3-dimethylaminopropyl) carbodiimide hydrochloride 'salt), and then add an appropriate amount of L-leucine methyl ester hydrochloride. The reaction mixture is at room temperature. Conditional reaction to form a water-insoluble hydrogel. This patent application focuses on 'adding another 9 aery 1 ufea derivative' in HA's EDC cross-linking procedure to obtain a modification. Of hyaluronic acid (Hydrogel).

Kuo et al., U.S. Patent No. 5,3,5,8,83, disclose a method for producing water-insoluble gelatine gels, films, and sponges. The technical content of this patent is to use an EDC cross-linking agent, add it to the HA solution, and add the ethanol to the post-reaction mixture after an appropriate reaction time to precipitate and precipitate the HA after the chemical modification of EDC. This Shen Dianwu was washed and dried Then, water is added to this product to form a water-insoluble colloid.

Kuo et al., In US 5,502,08 1 patent, discloses a pharmaceutical activity substance, which is covalently bonded to a hyaluronic acid polymer through a covalent bond via the action of a carbodiimide compound. On the chain.

Kuo in U.S. Patent No. 6,0 13,679 discloses a method for preparing a water-insoluble hyaluronic acid derivative. The technical content of the patent is mainly to use carbodiimide compounds as chemical cross-linking agents for hyaluronic acid to form water-insoluble hyaluronic acid derivatives. DE BELDER et al. Discloses a method for preparing a gel (Gel) for preventing sticking of body tissues in WO 86/009 1 2 patent. This gum system consists of a polysaccharide material containing -COOH group, hyaluronic acid, and poly-functional epoxide cross-linked hyaluronic acid to form a cross-linked hyaluronic acid colloid. M alson et al., WO 86/00079, disclose a method for preparing a crosslinked hyaluronic acid colloid. The scope of this patent application is a sterilized, hyaluronic acid glue without pyrogen-free · 593341 body. This glue system uses a polyfunctional epoxy compound, or a halohydrin, or It is obtained by the reaction of epihalohydrin or halide as a cross-linking agent of hyaluronic acid. * '

Mai son et al. Disclosed a method for producing cross-linked hyaluronic acid colloid in WO 90/0940 1 patent. The main feature of this patented technology is the use of phosphorus-containing reagent as a cross-linking agent for hyaluronic acid. .

Malson et al., U.S. Patent No. 5,783,69 1 discloses a method for preparing a cross-linked transparent cinnamic acid colloid using a phosphorus-containing compound as a hyaluronic acid cross-linking agent.

Malson et al., In US Pat. No. 4,716,154, discloses a method for producing a cross-linked hyaluronic acid colloid. This HA colloid is suitable as a vitreous substitute for ophthalmic retinal surgery. The main feature of the patented technology is to use polyfunctional epoxide or halohydrin or epihalohydrin or halide as the cross-linking agent of hyaluronic acid to prepare the cross-linking agent. Chain hyaluronic acid colloid. It can be found in Example φ of this patent that Malson et al. Used a high solid content (Solid content) HA solution and added an epoxy compound, such as BDDE, to an alkaline HA solution. It was found through experiments that the HA solution must be in the solid form of HA. When the content exceeds I 3 · 3% or more, the reaction temperature exceeds 50 ° C can form _ A colloid.

Nobuhiko et al. (Journal of controlled Release, 25, 1993, pl33-143) discloses a method for preparing cross-linked hyaluronic acid containing phospholipid microparticles (lipid mi c ro s p h e r e s). 11 593341

Nobuhiko et al. Dissolved hyaluronic acid in IN NaOH solution to form a basic solution of ΗA solid content of about 20 wt%, and then added an appropriate amount of PGPGE (Polyglycerol polyglycidyl ether) epoxy compound, in which the repeating units of PGPGE and HA (Repeating unit) The stoichiometric molar ratio is about 1.0. This reaction mixture reacts at 60 ° C for 15 minutes to form a crosslinked ΗΑ colloid.

Nobuhiko et al. (Journal of controlled Release, 22, 1992, P105-106) discloses a method for preparing cross-linked hyaluronic acid colloids. Nobuhiko et al. Dissolved hyaluronic acid in NaOH solution to form an alkaline HA solution with a solid content of about 20 wt%, and then added EGDGE (Ethylene glycol diglycidyl ether) or PGPGE epoxy compound which had been dissolved in ethanol, at 60 ° After reacting at a temperature of 15 minutes at a temperature of C, a crosslinked HA colloid can be obtained. 3 & 1 & 25, et al., In 115 4,582,865 (19 8 6) and 1; 5 4,605,691 (1 986), disclose a method for producing a crosslinked hyaluronic acid colloid. The main feature of the patented technology of Balazs et al. Is the use of HA solution, under the condition of pH 値 exceeding 9.0, using divinyl sulfone as the chemical cross-linking agent of HA to obtain cross-linked hyaluronic acid colloid .

Hamilton et al. In US 4,93 7,2 70 patent discloses a method for chemically cross-linking hyaluronic acid using EDC and L-leucine methyl ester ^ hlofide to prepare non-water soluble Sex Η A water glue method. anxious.,

Miller et al., In US 5,760,200 patent, discloses a method for preparing a water-insoluble polysaccharide derivative. The patent content is the use of polysaccharide materials 12 593341, such as hyaluronic acid, in an aqueous solution of polysaccharide materials, and acidic. Under pH 値 conditions, EDC and L'leucine methyl ester chloride were added to chemically cross-link HA to form a water-insoluble HA colloid. Alginate, a polysaccharide obtained from plants, is the main component of the colloidal structure of 'P' plants. Its composition is I, 4-linked-β-D-mannuronic acid (l, 4-linked β-D-mannuronic acid (M)) and 1,4-linked α-L-guluronic acid (G). A copolymer of monosaccharide molecules.

Thompson et al., U.S. Patent No. 5,5,63,186, disclose a method for producing a cross-linked alginate gel. The main technical content of this patent is the use of alginate polysaccharide polymers, dissolved in water to form alginate colloids. This colloid can use divalent metal ions, or dialdehydes or diamines. The colloidal cross-linking reaction is performed to obtain a cross-linked alginate colloid.

Desai et al., In US 5,334,640, disclose a method for making crosslinked biocompatible materials. Desai et al. Dissolved alginate sodium salt (SOdiumsa 11) in water, then added PEG diacrylate, and the reaction mixture was treated with light to photocrosslink alginate. material.

Patrick et al., In US Pat. No. 5,70,270, disclose a method for producing microcapsuie of crosslinkabU polysaccharide material. One of the technical examples of this patent is described as follows: · The alginate material is vacuum dried first, and the dried powder is suspended in Chlorine A, and the excess acryloyl chloride and triethyl chloride are added. Triethylamine. The reaction is carried out under reflux for 24 hours. After the reaction, alginate acrylate is filtered out, washed with ethanol and dried. This material is made by the emulsification method (Emul si fi cation). Microcapsules are then irradiated with UV light to form photo-crosslinked alginate microcapsules. [Guchi et al., In US 5,811,531 patent, discloses a cross-linked polysaccharide material absorbent (Absorbent). System of law. Iguchi et al. Used 100 parts of xanthan gum polysaccharide material and added 4 parts of a cross-linking agent aqueous solution. The cross-linking agent was composed of ethylene glycol diglycidyl ether and methanol. . The mixture of Gutton gum and cross-linking agent is heated at 14 ° C for 20 minutes to form a cross-linked Gutton gum absorbent (Absorbent). Using a similar method, Iguchi et al. Can also prepare cross-linked alginates, fruit Gum and guar gum and other polysaccharide materials.

Hamdi (J. Control. Res. 55: pl93-201) and others have disclosed a method for preparing cross-linked microspheres. Hamdi et al. Used soluble starch to dissolve in 2M NaOH solution by mechanical stirring. Stir the aqueous alkaline starch solution uniformly, and then emulsify it in a cyclohexane-chloroform mixed solvent (4: 1, Wv) containing 0.5% (ν / ν) sorbitane monoeleate emulsifier. The emulsion was poured into a glass reaction tank, and 2.5% epichlorohydrin (€ 0 丨 (: 111〇1 * 〇1 ^ (11 ^ 11)) was continuously stirred under the condition of 600′111 at 40 ° C. After reacting for 18 hours, after centrifugation and washing, cross-linked starch particles can be obtained. 14 593341 Chitin is the second most abundant organic substance in nature after cellulose. It mainly exists in carapace, cartilage, and insects. In the exoskeleton and fungal cell wall, chitin is a straight chain formed by the monomers gkicosamine and N-acetyN D-glucosamine with a / 3-β4 chain High molecular polysaccharide polymer, containing about 2000-3000 monomers, molecular weight of about two million, depending on its manufacturing conditions and sources. Chitin will be removed in part or all by the high temperature hot alkali deacetylation Chitosan becomes a free amine group to obtain chitosan. Chitin and chitosan are natural polymers with good histocompatibility and almost non-toxicity (half lethal dose, LD 5 Q = 16 g / kg), and it is biodegradable. In addition to many industrial applications, it can also be used for packaging and medicine due to its antibacterial effect. And its biodegradability can decompose the film after use, reducing its impact on the environment. These special Decomposability can make it potentially useful in packaging materials, agricultural films, biomedical materials, pharmaceutical materials, etc.

Unger et al., U.S. Patent 5,5 2,5,710, disclose a method for preparing a cross-linked, highly porous chitosan polysaccharide material. Unger et al. Dissolved the chitosan flake material in an acid solution to form a viscous solution. The gel was sliced and placed in a NaOH solution for 24 hours. The colloidal sheet material is then immersed in toluene (Toluene), and 2,4-TDI is added as a cross-linking agent (cross- 15 593341 1 inker). After the cross-linking, the chitosan is dried in a vacuum oven and then ground. Broken into powder, this cross-linked chitosan powder has extremely high pore volume.

Roy, U.S. Patent No. 5,770,712 discloses a method for preparing crosslinked hydrogel bead from chitosan polysaccharide material. Roy et al. Suspended the chitosan round beads in aqueous phase (aque 〇ussuspensi ο η) soaked in 100% ethanol solvent, and then added 1,4-butanediol diglycidyl ether to chitin containing The glycan globular alcohol solvent was reacted at room temperature for 24 hours. After the reaction, the alcoholic solvent is used to remove unreacted epoxy compounds, and the chitosan beads are dispersed in the aqueous solution, and then diethylamine (DEA) is added, and the reaction is performed at room temperature for about 16 hours. A DEA-activated, cross-linked chitosan bead was obtained. Chondroitin sulfate is obtained from the cartilage of cattle and fish. It is one of the largest structural molecules in the body (called Glycosaminoglycan). A component in connective tissue. Chondroitin sulfate is a polymer composed of N-acetyl-D-glucosamine and glucuronic acid. It is also called galactosaminoglucuronoglycan sulfate 〇 carotidin sulfate helps cartilage to absorb water and bring in nutrients, which acts like a buffer. It also protects existing cartilage from premature damage.

Sakurai et al., In U.S. Patent No. 4,863,907, disclose a method for producing a cross-linked glycosamioglycan polysaccharide material. The example of 593341 in this patent is as follows: a 12.5% chondroitin sulfate sodium salt solution is prepared in a 0.75N NaOH solution, and an appropriate amount of ethanol is added to form a viscous precipitate, which is recovered after separation. Add the epichlorohydrin to this viscous precipitate, stir it evenly, and then react at 20 ° C for 24 hours to obtain the cross-linked chondroitin sulfate polysaccharide material.

Matsuda et al., In US Pat. No. 5,462,976, disclose a method for making a photo-crosslinked glycosaminoglycan (GAG) derivative material. The feature of this patented technology is that in the DMF (dimethyl formamide) solution of n-butylamine GAG, H is added to anhydrous pyridine, then cinnamoyl chloride is added, and the reaction is performed at 75 ° C for 2 hours, and then added An alcoholic solvent precipitates the reaction product. This product is washed with ethanol and then irradiated with light at a wavelength of 270 nm to perform a photo-crosslinking reaction. [Summary of the Invention] It is at least one or a mixture selected from the group consisting of 1,4-dioxane, chloroform, methylene chloride, N, N -N, N-dimethylformamide (DMF), N, N-dimethyl Acetamide (DMAc), ethyl acetate, acetone , Methyl ethyl ketone (MEK), methanol, ethanol, propanol, isopropanol, and butanol. 17 593341 The above-mentioned formed film-like, powdery, or sheet-like, or fibrous, or spherical insoluble polysaccharide materials are washed with a mixed organic solvent containing water, and then washed with clean water. The dried water-insoluble polysaccharide material can be used in medicine or cosmetics. The composition of the above-mentioned water-containing mixed organic solvent includes water and an organic solvent, and the organic solvent may be acetone, methyl ethyl ketone (MEMS), methanol, ethanol, propanol, isopropyl alcohol Isopropanol, butanol or a mixture thereof, the weight fraction of the organic solvent in the cleaning solution occupies 50 to 100%, and the ketones and alcohol solvents can be used in any ratio. The temperature of the cleaning solution is 15 ° C -50 ° C. The non-water-soluble polysaccharide materials after the cleaning solution is washed, and then cleaned with clean water at a temperature of 25 ° C-50 ° C, and then further used. Hot air drying below 60 ° C, or Radiation heating drying, or Vacuum drying. [Embodiments] Examples The following examples further illustrate the present invention, but these examples are only for illustration, and the scope of the present invention is not limited thereto. Example (1): Preparation of non-water-soluble, cross-linked hyaluronic acid polysaccharide material. Sodium Hyaluronate polysaccharide material powder 0.1 g (containing 1.0 milli-equivalent [meq] Hydoxy group is dissolved in 10ml of distilled water, and stirred at room temperature for 18 593341 to form a 1% solid content hyaluronic acid solution. Then use 1 · 0 N NaOH or 1. ON HC1 solution to adjust the pH of the hyaluronic acid solution, as shown in the following table (1A). Preheat this hyaluronic acid solution to 35t, and then add a quantitative amount of ethylene glycol diglycidyl ether (EGDGE) epoxy compound to the hyaluronic acid solution (as shown in Table 1 A), in which EGDGE is added in the equivalent of milli equivalents (Meq) and hyaluronic acid hydroxyl group (Hydroxy group), the stoichiometric ratio is 4 meq: 1 meq, stir well, at the set cross-linking temperature and time (as shown in Table 1 A), continue to stir for transparency Acid-chain reaction. Then the EGDGE epoxy compound cross-linked hyaluronic acid solution is poured into a flat mold made of Teflon material, dried in a 35 ° C hot oven to form a film, and the dried hyaluronic acid film Put it into a cleaning solution (80 wt% acetone aqueous solution), and then put it into distilled water to wash it. The polysaccharide hyaluronic acid film after drying in a 35t hot oven is carried out in a 0.02M PBS solution. Solubility test (results are shown in Table 1 B). Solubility test results: According to the results of this experiment, it was found that the solution formed by adding polysaccharide hyaluronic acid material to the saccharine, adding EGDGE epoxy compound cross-linking agent, when the pH of the hyaluronic acid solution was 3.0-7 · In the range of 5, when the epoxy compound cross-linking reaction time is more than 0.5 hours, the thin film formed after drying the $ 593341 hyaluronic acid solution after the cross-linking is immersed in a 0.02M PBS solution for 24 hours. Will dissolve (as shown in Table 1B), forming a water-insoluble, cross-linked hyaluronic acid film material. 593341 Table (ΙΑ) Sample 1A 1B 1C 1D 1E iF Weight of sodium hyaluronate (gram) 0.1 0.1 0.1 0.1 0.1 0.1 Milli-equivalent number of hydroxyl group of hyaluronic sodium (m equ valent) ) 1 1 1 1 1 1 Hyaluronic acid sodium salt solution solid content (Solid content 9 wt%) 1 1 1 1 1 1 Hyaluronic acid solution pH 値 3.0 before the cross-linking reaction Soil 0 · 2 3.5 Soil 0.2 7.5 Soil 0.2 10.0 Soil 0.2 11.5 Soil 0.2 12_5 Soil 0.2 EGDGE added weight (g) 0.448 0.448 0.448 0.448 0.448 0.448 EGDGE added millequivalents (meq) 4 4 4 4 4 4 HA solution cross-linking reaction temperature (° c) / time (minutes) ), (Sample number) 35/30 (1Α1) 35/30 (1B1) 35/30 (1C1) 35/30 (1D1) 35/30 (1E1) 35/30 (1F1) Crosslinking reaction temperature of HA solution ( ° c) / time (hours), (sample number) 35/1 (1Α2) 35/1 (1Β2) 35/1 (1C2) 35/1 (1D2) 35/1 (1E2) 35/1 (1F2) Crosslinking reaction temperature of HA solution (° c) / time (hour), (sample number) 35/2 (1F3) 35/2 (1G3) Crosslinking reaction temperature of HA solution (° c) / time (hour), ( Sample number) 35/24 (1F4) 35/24 (1F4) Table (I Β) EGDGE cross-linking The solubility test sample of the formed ΗΑ film in 0.02M 593341 PBS solution was placed in 25T :, 0.02M (1A1) (1B1) (1C1) (1D1) (1E1) (LF1) insoluble insoluble for 24 hours in PBS solution Dissolve dissolve dissolve sample at 25 ° C, 0.02M (1A2) (1B2) (1C2) (1D2) (1E2) (1F2) in PBS solution for 24 hours. 0.02M — —_ (1F3) (1G3) Dissolve and dissolve samples in PBS solution for 24 hours and place at 25 ° C. 0.02M —- (1F4) (1F4) Dissolve and dissolve samples in PBS solution for 24 hours. Example (2): Non Method for preparing water-soluble, cross-linked hyaluronic acid polysaccharide material: take 0.1 g of sodium hyaluronate polysaccharide material powder (containing 1.0 milliequivalent hydroxyl group), dissolve in 10 ml of distilled water, and stir at room temperature After homogenization, a 1% hyaluronic acid solution is formed. Then use 1.0 N NaOH or 1 · NO HC1 solution to adjust the φ pH 値 of the hyaluronic acid solution, as shown in the following table (2). Preheat this hyaluronic acid solution to 30t, and then add a quantitative amount of EGDGE epoxy compound to the hyaluronic acid solution, as shown in Table (2), where the milli-equivalents of EGDGE and the milli-equivalents of hyaluronic acid are added. The stoichiometric ratio is 2: 1, after stirring evenly at the cross-linking temperature of 30 ° C for 4 hours, then the hyaluronic acid solution after cross-linking at different pH will use 1.0 N NaOH or 1. ON HC1 solution, Adjust the pH of the hyaluronic acid solution to 6.5-7.5 22 593341 is close to a neutral state, and then pour these hyaluronic acid solutions into a Teflon flat mold and dry in a hot oven at 30 ° C for 1 to 2 hours. Film, the dried hyaluronic acid film is placed in a cleaning solution (a composition containing 80% by weight of an aqueous solution of propyl hydrazone), and then placed in steamed water to wash the polysaccharide after drying in a hot oven at 3 5 ° C Hyaluronic acid-like film was tested for solubility in 0.02M PBS solution, and the results are shown in Table (2). Solubility and DSC thermal analysis test results: It is found from the experimental results that the solution formed by dissolving the hyaluronic acid material with water is added with EGDGE cross-linking agent. When the pH of the hyaluronic acid solution is between 2.5 and 7.1, the epoxy compounds cross-link. The film formed after drying the hyaluronic acid solution after the chain is immersed in 0.02M PBS solution for 48 hours and will not dissolve (as shown in Table 2), forming a water-insoluble, cross-linked hyaluronic acid film material . In addition, the EGDGE cross-linked hyaluronic acid solution was dried and washed. The resulting film was subjected to thermal analysis using DSC (Differential Sacnning Calorimetry) thermal analysis. The experimental results found that the uncross-linked hyaluronic acid film material has The main melting point has a temperature of 166 ° C (Tm2). It can be clearly seen from Table (2) that the melting point of the hyaluronic acid film material (Sample Nos. 21A, 21B) obtained after the EGDGE chemical cross-linking in the pH range of the hyaluronic acid solution in the range of 2.5-7.1 (Table 2 ), 23 593341 have been significantly improved due to the good cross-linking effect. This result shows that the pH of the present hyaluronic acid solution is between 2.5 and 7.1 and the epoxy compound is cross-linked to obtain a better cross-linking effect. The melting points (Table 2) of hyaluronic acid film materials obtained with PH 値 under the cross-linking conditions of 10.2 and 12.6 (sample number 21F) are significantly lower than those of non-crosslinked hyaluronic acid film materials.

24 593341 Table (2) Sample Unlinked HA film 21A 21B 21C 21D 21E 21F Hyaluronic acid sodium salt hydroxyl equivalents 1 1 1 1 1 1 pH ≥ 2.5 of hyaluronic acid solution before crosslinking reaction Soil (λ 2 3.3 ± 0 · 2 4 · 5 Soil 0.2 7.1 Soil 0.2 10 · 2 Soil 0.2 2 12.6 Soil 0 · 2 EGDGE added weight (g) 0.224 0.224 0.224 0.224 0.224 0.224 0.224 EGDGE added milli-equivalent 2 ' 2 2 2 2 2 Cross-linking reaction temperature (° c) / time (hours) 30/4 30/4 30/4 30/4 30/4 30/4 After the cross-linking, the hyaluronic acid solution is placed in the mold and dried. Temperature fc) / time (hours) 30/12 30/12 30/12 30/12 30/12 30/12 The sample is placed at 37 ° C and dissolved in 0.02M PBS solution for 48 hours. Insoluble and insoluble. Insoluble and soluble within 24 hours. Immediately dissolve DSC: Tm2 ° C 166 185 184 — 173 159 138 Example (3): Method for preparing water-insoluble, cross-linked hyaluronic acid polysaccharide material 25 593341 Take the powder of hyaluronic acid sodium salt polysaccharide material. · I grams, dissolved in 10 ml of distilled water, and stirred at room temperature to form a 1% solids hyaluronic acid solution. Then use 1.0 N NaOH or 1 · ON HC1 solution to adjust the pH of the hyaluronic acid solution, as shown in the following table (3). Preheat this hyaluronic acid solution to 30 ° C, and then add quantitative EGDGE to the hyaluronic acid solution, as shown in Table (3), where the number of milli-equivalents added to EGDGE and the number of milli-equivalents of hyaluronic acid are stoichiometric. The ratio is 6: 1. After homogeneous stirring, react at a cross-linking temperature of 3 5 ° C. After 4 hours, adjust the hyaluronic acid solution after cross-linking at different pH using 1.0 N NaOH or 1.0 N HC1 solution to adjust The pH of the hyaluronic acid solution is approximately 6.5-7.5, and the hyaluronic acid solution is poured into a Teflon flat mold, and dried in a hot oven at 35 ° C for 1 to 2 hours to form a film and dry. The subsequent hyaluronic acid film is placed in a cleaning solution (80 wt% aqueous acetone solution) and then washed and then placed in steamed water. The polysaccharide hyaluronic acid is dried in a hot oven at 35 ° C. The film was tested for solubility in a 0.02M PBS solution, and the results are shown in Table (3). Solubility and DSC test analysis results: The experimental results found that when the hyaluronic acid solution was added with EGDGE cross-linking agent, when the pH of the hyaluronic acid solution was in the range of 3 · 3-7 · 4 '26 593341 The film formed by drying the acid solution will not dissolve in 0.02M PBS solution for 72 hours (as shown in Table 3), forming a water-insoluble, cross-linked hyaluronic acid film material. The EG DGE cross-linked hyaluronic acid solution was dried and washed. The DSC thermal analysis experiment found that the pH value of the hyaluronic acid solution was EGDGE chemically cross-linked in the range of 3.3-7.4. The melting point of the hyaluronic acid film material (sample No. 3 A, 3B, 3C) is significantly higher than that of the uncrosslinked hyaluronic acid film material because of its good cross-linking effect. Table (3) Sample (Unsampled) HA film 3A 3B 3C 3D 3E Weight (g) of sodium hyaluronate —- 0.1 0.1 0.1 0.1 0.1 Number of milli-equivalents of hyaluronate sodium salt — 1 1 1 1 1 Solid content of sodium hyaluronate solution — 1 1 1 1 1 Hyaluronic acid solution PH 値 before cross-linking reaction — 3.3 soil 0.2 4.5 soil 0 · 2 7 · 4 soil 0.2 10.5 soil 0 · 2 12.8 soil 0 · 2 EGOGE added weight (g) — 0.672 0.672 0.672 0.672 0.672 EGDGE added milli-equivalent --- 6 6 6 6 6 HA solution cross-linking reaction temperature (° c) / time (hour) — 35/4 35/4 35/4 35/4 35/4 27 The sample is placed in 25 ° C '0.02M PBS solution for 72 hours and immediately insoluble and insoluble. Insoluble and soluble within 24 hours. DSC: Tm2 ° C 170 187 181 175 158 593341 Example (4) ): Water-insoluble, cross-linking alginate polysaccharides. Preparation method of materials: Take 0.2 g of alginate sodium salt (sodium salt) polysaccharide material powder (containing 2.0 milli-equivalent basis), dissolve 20 ml of saturated water was stirred and stirred at room temperature to form a 1% alginate solution. Then use 1 · N NaOH or 1.0N HC1 solution to adjust the pH of the alginate solution, as shown in Table (4) below. Then preheat this alginate solution to a predetermined temperature of 35 ° C. Then add a certain amount of EGDGE epoxy compound to the alginate solution, as shown in Table (4), stir evenly at the set cross-linking temperature and Under time, as shown in Table (4), the alginate polysaccharide material cross-linking reaction was continued with stirring. The alginate material solution after the EG DGE epoxy compound is cross-linked is poured into a flat mold made of Teflon material, and dried in a hot oven at a temperature of 35 ° C to form a film. The dried alginate film It was washed in a cleaning solution, and then washed in distilled water. The alginate film after drying at 35 ° C was subjected to a solubility test (in a 0.02M PBS solution). The results are shown in Table (4). The experimental results show that the alginate solution is added to the EGDGE cross-linking agent. When the pH of the alginate solution is in the range of 3 · 8-28 593341 7.2, the brown alga formed after the cross-linked alginate solution is dried Acid salt film, soaked in 0.02 M PBS solution for more than 48 hours will not dissolve, forming an insoluble alginate film material. Table (4) Sample (Unsampled) Alginate Alkali 4A 4B 4C 4D 4E 丨 Alginate sodium salt weight (g) — 0.2 0.2 0.2 0.2 0.2 Alginate hydroxyl equivalent number 2 2 2 2 2 Alginate solution solids 1 1 1 1 1 pH of the alginate solution before cross-linking 値 — 3 · 8 soil 0.2 5 · 0 soil 0 · 2 7.2 soil 0.2 10 · 2 soil 0 · 2 12.2 soil 0 · 2 EGDGE added weight m, μ w 0.224 0.224 0.224 0.224 0.224 EGDGE added milli-equivalent — 2 2 2 2 2 Cross-linking reaction temperature (° c) / time (minutes) 35 / 4hr 35 / 4hr 35 / 4hr 35 / 4hr 35 / 4hr The sample was placed in 25 ° C, and dissolved in 0.02M PBS solution for more than 2 days. Insoluble for 2 days or more. Insoluble for 2 days or more. Insoluble and dissolved immediately. Example 5: Insoluble, cross-linked cartilage Method for preparing chondroitin sulfate polysaccharide materials 29 593341 Take chondroitin-6-sulfate polysaccharide material powder 0.503 (containing 3 milli-equivalent hydroxyl group), dissolved in 10ml The distilled water was stirred well at room temperature to form a 5% chondroitin-6-sulfate solution. Then use 1.0 N NaOH or 1.0 N HC1 solution to adjust the pH of chondroitin-6-sulfate solution, as shown in the following table (5). Preheat this chondroitin-6-sulfate solution to a predetermined temperature of 35 ° C, and then add a certain amount of EGDGE epoxy compound to the chondroitin-6-sulfate solution, as shown in Table (5), stir well. At the set cross-linking temperature and time, as shown in Table (5), continue to stir for chondroitin-6-sulfate cross-linking reaction. The chondroitin-6-sulfate solution after the EGDGE epoxy compound is cross-linked is poured into a flat mold made of Teflon material, and dried to form a film in a 3 5 t hot oven. The dried chondroitin-6 -The sulfate film was washed in a cleaning solution, and then washed in distilled water. After 35t of dried chondroitin-6-sulfate e film, the solubility test (in a 0.02M PBS solution) was performed. The results are shown in the table ( 5) shown. The experimental results show that when chondroitin-6-sulfate solution is added to the EGDGE cross-linking agent, when the pH of the chondroitin_sulfate solution is in the range of 3 · 7-7 · 0, the chondroitin-6-sulfate after cross-linking The film formed by drying the salt solution will not dissolve after being soaked in a PBS solution of 0.02 M for more than 48 hours, forming a water-insoluble chondroitin-6-sulfate film material. 30 Table (5) Sample (Unsampled) Chondroitin-6-sulfate m ΓΤΠ 5A 5B 5B 5C 5C Chondroitin-6-sulfate weight (g) 0.503 0.503 0.503 0.503 0.503 Chondroitin-6-sulfate Number of hydroxyl equivalents 3 3 3 3 3 Chondroitin-6-sulfate solution solids content-5 5 5 5 5 PH of chain reaction chondroitin-6-sulfate solution 値 3.7 0.2 0.2 5 · 5 00 · 2 7 · 2 ± 0 · 2 9.5 Soil 0 · 2 12 · 2 Soil 0 · 2 Weight of EGDGE added (g) 0.336 0.336 0.336 0.336 0.336 Milli equivalent of EGDGE added 3 3 3 3 3 Cross-linking reaction temperature / time 35 〇C * 4hr 35 ° C * 4hr 35 ° C * 4hr 35 ° C * 4hr 35 ° C 氺 4hr The sample was placed in 25 ° C and dissolved in 0.02M PBS solution for more than 2 days and insoluble for 2 days or more. Insoluble immediately dissolve immediately 593341 Example (6): Preparation of non-water-soluble, cross-linked pectin polysaccharide material 0.352 (containing 4 milliequivalent hydroxyl groups) of pectin polysaccharide material powder, soluble in 17.6 After stirring in ml of distilled water at room temperature, a 2% solid pectin solution was formed. Then use 1.0 N NaOH or 1.0 N HC1 solution to adjust the pH of the pectin solution, as shown in the following table (6). Preheat this pectin solution to a predetermined temperature of 3 5 t, and then add a certain amount of EGDGE epoxy compound to the pectin solution. As shown in Table (6) 31 593341, stir well and set the cross-linking temperature and time. Next, as shown in Table (6), stirring was continued to perform a pectin cross-linking reaction. The EGDG-E epoxy compound cross-linked pectin solution is poured into a flat mold made of Teflon material, dried in a 35t hot oven to form a film, and the dried pectin film is placed in a cleaning solution for cleaning. Put it into distilled water for washing, and then dry the dried pectin film, and perform a solubility test (in a 0.02M PBS solution). The results are shown in Table (6). The experimental results found that the EGDGE cross-linking agent was added to the pectin solution. When the pH of the pectin solution was in the range of 2.9-7.5, the film formed after drying the cross-linked pectin solution was immersed in 0.02M PBS. It will not dissolve in the solution for more than 24 hours, forming a water-insoluble pectin film material. Table (Sample) Uncrosslinked pectin film 6A 6B 6C 6D 6E Weight (g) of pectin 0.352 0.352 0.352 0.352 0.352 0.352 Pectin hydroxyl equivalents-4 4 4 4 4 Pectin solution Solid content (wt%) 2% 2% 2% 2% 2% 2% 2% pH value of pectin solution before crosslinking reaction 値 2.9 3.81 5.8 7.5 12.3 EGDGE added weight (g) 0.448 0.448 0.448 0.448 0.448 32 EGDGE added equivalent Number 4 4 4 4 4 Crosslinking reaction temperature (° C) / time (minutes) (sample number) 35 ° C * 4hr (PEF1A) 35 ° C * 4hr (PEF1B) 35 ° C * 4hr (PEF1C) 35 ° C * 4hr (PEF1D) 35 ° C * 4hr (PEF1E) The sample is placed in 25 samples, dissolved in 0.02M PBS solution for more than 3 days, insoluble for 3 days or more, insoluble for more than 3 days, insoluble for more than 3 days, insoluble for more than 24 days, dissolved in 593341. (Example (7): Preparation of water-insoluble, cross-linked alginate polysaccharide material. Take 0.1 g of alginate sodium salt polysaccharide material powder (containing 1.0 milli-equivalent hydroxyl group) and dissolve it in 10 ml of saturated water. After stirring at room temperature, an alginate solution with a solid content of 1% is formed. Then use a HC1 solution of 1.0N to adjust the permeability. The pH of the hyaluronic acid solution is shown in the following table (7). Then preheat this alginate solution to a predetermined temperature of 35 ° C, and then add different doses of EGDGE epoxy compound to the alginate solution, as shown in the table. As shown in (7), stir well. At the set cross-linking temperature and time, as shown in Table (7), continue to stir to carry out the alginate polysaccharide material cross-linking reaction. After the EGDGE epoxy compound is cross-linked, The alginate solution is poured into a 33 593341 flat mold made of Teflon material, and dried to form a film in a 40 ° C hot oven. The dried alginate film is placed in a cleaning solution, and then placed in distilled water. The alginate film was cleaned and dried in the medium, and the solubility test was performed, and the results are shown in Table (7). The experimental results showed that the EGDGE cross-linking agent was added to the alginate solution, and the EGDGE epoxy was added to the alginate solution. When the compound's milli-equivalent number is 1/5 or more of the alginate, the thin film formed after the cross-linked alginate solution is dried will not dissolve in the 0.02M PBS solution for more than 24 hours. To form a water-insoluble film Table (7) Sample (Sample) Uncrosslinked alginate film 7A 7B 7C 7D 7E Alginate film weight M (g) 0.1 0.1 0.1 0.1 0.1 Alginate hydroxyl equivalent number _ 1 1 1 1 1 Solid content of alginate solution (%) 1 1 1 1 1 pH of alginate solution before cross-linking reaction 値 --- 3.5 soil 0 · 2 3.5 soil 0 · 2 3 · 5 soil 0.2 3.5 ± 0.2 3.5 soil 0.2 EGDGE added weight (g) 0.002 0.011 0.022 0.112 0.224 EGDGE added milli-equivalent --- 0.02 0.1 0.2 1 2 Cross-linking reaction temperature (° c) / time (minutes) 35 ° C * 24hr 35 ° C * 24hr 35 〇C * 24hr 35 ° C * 24hr 35 ° C * 24hr 34 The sample is placed in 25t, 0. 02M PBS solution for 24 hours to dissolve, dissolve, dissolve, insoluble, insoluble 593341. Example (8): Water-insoluble chondroitin-6 -Chondroitin-6-sulfate Polysaccharide material preparation method Chondroitin-6-sulfate polysaccharide material powder 0.5 · 3 (containing 3 equivalents of the base), dissolved in 10 mM steam Saturated water, stirred evenly at room temperature to form a 5% chondroitin-6-sulfate solution. Then use 1 · 0 N NaOH or 1 · 0N HC1 solution to adjust the pH of the chondroitin-6-sulfate solution, as shown in the following table (8). Preheat this chondroitin-6-sulfate solution to a predetermined temperature of 35t :, then add a certain amount of EGDGE epoxy compound to the chondroitin-6-sulfate solution, as shown in Table (8), stir well, and At the set cross-linking temperature and time, as shown in Table (8), the chondroitin-6-sulfate cross-linking reaction was continued with stirring. The chondroitin-6-sulfate solution after the EGDGE epoxy compound is cross-linked is poured into a flat mold made of Teflon material, and dried in a hot oven at 35 ° C to form a film 'dried chondroitin- The 6-sulfate film was placed in a cleaning solution and washed with distilled water. The chondroitin-6-sulfate film after drying at 35 ° C was tested for solubility (in a 0.02M PBS solution). The results are shown in the table. (8). The experimental results show that when chondroitin-6-sulfate solution is added with EGDGE cross-linking agent, 35 593341 when the pH of chondroitin-6-sulfate solution is in the range of 3.7-7.0 and the solid form of chondroitin-6-sulfate solution When the content is above 0.1%, the film formed after the cross-linked chondroitin-6-sulfate solution is dried and immersed in 0.02 M PBS solution for more than 48 hours will not dissolve, forming a water-insoluble chondroitin -6-sulfate film material.

Table (8) Xia style (Sample) 8A 8B 8C 8D 8E Chondroitin-6-sulfate weight (g) 0.503 0.503 0.503 0.503 0.503 Chondroitin-6-sulfate hydroxyl equivalents 3 3 3 3 3 Chondroitin- Solid content of 6-sulfate solution 0.05 0.1 1 5 10 Cross-linking reaction chondroitin-6 · sulfate solution pH 値 3.2 soil 0.2 3.2 soil 0.2 0.2 3.2 0.2 ± 0.2 3.2 soil 0.2 EGDGE added weight ( G) 0.336 0.336 0.336 0.336 0.336 EGDGE added milli-equivalents 3 3 3 3 3 Crosslinking reaction temperature / time 35 ° C * 8hr 35 ° C * 8hr 35 ° C * 8hr 35 ° C * 8hr 35 ° C * 8hr Test Samples were immediately dissolved in 0.02M PBS solution at 25 ° C for more than 2 days, insoluble for 2 days or more, insoluble for 2 days or more, and insoluble for 2 days or more. Example (9): Preparation of non-water-soluble hyaluronic acid polysaccharide materials. Transparent 0.1 g of sodium polysaccharide powder (containing 1.0 milli-equivalent hydroxyl group) was dissolved in 10 ml of distilled water and stirred at room temperature to form a 1% solid solution of hyaluronic acid. Then use 36 593341 1. ON HC1 solution, adjust the pH of the hyaluronic acid solution, as shown in the following table (9). Then preheat this hyaluronic acid solution to 3 (rc), and then add quantitative different kinds of epoxy compounds to the hyaluronic acid solution, as shown in Table (9), where the number of milli-equivalents of epoxy compounds and hyaluronic acid The stoichiometric ratio of acidic hydroxyl equivalents was kept at 1: 1, and after stirring for 4 hours at a cross-linking temperature of 30 ° C, these hyaluronic acid solutions were poured into a Teflon flat mold at 3 5 The film is formed by drying in a hot oven at ° C for an hour. The dried hyaluronic acid film is placed in a cleaning solution (70 wt% acetone aqueous solution) and then washed in distilled water. The dried polysaccharides are transparent. Soluble acid film was tested for solubility in 0.02M PBS solution, and the results are shown in Table (9). From the experimental results, it was found that the hyaluronic acid solution was added to various poly-functional epoxides used in this example. Cross-linking agent, the film formed by drying the cross-linked hyaluronic acid solution, it will not dissolve after immersing in 0 · 0 2 M PBS solution for more than 24 hours, forming a water-insoluble hyaluronic acid film Material Table (9) Sample 9A 9B 9C 9D 9E 9F Weight of sodium hyaluronate (g) 0.1 0.1 0.1 0.1 0.1 0.1 Number of hydroxyl equivalents of sodium hyaluronate 1 1 1 1 1 1 37 593341 Solid content of sodium hyaluronate solution 1 1 1 1 1 1 Hyaluronic acid solution pH 値 3.3 soil 0.2 3.3 ± 0.2 3.3 soil 0.2 7 · 3 soil 0.2 3.3 soil 0.2 6.5 ± 0.2 BDDE added weight (g) / milli-equivalent 0.101 / 1 — — — — — — EGDGE added weight (g) / milli-equivalent — — 0.112 / 1 — — — — — Ex-861 (difunctional epoxy compound) added weight (g) / milli-equivalent 0.587 / 1 0.587 / 1 EX-411 (tetrafunctional epoxy compound) added weight (g) / milli-equivalent 0.231 / 1 0.231 / 1 HA solution cross-linking reaction temperature (° c) / time (hour) 30/4 30 / 4 30/4 30/4 30/4 30/4 The sample is placed at 25 ° C, 0.02M PBS solution for 24 hours. Insoluble, insoluble, insoluble, insoluble, insoluble Note: * Ex-861 (di-epoxide) Polyethylene glycol diglycidyl

ether, WPE (weight per epoxy equivalent) = 587 * EX-41 l (Tetra-epoxide) Pentaerythritol Poly glycidyl Ether: WPE (weight per epoxy equivalent) = 231 38 593341 Example (ten) · Water-insoluble hyaluronic acid Preparation of Polysaccharide Materials-Method of taking 0.1 · 1g of sodium hyaluronate polysaccharide material powder (containing 1.0 milli-equivalent hydroxyl group), dissolving in 10m 1 of distilled water, and stirring evenly at room temperature to form 1% Hyaluronic Acid Solution. Then use HC1 solution of 1.0N to adjust the pH of the hyaluronic acid solution, as shown in the following table (10). Preheat this hyaluronic acid solution to different temperatures, as shown in Table (10), and then add a quantitative amount of EGDGE epoxy compound to the hyaluronic acid solution, as shown in Table (10), where the epoxy The stoichiometric ratio of milli-equivalents of the compound (epoxide) added to the milli-equivalents of hyaluronic acid was kept at 2: 1, and after stirring for 6 hours at different cross-linking temperatures, the mixture was poured into the hyaluronic acid solution. In a Teflon flat mold, the dried hyaluronic acid film is placed in a cleaning solution (a 60 wt% acetone aqueous solution), and then washed in distilled ® water. The dried polysaccharide hyaluronic acid film The solubility test in 0.02M PBS solution was performed. The results are shown in Table (10). The experimental results showed that the hyaluronic acid solution was added with the EGDGE cross-linking agent, and the cross-linking reaction was performed at a temperature of 20-45 ° C. The film formed by drying the hyaluronic acid solution will not dissolve after being soaked in 0.02M PBS solution for more than 72 hours, forming a water-insoluble hyaluronic acid film material. 39 Table (10) Sample 10A 10B 10C 10D 10E 10F Weight (g) of sodium hyaluronate 0.1 0.1 0.1 0.1 0.1 0.1 Number of hydroxyl equivalents of sodium hyaluronate 1 1 1 1 1 1 Hyaluronic The solid content of the sodium salt solution 1 1 1 1 1 1 1 pH of the hyaluronic acid solution before the cross-linking reaction 値 3_5 soil 0 · 2 6.5 ± 0 · 2 3.3 soil 0.2 7J soil 0.2 3.1 soil 0.2 7.3 soil 0.2 EGDGE added weight (G) 0.22 0.22 0.22 0.22 0.22 0.22 Milli-equivalents of EGDGE added 2 2 2 2 2 2 Cross-linking reaction temperature and time 20 ° C 20 ° C 35 ° C 35 ° C 45 ° C 45 ° C * 6hr * 6hr * 6hr * 6hr 氺 6hr * 6hr The sample is placed at 37 ° C, 0.02MPBS for 3 days, not 3 days, 3 days, 3 days, 3 days, and 3 days. Alginate polysaccharide material solution is prepared by freeze-drying porous porous substrate. Alginate polysaccharide material powder is taken, dissolved in distilled water, and stirred to form a solid form at 50-60 ° C. 1%, 2%, 3% solution. Then use 1 · N HC1 solution to adjust the pH of the solution to about 4.0. Quantitative EGDGE epoxy compound is added to the polysaccharide solution, so that the stoichiometric ratio of the milli-equivalent number of the epoxy compound to the milli-equivalent number of the polysaccharide hydroxyl group is maintained at 1: 1. After homogeneous stirring, react at a constant temperature of 3 5 ° C for 6 hours, and then pour the 40 593341 chain reaction polysaccharide solution into the mold. After freeze-drying, an open cell type can be obtained. Porous cross-linked polysaccharide substrate. After freeze-drying, the porous cross-linked alginate substrate was washed in a cleaning solution (80 wt% acetone in water), and then washed in distilled water. The dried cross-linked alginate substrate was tested for solubility in a 0.02M PBS solution. The results are shown in Table (Π). From the experimental results, it was found that the porous cross-linked alginate substrate did not dissolve in the PBS solution for more than 24 hours, forming a water-insoluble porous alginate substrate. Table (11) Sample 11A 11B 11C 11D 11E 11F Alginate sodium salt solution 1 1 2 2 3 3 pH of alginate solution after adding EGDGE cross-linking agent 3.5 0.2 0.2 6.5 0.2 0.2 3.3 土0 · 2 7.3 soil 0.2 3.1 soil 0.2 7 · 3 soil 0.2 The porous cross-linked alginate substrate sample is placed at 37 ° C, 0.02 MPBS solution for 24 hours, insoluble for 24 hours, insoluble for 24 hours, insoluble for 24 hours, insoluble for 24 hours, insoluble for 24 hours Insoluble Example (12): Hyaluronic acid polysaccharide material solution after cross-linking is used to prepare porous porous substrates by freeze-drying method. • Hyaluronic acid sodium salt polysaccharide material powder is taken, dissolved in steam-saturated water, After stirring under warm conditions, a solid content of 1%, 2%, and 3% was formed. Then use 1.0N HC1 solution to adjust the pH of the solution to 4.5. Quantitative EGDGE epoxy compound is added to the polysaccharide solution, so that the stoichiometric ratio of the milli-equivalent number of the epoxy compound to the milli-equivalent number of the polysaccharide hydroxyl group is maintained at 1 ·· 1. After stirring well, react at constant temperature of 35 ° C for 6 hours, and then pour the polysaccharide solution after cross-linking reaction into the mold. After freeze-drying, a porous cross-linked polysaccharide group with open pores can be obtained. material. Transparent after freeze-drying 41 593341 The porous sodium cross-linking substrate of high quality is placed in a cleaning solution (80 wt% acetone aqueous solution), and then washed in distilled water. After drying, the porous cross-linked hyaluronate sodium salt substrate was tested for solubility in a 0.02M P ^ S solution, and the results are shown in Table (12). According to the experimental results, it was found that the porous cross-linked hyaluronic acid sodium salt substrate will not dissolve after being immersed in a 0.02 M PBS solution for more than 24 hours, forming a '-insoluble porous hyaluronic acid substrate. Table (12) Sample 12A 12B 12C 12D 12E 12F HA solid content 1 1 2 2 3 3 Add EGDGE crosslinker 3 · 5 soil 6 · 5 ± 3.3 soil 7 · 3 soil 3.1 soil 7.5 soil PH of HA solution 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Porous cross-linked HA sample 24 hours 24 hours 24 hours 24 hours 24 hours 24 hours at 37 ° C, 0.02M from time to time from time to time from time to time (13): Chondroitin-4-siilfate material solution after cross-linking Preparation of porous porous substrate using freeze-drying method Take chondroitin sulfate material powder, dissolve in distilled water, and at room temperature After stirring under the conditions, a solid content solution of 5% and 10% is formed. #Use K0N HC1 to adjust the pH of chondroitin-4-sulfate solution (as shown in Table 13). Quantitative EGDGE epoxide was added to the chondroitin sulfate solution to keep the stoichiometric ratio of milli-equivalents of EGDGE epoxy compounds to milli-equivalents of polysaccharide hydroxyl groups to 4: 1. Stir evenly at 3 5 ° C. Reaction at constant temperature for 2 hours. Then pour the chondroitin-4-sulfate solution after the cross-linking reaction into 42 593341 mold. After freeze-drying, an open hole shape can be obtained. Porous cross-linked chondroitin-4-sulfate substrate. The freeze-dried chondroitin sulfate e porous cross-linked substrate was placed in a cleaning solution (composed of 80 wt% acetone aqueous solution), and then washed in distilled water. The dried cross-linked chondroitin-4-sulfate substrate was tested for solubility in a 0.02M PBS solution. The results are shown in Table (13). According to the experimental results, it was found that the porous cross-linked chondroitin-4-sulfate substrate does not dissolve after being immersed in a PBS solution of 0.02M for more than 24 hours, forming a water-insoluble porous chondroitin-4-sulfate substrate . Table (13) Sample 13A 13B 13C 13D 13E 13F C solid content of chondroitin-4-sulfate solution 5 5 5 10 10 10 pH of chondroitin-4-sulfate solution after adding EGDGE crosslinker 剂2.5 ± 0.2 4.6 soil 0.2 6.5 ± 0.2 2.4 soil 0.2 3.9 soil 0.2 6 · 3 soil 0.2 Porous cross-linked chondroitin-4-sulfate sample at 37 ° C, 0.02M PBS solution 24 hours insoluble 24 hours insoluble 24 Insoluble for 24 hours Insoluble for 24 hours Insoluble for 24 hours Insoluble for 24 hours Insoluble 43 Example (14): Preparation of water-insoluble, cross-linked alginate fibers 0.1 g of alginate sodium salt powder (containing 1.0 milliequivalent) Hydroxyl), dissolved in 10 ml of distilled water, and stirred at 50-60 ° C to form a 1% solid solution. Then use 1.0N 'HC1 solution and adjust the pH of the solution to about 4.0. Then add a quantitative amount of EGDGE epoxy compound to the polysaccharide solution, so that the stoichiometric ratio of the number of milli-equivalents of the epoxy compound to the number of milli-equivalents of the hydroxyl groups of the polysaccharide remains at 1: 2. After stirring well, the reaction was performed at a constant temperature of 3 5 t for 6 hours. Then, the alginate sodium salt solution after the cross-linking reaction was extruded into 95% alcohol with monofilament fibers (Monofilament fiber) with injection needles of different sizes. The alginic acid fibers were taken out and dried in a desiccator to store fibrous substrates with different diameters ranging from 1 μm to 50 μm. The alginate fiber after the epoxy compound is cross-linked will not dissolve after being immersed in 0.02M PBS solution for more than 48 hours to form a kind of insoluble alginate fiber. Example (15): Preparation of non-water-soluble, cross-linked pectin fibers. 0.352 (containing 4 milliequivalent hydroxyl groups) of M gum polysaccharide powder was dissolved in 17.6 ml of distilled water at room temperature. After stirring, a 2% pectin solution was formed. Then use 1 · N NaOH or 1.0N HC1 solution to adjust the pH of the solution to 3.5. Quantitative EGDGE epoxy compound was added to the polysaccharide solution, so that the molar ratio of epoxy compound to the equivalent of hydroxyl polysaccharide hydroxyl 593341 was kept at 2: 1. After stirring well, react at a constant temperature of 3 5 ° C for 6 hours. Then, the pectin solution after the cross-linking reaction was extruded into a single fiber in 95% alcohol with injection needles of different sizes. Then take out this fruit Qing fiber, store it in a desiccator, and save it to obtain pectin fiber-like substrates with different diameters ranging from 1 mm to 50 μm. Such pectin fibers after being cross-linked by epoxy compounds will not dissolve after being soaked in 0.02M PBS solution for more than 48 hours, forming a water-insoluble pectin fiber. Example 45 (sixteen): Preparation of water-insoluble, cross-linked HA fibers. Hyaluronic acid sodium salt polysaccharide material powder 0.1 g (containing factory 0 milli-equivalent hydroxyl group) was dissolved in 10 m 1 of Stir in water and stir at 30-3 5 ° C to form a 1% HA solution. Then use 1. ON solution of H C 1 and adjust the ρ 溶液 of the solution to about 4 · 5. Then add a certain amount of EGDGE epoxy compound to the ΗΑ solution, so that the stoichiometric ratio of the milli-equivalent number of EGDGE epoxy compound to the milli-equivalent number of polysaccharide hydroxyl group is maintained at 3: 1. After stirring well, react at a constant temperature of 35 ° C for 2 hours. The hyaluronic acid solution after the EGDGE cross-linking reaction was then pressed into 95% alcohol with single-sized injection needles to form single fibers. This ΗA fiber was taken out and dried in a desiccator, and then stored to obtain HA fiber-like substrates having different diameters of 1πΐΓη · 50 μm in different thicknesses. Such EG fibers after EGDGE cross-linking will not dissolve after being soaked in a PBS solution of 0. 02M for more than 48 hours, forming a kind of insoluble HF fibers. Example (seventeen) ································································································· C-60 ° C after stirring to form a solid content of 3%, a solution. Then: use 1 · ON solution of HC1, adjust the pH of the solution to about 3.0. A quantitative amount of the EGDGE epoxy compound was added to the polysaccharide solution so that the stoichiometric ratio of the milli-equivalent number of the epoxy compound to the milli-equivalent number of the polysaccharide hydroxyl group was maintained at 2: 1. Stir well and react at a constant temperature of 3 5 ° C for 6 hours. Then the alginate polysaccharide solution after the EGDGE cross-linking reaction is dropped into an alcohol or liquid nitrogen with a syringe needle of different sizes using a syringe pump, and freeze-dried To obtain sphere porous porous polysaccharide cross-linked substrates with different diameters of 593341 2 mm-2 〇 Ο μ m. The alginate spherical porous substrate after EGDGE cross-linking will not dissolve for more than 48 hours after being soaked in 0.02M PBS solution, forming a water-insoluble alginate spherical porous substrate. material. Example (eighteen): HA polysaccharide material solution after cross-linking was prepared by using a freeze-drying method to prepare spherical porous substrates. HA polysaccharide material powder was taken, dissolved in distilled water, and mixed at 30 ° C to form uniformly. 2.5% solids solution. Then use 1. ON HC1 solution, adjust the pH of the solution to about 5.0. Then add a certain amount of BDDE epoxy compound to the polysaccharide solution, so that the stoichiometric ratio of the number of milli-equivalents of the epoxy compound to the number of milli-equivalents of the hydroxy group of the polysaccharide is maintained at 3: 1. After homogeneous stirring, react at a constant temperature of 35 ° C for 3 hours. Then the HA polysaccharide solution after the BDDE cross-linking reaction is dropped into the alcohol or liquid nitrogen with a syringe needle of no size, and then freeze-dried, that is, Sphere porous HA crosslinked substrates with different diameters ranging from 2 mm to 200 μm can be obtained. This HA spherical porous substrate after BDDE cross-linking will not dissolve after being immersed in 0.02M PBS solution for more than 48 hours, forming a non-water soluble HA spherical porous substrate. Example (nineteen): Chondroitin-4-sulfate material solution after cross-linking was prepared using a freeze-drying method to prepare spherical porous substrates. Chondroitin-4-sulfate material powder was taken and dissolved in distilled water. After stirring at ° C, a 5% solids solution was formed. Then use 1.0N HC1 solution to adjust the pH of the solution to 4.0. Quantitative EG DGE epoxy compound was added to the chondroitin-4-sulfate solution, so that the ratio of the stoichiometric number of milli-equivalents of the EGDGE epoxy compound to the number of hydroxyl equivalents of the chondroitin-4-sulfate was kept at 47 593341. twenty one. After stirring well, react at a constant temperature of 35 ° C for 2 hours, and then pass the EGDGE solution to the chondroitin-4-sulfate solution. Use a syringe pump to drop alcohol or liquid nitrogen with different sizes of injection needles and freeze-dry them. To obtain sphea porous chondroitin-4-sulfate substrates with different diameters from 2mm to 200pm. The chondroitin-4-sulfate spherical porous substrate after EG DGE 'cross-linking will not dissolve in PBS solution for more than 48 hours, forming an insoluble chondroitin- 4-sulfate spherical porous substrate. Example (twenty): A solution of cross-linked consequence gum material was prepared by using a freeze-drying method to prepare a spherical porous cavity substrate. Pectin polysaccharide material powder 0.352 (containing 4 milli-equivalent hydroxyl groups) was dissolved in 17.6 ml of distilled water. Stir at room temperature to form a 2% pectin solution. Then use 1.0N HC1 solution to adjust the pH of the solution to about 5.5. Quantitative EGDGE epoxy compound was added to the polysaccharide solution so that the stoichiometric ratio of the milli-equivalent number of the epoxy compound to the milli-equivalent number of the polysaccharide hydroxyl group was maintained at 2: 1, and the mixture was uniformly stirred at a constant temperature of 3 5 ° C. The reaction took 4 hours. Then, the pectin solution after the cross-linking reaction is dropped into alcohol, acetone or liquid nitrogen with injection needles of different sizes by using a syring pump, and freeze-dried to obtain spherical porous holes with different diameters of 2mm-200gm. Pectin substrate. This pectin spherical porous substrate after EGDGE cross-linking will not dissolve after being soaked in 0.02M PBS solution for more than 48 hours, forming a water-insoluble pectin spherical porous substrate. 48 593341 In the embodiment of the present invention, by using various polysaccharide materials at different pH values, types of cross-linking agents, cross-linking temperature, and cross-linking reaction time and ancient drying methods, good quality water-insoluble polysaccharide-based groups can be obtained material. However, the present invention is not limited to the above embodiments, but should be based on the scope of patent applications. [Schematic description] 49

Claims (1)

593341 Amended and replaced the scope of the patent application: June 1 a 1 · A method for manufacturing water-insoluble polysaccharide materials, including the following steps: (a) providing an aqueous solution containing hydroxy polysaccharides; (b) adjusting the Hydroxyl polysaccharide-containing aqueous solution is printed on 2-8 of Azadirachski; (c) Add a polyfunctional epoxy compound to the hydroxyl polysaccharide-containing aqueous solution to perform a cross-linking reaction; (d) Make as much as possible after cross-linking The carbohydrate solution is subjected to a shaping process to obtain a non-aqueous soluble polysaccharide material. 2. The method according to item 1 of the scope of patent application, wherein step (d) is to place the cross-linked polysaccharide solution into a mold and dry it to form a water-insoluble polysaccharide film-like material. 3. The method according to item 1 of the scope of patent application, wherein the step (d) is to place the polysaccharide material solution after cross-linking into a mold, and then freeze-dry to make it porous. Structure of water-insoluble polysaccharides matrix (water-insoluble polysaccharides matrix). 4. The method according to item 1 of the scope of patent application, wherein step (d) is adding an organic solvent to the polysaccharide material solution after cross-linking to precipitate the polysaccharide material after cross-linking, and then filtering A powdery (Podwer) or sheet (Sileet) water-insoluble polysaccharide material is obtained. 50 593341 5 · The method according to item 1 of the scope of patent application, wherein the step (d) is to extrude the solution of the polysaccharide material after cross-linking into a coagulating solution (Coagulant) containing organic solvents, and form fibers therein. Fiber-insoluble polysaccharide material. (3. The method according to item 5 of the scope of patent application, wherein step (d) is a method of intermittent extrusion of the polysaccharide material solution after cross-linking to form spherical particles. The method according to any one of items 2 to 6, wherein the film-like or powder-like, or flake-like, or fibrous, or spherical particles are insoluble polysaccharide materials, washed with a mixed organic solvent containing water, and then used The water-insoluble polysaccharide material after being washed with clean water and dried can be applied to medicine or cosmetics. 8. For the method in the first item of the patent application, wherein the hydroxyl-containing polysaccharide in step (a) is selected from the group consisting of The following groups: Hyaluronic acid, Carboxymethylcellulose, Starch, Alginate 'Chondroitin-4-Sulfate, Cartilage Chondroitin-6-Sulfate, Xanthane Gum, Chitosan, Pectin, Aga0, Carrageenan, and Guar Tree 51 593341 Guar gum (9. If applying for a patent The method of the first item, wherein step (a) is to dissolve the hydroxyl-containing polysaccharides in water to form a uniform aqueous solution, and the weight solids content of the hydroxyl-containing polysaccharides (s 〇1 idc ο ntent, wt%) is 〇 2 wt,%-10 wt% o 10. The method according to item 1 of the scope of patent application, wherein step (b) is to adjust the pH 于 in the range of 2.5-7 · 5. Item, wherein the epoxy compound in step (c) is selected from the group consisting of 1,4-butanediol diglycidyl ether (BDDE), ethylene glycol diglycidyl ether Glyceryl ether (ethylene glycol diglycidyl ether; EGDGE), 1,6-hexanediol diglycidyl ether (1,6-1 ^ \ & 116 (1 丨 〇1 (! 丨 213 ^ 丨 8) ^ 161: 1161 *), Polyethylene glycol diglycidyl ether, Polypropylene glycol diglycidyl ether, Polytetramethylene glycol digylcidyl ether, Neopentyl Glycol diglycidyl ether > Polyglycerol Polyglycerol. 52 593341 polyglycerol polyglycidyl ether, diglycerol polyglycidyl ether: glycerol poiygiycidyl ether, triglyceride polyglycidyl ether tri-methylolpropane polyglycidyl ether), pentaerythritol polyglycidyl ether, and sorbitoi polyglycidyl ether. 12. The method according to item 3 of the patent claim 3, wherein in step (c), the epoxy compound is added in an amount of 0.1 to 8 with respect to the equivalent of 1 equivalent in the polyacetic acid group. O equivalent. 1 3 · The method according to item 11 of the scope of patent application, wherein the preferred addition amount of the epoxy compound is 0. 2-6 · 0 equivalent. 1 4 · The method according to item 1 of the scope of patent application, wherein the temperature of the cross-linking reaction in step (c) is still "X :, the time of the inflammation chain reaction is 10 minutes to 12 hours. 53 593341 1 6 · If applying The method of item 3 of the patent, wherein the polysaccharide solution after the cross-linking is placed in a mold of an appropriate shape, and then a freeze-drying method is used to produce a porous structure, and the pore morphology is interconnected. (Interconnective) structure of water-insoluble polysaccharide base material. 1 7 · The method according to item 4 of the patent application, in which the polysaccharide solution after the cross-linking is poured into a coagulation solution containing organic solvent, and is stirred. Next, the cross-linked polysaccharides are precipitated out, and then filtered to obtain powder or sheet-shaped water-insoluble polysaccharide materials. 1 8 · As in the fifth item of the patent application A method in which a solution of a polysaccharide material after cross-linking is continuously extruded into a coagulant containing an organic solvent using an extruder to form a fiber-like non-water-soluble polysaccharide material, which is formed after drying. Diameter between 1 ηΐϊη -50μιη fibrous insoluble polysaccharide material. I 9 · The method according to item 6 of the patent application, wherein the cross-linked polysaccharide material solution is intermittently extruded into a coagulant to form a diameter medium The spherical shape is from 1 to 50 μm. 20. The method according to item 18 of the patent application, which contains 54 593341 organic solvent. The coagulant composition includes water and organic solvents, where the weight fraction of organic solvents (weight fraction) occupies 6 (1-10 0%. 2 1. The method according to item 20 of the patent application, wherein the organic solvent is selected from the following group: 1,4-dioxane, Chloroform, methylene chloride, N, N-dimethylformamide (N, N_dimethylformamide; DMF), N, N-dimethylacetamide (N, N-dimethylacetamide; DMAc), ethyl acetate, acetone, methyl ethyl ketone (MEK), methanol, ethanol, propanol, isopropanol, and butane Alcohol (butanol) ° 22 · as the 18th in the patent application The method, wherein the solution of cross-linking polysaccharides to a coagulation liquid formed of the solidification temperature of 5-60 ° C. 23. The method according to item 7 of the scope of patent application, wherein the powdery, or film-like, or sheet-like, or fibrous or spherical granular water-insoluble polysaccharide material is washed with a mixed organic solvent containing water, and then used Clean water, including water-containing mixed organic solvent group 55 593341 Ingredients include water and organic solvents, the organic solvent is acetone, methyl ethyl ketone (ME K), < methanol, ethanol (Ethanol), propanol, isopropanol, or butanol or a mixture thereof, the weight fraction of the organic solvent in the cleaning solution accounts for 50-100%, and the ketones It can be used in any proportion with alcohol solvents. The temperature of the cleaning solution is 15t -50 ° C. The non-water-soluble polysaccharide materials after the cleaning solution is washed, and then the temperature is 2 5 ° C -5 0T: clean water. . 24. The method according to item 23 of the patent application, wherein the powdery, or film, or sheet, or fibrous or spherical granular non-aqueous powder is washed with a mixed organic solvent containing water and then cleaned with clean water. The water-soluble polysaccharide materials are further dried by hot air drying at a temperature below 60 ° C, radiation heating drying, or vacuum drying.