CN117720748A - Preparation method of crosslinked hydrogel - Google Patents
Preparation method of crosslinked hydrogel Download PDFInfo
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- CN117720748A CN117720748A CN202311731839.8A CN202311731839A CN117720748A CN 117720748 A CN117720748 A CN 117720748A CN 202311731839 A CN202311731839 A CN 202311731839A CN 117720748 A CN117720748 A CN 117720748A
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- 239000000017 hydrogel Substances 0.000 title claims abstract description 42
- 238000002360 preparation method Methods 0.000 title abstract description 14
- 238000006243 chemical reaction Methods 0.000 claims abstract description 65
- 229920001661 Chitosan Polymers 0.000 claims abstract description 46
- 238000004132 cross linking Methods 0.000 claims abstract description 46
- 230000001954 sterilising effect Effects 0.000 claims abstract description 36
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 36
- 239000000463 material Substances 0.000 claims abstract description 23
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 17
- 150000001875 compounds Chemical class 0.000 claims abstract description 10
- 238000002156 mixing Methods 0.000 claims abstract description 9
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 7
- 230000008859 change Effects 0.000 claims abstract description 6
- 229920000642 polymer Polymers 0.000 claims abstract description 4
- 238000000034 method Methods 0.000 claims description 16
- 238000006467 substitution reaction Methods 0.000 claims description 16
- 230000003204 osmotic effect Effects 0.000 claims description 13
- 239000003795 chemical substances by application Substances 0.000 claims description 12
- 239000007822 coupling agent Substances 0.000 claims description 9
- RBACIKXCRWGCBB-UHFFFAOYSA-N 1,2-Epoxybutane Chemical compound CCC1CO1 RBACIKXCRWGCBB-UHFFFAOYSA-N 0.000 claims description 8
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 claims description 8
- 230000035484 reaction time Effects 0.000 claims description 8
- BMTZEAOGFDXDAD-UHFFFAOYSA-M 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholin-4-ium;chloride Chemical compound [Cl-].COC1=NC(OC)=NC([N+]2(C)CCOCC2)=N1 BMTZEAOGFDXDAD-UHFFFAOYSA-M 0.000 claims description 6
- 229920002385 Sodium hyaluronate Polymers 0.000 claims description 6
- 230000003213 activating effect Effects 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 229940010747 sodium hyaluronate Drugs 0.000 claims description 6
- YWIVKILSMZOHHF-QJZPQSOGSA-N sodium;(2s,3s,4s,5r,6r)-6-[(2s,3r,4r,5s,6r)-3-acetamido-2-[(2s,3s,4r,5r,6r)-6-[(2r,3r,4r,5s,6r)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2- Chemical compound [Na+].CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 YWIVKILSMZOHHF-QJZPQSOGSA-N 0.000 claims description 6
- SYURNNNQIFDVCA-UHFFFAOYSA-N 2-propyloxirane Chemical compound CCCC1CO1 SYURNNNQIFDVCA-UHFFFAOYSA-N 0.000 claims description 5
- 108010022355 Fibroins Proteins 0.000 claims description 5
- 108010010803 Gelatin Proteins 0.000 claims description 5
- 238000000502 dialysis Methods 0.000 claims description 5
- 229920000159 gelatin Polymers 0.000 claims description 5
- 239000008273 gelatin Substances 0.000 claims description 5
- 235000019322 gelatine Nutrition 0.000 claims description 5
- 235000011852 gelatine desserts Nutrition 0.000 claims description 5
- 239000012535 impurity Substances 0.000 claims description 5
- 239000012266 salt solution Substances 0.000 claims description 5
- 229920002101 Chitin Polymers 0.000 claims description 4
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- 238000000746 purification Methods 0.000 claims description 3
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims description 2
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 2
- 108010020346 Polyglutamic Acid Proteins 0.000 claims description 2
- 239000012190 activator Substances 0.000 claims description 2
- 125000003277 amino group Chemical group 0.000 claims description 2
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- 238000000265 homogenisation Methods 0.000 claims description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 2
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 claims description 2
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- 235000010413 sodium alginate Nutrition 0.000 claims description 2
- 239000000661 sodium alginate Substances 0.000 claims description 2
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- 238000004519 manufacturing process Methods 0.000 claims 5
- 125000002730 succinyl group Chemical group C(CCC(=O)*)(=O)* 0.000 claims 2
- -1 salt compound Chemical class 0.000 abstract description 16
- 229920002521 macromolecule Polymers 0.000 abstract description 4
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- 239000000843 powder Substances 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
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- WCDDVEOXEIYWFB-VXORFPGASA-N (2s,3s,4r,5r,6r)-3-[(2s,3r,5s,6r)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O)[C@H](O)[C@H]1O WCDDVEOXEIYWFB-VXORFPGASA-N 0.000 description 7
- 229940014041 hyaluronate Drugs 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
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- 239000011780 sodium chloride Substances 0.000 description 5
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- NMHMNPHRMNGLLB-UHFFFAOYSA-N phloretic acid Chemical compound OC(=O)CCC1=CC=C(O)C=C1 NMHMNPHRMNGLLB-UHFFFAOYSA-N 0.000 description 4
- 239000002994 raw material Substances 0.000 description 4
- 239000003513 alkali Substances 0.000 description 3
- 230000006196 deacetylation Effects 0.000 description 3
- 238000003381 deacetylation reaction Methods 0.000 description 3
- 238000000921 elemental analysis Methods 0.000 description 3
- 229910052739 hydrogen Inorganic materials 0.000 description 3
- 230000002209 hydrophobic effect Effects 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 125000004080 3-carboxypropanoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C(O[H])=O 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- LVXHNCUCBXIIPE-UHFFFAOYSA-L disodium;hydrogen phosphate;hydrate Chemical compound O.[Na+].[Na+].OP([O-])([O-])=O LVXHNCUCBXIIPE-UHFFFAOYSA-L 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 230000023597 hemostasis Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
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- 229910000403 monosodium phosphate Inorganic materials 0.000 description 2
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- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 2
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 description 1
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical group N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 1
- 239000002262 Schiff base Substances 0.000 description 1
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- 239000003431 cross linking reagent Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
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- Medicinal Preparation (AREA)
Abstract
The invention relates to the technical field of biomedical polymers, in particular to a preparation method of crosslinked hydrogel, which comprises the following steps: uniformly mixing a carboxyl high molecular compound, a hydrophobically modified chitosan derivative, a salt compound and a condensing agent in a solution state to obtain a reaction material, carrying out a crosslinking reaction on the reaction material under a variable temperature condition, and carrying out moist heat sterilization after the crosslinking reaction is finished to obtain crosslinked hydrogel; the ratio of the mol number of carboxyl in the carboxyl macromolecular compound to the mol number of amino in the hydrophobically modified chitosan derivative is 1:0.015-0.5; the temperature change condition is that the reaction is continued at the second crosslinking temperature after the reaction at the first crosslinking temperature, and the absolute value of the difference value between the front reaction temperature and the rear reaction temperature is more than 5; the crosslinked hydrogel provided by the invention has good sterilization stability and excellent stretching ductility.
Description
Technical Field
The invention relates to the technical field of biomedical polymers, in particular to a preparation method of crosslinked hydrogel.
Background
Natural polymer materials, such as hyaluronic acid, chitosan, gelatin, collagen, silk fibroin and the like, are widely applied to the preparation of hydrogels in the biomedical field due to good biocompatibility, degradability and easy modification. However, in performing clinical transformations, the sterilization process of the product needs to be considered. Considering the sufficiency and cost of sterilization, the wet heat sterilization is a preferred sterilization mode, however, the natural polymer material has heat sensitivity, and can cause the breakage of molecular chains under the sterilization condition, so that the obvious reduction of mechanical strength is brought, and the sterilization stability of the product is always an important research problem of industrial production.
The chitosan and the derivatives thereof have strong application potential in the biomedical field, wherein the hydrophobic modified chitosan not only solves the problem of water solubility of chitosan, but also can be used for drug slow release, hemostasis, temperature-sensitive gel and related crosslinking reaction due to the hydrophobic effect. For example, chinese patent CN116854998A adopts carboxylated hydroxybutyl chitosan and other aqueous solution crosslinking of oxidized polysaccharide with dialdehyde groups to form double-network crosslinked hydrogel through schiff base reaction, improving chemical stability of single gel network, but the preparation process requires strict control of factors such as mixing time, operating temperature, and the like, and the operation is relatively complex. Literature (Materials Science & Engineering C,2019, 104:10993) uses dodecyl-modified chitosan to crosslink with formylated dextran in situ, and has been used for hemostasis, gel strength has been studied, but the thermal stability of the gel is unknown. In addition, the injection using the pre-gel solution may cause problems such as uneven mixing and inconsistent injection.
In summary, the following disadvantages remain in the related crosslinking technology: (1) a multiple crosslinking mode is adopted, so that the operation is complex; (2) The thermal stability of the gel, especially the wet heat sterilization stability, is unknown; (3) The pre-gel solution is adopted for injection, which may cause problems of uneven mixing, incoherence of injection and the like. In view of the above-mentioned shortcomings, the technical scheme of the invention is specifically proposed.
Disclosure of Invention
In order to solve the technical problems, a preparation method of the crosslinked hydrogel is provided. According to the invention, the carboxyl macromolecular compound and the hydrophobically modified chitosan derivative are crosslinked, the final product is obtained after wet heat sterilization, the degree of tightness of a crosslinked gel network is regulated and controlled through the variable-temperature crosslinking reaction in a salt solution, and the final product obtained after high heat sterilization has good heat stability and excellent stretching ductility; the method of the invention has simple operation and can be used immediately.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
a method for preparing a crosslinked hydrogel comprising the steps of:
uniformly mixing a carboxyl high molecular compound, a hydrophobically modified chitosan derivative, a salt compound and a condensing agent in a salt solution to obtain a reaction material, carrying out a crosslinking reaction on the reaction material under a variable temperature condition, and carrying out moist heat sterilization after the crosslinking reaction is finished to obtain crosslinked hydrogel;
the ratio of the mol number of carboxyl in the carboxyl high molecular compound to the mol number of amino in the hydrophobically modified chitosan derivative to the mol number of the condensing agent is 1:0.015-0.5:0.05-0.4;
the temperature change condition is that the reaction is continued at a second crosslinking temperature after the reaction at the first crosslinking temperature, so that the first crosslinking temperature is T1, the second crosslinking temperature is T2, and the temperature difference between the first crosslinking temperature and the second crosslinking temperature is T, and then T= |T1-T2| and T is more than 5 are satisfied.
Further, the first crosslinking temperature is 0-15 ℃, the reaction time is 12-30 h, the second crosslinking temperature is 16-50 ℃, and the reaction time is 24-72 h;
or the first crosslinking temperature is 16-50 ℃, the reaction time is 1-24 h, and the second crosslinking temperature is 0-15 ℃ and the reaction time is 24-72 h. Preferably, the temperature-variable crosslinking reaction is carried out at low temperature and then at high temperature, and the activity of the condensing agent can be reserved by the low-temperature reaction, so that the method is fully used for the second-step high-temperature crosslinking, and is more beneficial to regulating and controlling the tightness degree of a gel network.
Further, the carboxyl polymer compound is selected from one or more of sodium hyaluronate, carboxymethyl chitosan, polyglutamic acid, carboxymethyl chitin, N-succinyl chitosan, sodium alginate, chondroitin sulfate, silk fibroin, succinyl silk fibroin, gelatin and succinyl gelatin.
Further, the hydrophobically modified chitosan derivative is a product obtained by substitution reaction of chitosan and an epoxy compound, and the total substitution degree of hydroxyl and amino sites in the molecular structure of the chitosan is 0.3-2; the epoxy compound is selected from 1, 2-epoxybutane and/or 1, 2-epoxypentane.
Further, the salt solution contains one or more of chloride salt, hydrogen phosphate dibasic salt and dihydrogen phosphate monobasic salt; the condensing agent comprises a coupling agent used alone or in combination with a coupling agent/activating agent; the coupling agent is selected from EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) or DMTMM (4- (4, 6-dimethoxy triazine-2-yl) -4-methylmorpholine hydrochloride), and the coupling agent/activator is selected from one or more of EDC/NHS (N-hydroxysuccinimide ester), EDC/HOBt (1-hydroxybenzotriazole) and EDC/sulfoNHS (N-hydroxy thiosuccinimide).
Further, the osmotic pressure of the reaction mass is 150-800mOsmol/kg.
Further, the method further comprises a purification treatment before the wet heat sterilization, wherein the purification treatment is specifically to remove small molecular impurities through dialysis treatment and then to carry out homogenization treatment.
Further, the temperature of the moist heat sterilization condition is 110-130 ℃ and the sterilization time is 8-20min.
Further, the pH value of the crosslinked hydrogel is 6.0-8.0, and the osmotic pressure is 200-400mOsmol/kg.
The beneficial technical effects are as follows:
according to the invention, the carboxyl macromolecular compound is taken as a basic raw material, the hydrophobic modified chitosan derivative is taken as a cross-linking agent, the condensing agent is used for coupling the two raw materials, the concentration of the raw materials is adjusted in a saline solution so as to change the osmotic pressure of a mixed reaction material, and the raw materials are crosslinked under a variable temperature condition, so that the tightness degree of a crosslinked gel network is regulated, the obtained hydrogel is subjected to terminal damp-heat sterilization, the dynamic viscosity retention rate of the sterilized hydrogel is high (up to more than 95 percent), the thermal stability of the carboxyl macromolecular compound is obviously improved, in addition, the crosslinked hydrogel has excellent tensile ductility, the uncomfortable feeling caused by discontinuous injection can be reduced during application, the crosslinked hydrogel has good biocompatibility, is used in an aseptic form immediately, and is convenient to operate; the method is easy to control and operate, is convenient for industrial production, and the obtained crosslinked hydrogel has application prospects in the biomedical fields of ophthalmology, medical science, orthopedics, wound repair and the like.
Drawings
FIG. 1 is a graph showing the ductility of crosslinked hydrogels for each case.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and fully with reference to the accompanying drawings, in which it is evident that the embodiments described are only some, but not all embodiments of the invention. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
The numerical values set forth in these examples do not limit the scope of the present invention unless specifically stated otherwise. Techniques, methods known to those of ordinary skill in the relevant art may not be discussed in detail, but should be considered part of the specification where appropriate. In all examples shown and discussed herein, any specific values should be construed as merely illustrative, and not a limitation. Thus, other examples of the exemplary embodiments may have different values.
It should be noted that the terms "first", "second", etc. are used to define the temperature of the crosslinking reaction, and are merely for convenience in describing the temperature change in the reaction step and distinguishing the reaction temperature and the time before and after the reaction, and unless otherwise stated, the terms have no special meaning, and therefore, should not be construed as limiting the scope of the present invention.
The experimental methods in the following examples, for which specific conditions are not noted, are generally determined according to national standards; if the national standard is not corresponding, the method is carried out according to the general international standard or the standard requirements set by related enterprises.
Preparation example 1
Dispersing chitosan with deacetylation degree of 70% and weight average molecular weight of 800kDa in 50% (w/w) potassium hydroxide alkaline aqueous solution, and activating at room temperature for 24 hours; then, squeezing alkali liquor to obtain wet powder, dispersing the wet powder in a 1, 2-epoxybutane system (chitosan dry powder: 1, 2-epoxybutane: isopropanol mass ratio=1:20:2), and reacting at 35 ℃ for 48 hours under stirring; after the reaction is finished, washing for a plurality of times by ethanol to remove the 1, 2-epoxybutane; then redissolving the squeezed dry matter in acetic acid solution, adjusting the pH to 6.5-7.0 by sodium hydroxide solution, and dialyzing in pure water for 3 days to obtain hydroxybutyl chitosan solution.
The percentage of each element (C, H, O, N) in the product was measured by an elemental analyzer (Thermo Scientific Flash 2000), and then the substitution degree was calculated from the molecular formula, and the substitution degree of the hydroxybutyl chitosan of this preparation example was 1.2 by the elemental analysis method. The hydroxybutyl chitosan in this example is designated HBCS-1.
The structural formula of the hydroxybutyl chitosan is as follows: [ C 6 H 11-a-b O 4 N·(C 2 H 3 O) a ·(C 4 H 9 O) b ·cH 2 O] n . The degree of substitution was calculated according to the following formula:
wherein: a is chitosan acetyl degree; b is the number of groups substituted with hydroxybutyl, i.e. the degree of substitution; c is the water content.
Preparation example 2
Dispersing chitosan with a deacetylation degree of 93% and a weight-average molecular weight of 300kDa in 50% (w/w) potassium hydroxide alkaline aqueous solution, and activating at room temperature for 24 hours; then, squeezing alkali liquor to obtain wet powder, dispersing the wet powder in a 1, 2-epoxypentane system (chitosan dry powder: 1, 2-epoxypentane: isopropanol mass ratio=1:25:5), and reacting at 40 ℃ for 72 hours under stirring; after the reaction is finished, washing for a plurality of times by ethanol to remove the 1, 2-epoxypentane; then redissolving the squeezed dry matter in acetic acid solution, regulating the pH value to 6.5-7.0 by using sodium hydroxide solution, and dialyzing in pure water for 3 days to obtain hydroxypentyl chitosan solution.
The substitution degree of the hydroxypentyl chitosan of this preparation example was obtained by measuring the percentage of each element (C, H, O, N) in the product by an elemental analyzer (Thermo Scientific Flash 2000), and then calculating the substitution degree according to the molecular formula, and obtaining the substitution degree of 2 by an elemental analysis method. The hydroxypentyl chitosan of this example was designated HACS.
The structural formula of the hydroxypentyl chitosan is as follows: [ C 6 H 11-a-b O 4 N·(C 2 H 3 O) a ·(C 5 H 11 O) b ·cH 2 O] n . The degree of substitution was calculated according to the following formula:
wherein: a is chitosan acetyl degree; b is the number of groups substituted by hydroxypentyl, i.e. the degree of substitution; c is the water content.
Preparation example 3
Dispersing chitosan with deacetylation degree of 85% and weight average molecular weight of 100kDa in 50% (w/w) potassium hydroxide alkaline aqueous solution, and activating at room temperature for 24 hours; then, squeezing alkali liquor to obtain wet powder, dispersing the wet powder in 1, 2-epoxybutane (chitosan dry powder: 1, 2-epoxybutane: isopropanol mass ratio=1:15:3), and reacting at 29 ℃ for 18 hours under stirring; after the reaction is finished, washing for a plurality of times by ethanol to remove the 1, 2-epoxybutane; then redissolving the squeezed dry matter in acetic acid solution, adjusting the pH to 6.5-7.0 by sodium hydroxide solution, and dialyzing in pure water for 3 days to obtain hydroxybutyl chitosan solution.
The percentage of each element (C, H, O, N) in the product was measured by an elemental analyzer (Thermo Scientific Flash 2000), and then the substitution degree was calculated from the molecular formula (same as in preparation example 1), and the substitution degree of hydroxybutyl chitosan of this preparation example was 0.3 by elemental analysis. The hydroxybutyl chitosan in this example is designated HBCS-2.
Example 1
The crosslinked hydrogel of this example is a crosslinked sodium hyaluronate with HBCS-1:
dissolving sodium Hyaluronate (HA) dry powder with weight average molecular weight of 1910kDa in PBS buffer solution (0.9 wt% sodium chloride, 0.04wt% disodium hydrogen phosphate monohydrate, 0.05wt% sodium dihydrogen phosphate) with pH=6.8, adding aqueous solution of HBCS-1, sodium chloride and DMTMM after dissolving uniformly, mixing uniformly to obtain reaction material, making the final concentration of HA in the reaction material be 8.5mg/mL, making the feeding mole ratio be HA (carboxyl) to HBCS-1 (amino) to DMTMM=1:0.07:0.15, and making the osmotic pressure of the reaction material be 300mOsmol/kg;
the reaction materials are subjected to crosslinking reaction under the temperature changing condition that: the reaction materials are firstly placed at 5 ℃ to react for 24 hours, and then placed at 25 ℃ to react for 48 hours;
after the crosslinking reaction is finished, placing the gel obtained by the reaction in a dialysis bag, and dialyzing in PBS buffer solution with pH value of 7.2 for 3 days to remove small molecular impurities; the resulting gel was then homogenized using a 300 mesh plate and filled into pre-filled syringes and subjected to heat-moisture sterilization at 121 ℃ for 15min to give the product crosslinked hydrogel (product pH 7.2, osmotic pressure 300 mOsmol/kg).
Example 2
The crosslinked hydrogel of this example is a crosslinked carboxymethyl chitin with HACS:
dissolving dry carboxymethyl chitin (CH, carboxylation degree 110%) with pure water uniformly, adding HACS water solution, sodium chloride (0.47 wt%) and coupling agent EDC, and activating agent NHS, mixing uniformly to obtain reaction material, making final concentration of CH in the reaction material be 40mg/mL, making feeding mole ratio be CH (carboxyl) and HACS (amino) and EDC: NHS=1:0.015:0.05:0.07, regulating pH to 5.5, and osmotic pressure of reaction material be 150mOsmol/kg;
the reaction materials are subjected to crosslinking reaction under the temperature changing condition that: the reaction materials are firstly placed at 30 ℃ to react for 8 hours, and then placed at 15 ℃ to react for 42 hours;
after the crosslinking reaction is finished, placing the gel obtained by the reaction in a dialysis bag, and dialyzing in PBS buffer solution with pH value of 6.0 for 3 days to remove small molecule impurities; the resulting gel was then homogenized using a 300 mesh plate and filled into pre-filled syringes and subjected to wet heat sterilization at 110℃for 20min to give the product crosslinked hydrogel (product pH 6.0, osmotic pressure 200 mOsmol/kg).
Example 3
The crosslinked hydrogel of this example is a crosslinked sodium hyaluronate with HBCS-2:
dissolving sodium Hyaluronate (HA) with weight average molecular weight of 1600kDa in PBS buffer solution (0.95 wt% sodium chloride, 0.02wt% disodium hydrogen phosphate monohydrate, 0.1wt% sodium dihydrogen phosphate) with pH=7.4, adding aqueous solution of HBCS-2, potassium chloride and DMTMM after dissolving uniformly, mixing uniformly to obtain reaction material, making the final concentration of HA in the reaction material be 16mg/mL, making the feeding mole ratio be HA (carboxyl) to HBCS-2 (amino) to DMTMM=1:0.5:0.36, and making the osmotic pressure of the reaction material be 800mOsmol/kg;
the reaction materials are subjected to crosslinking reaction under the temperature changing condition that: the reaction materials are firstly placed at 50 ℃ for reaction for 2 hours, and then placed at 10 ℃ for reaction for 48 hours;
after the crosslinking reaction is finished, placing the gel obtained by the reaction in a dialysis bag, and dialyzing in PBS buffer solution with pH value of 8.0 for 3 days to remove small molecular impurities; the resulting gel was then homogenized using a 300 mesh plate and filled into pre-filled syringes and subjected to heat-moisture sterilization at 125℃for 8min to give a crosslinked hydrogel product (product pH 8.0, osmotic pressure 400 mOsmol/kg).
Comparative example 1
The crosslinked hydrogel of this comparative example was prepared in the same manner as in example 1, except that the reaction mass was allowed to react at 5℃for 72 hours.
Comparative example 2
The crosslinked hydrogel of this comparative example was prepared in the same manner as in example 1, except that the reaction mass was allowed to react at 25℃for 72 hours.
Comparative example 3
The crosslinked hydrogel of this comparative example was prepared in the same manner as in example 1, except that the osmotic pressure of the reaction mass was 1000mOsmol/kg (osmotic pressure was adjusted by adjusting the sodium chloride content therein).
Comparative example 4
The crosslinked hydrogel of this comparative example was prepared in the same manner as in example 1, except that the molar ratio of HA (carboxyl group) to HBCS-1 (amino group) to dmtmm=1:1.2:0.15 was charged.
Comparative example 5
The crosslinked hydrogel of this comparative example was prepared in the same manner as in example 1, except that HBCS-1 was replaced with water-soluble carboxymethyl chitosan.
Test example 1
This example is an evaluation of the effect of sterilization stability of the above examples and comparative examples.
The testing method comprises the following steps: the dynamic viscosity was measured using a HAAKE MARS III rheometer, wherein the plate diameter was 35mm, and the dynamic viscosity at 25 ℃, 0.25Hz, sterilization retention (%) = dynamic viscosity after sterilization/dynamic viscosity before sterilization x 100%.
TABLE 1 dynamic viscosity Change before and after Sterilization in examples
As shown in Table 1, the crosslinked hydrogel prepared by the method of examples 1-3 has a wide mechanical strength range, good sterilization stability, and a sterilization retention rate of more than 85%, and the sterilization effect on the mechanical properties of the gel is small, so that the crosslinked hydrogel is suitable for subsequent industrial production.
Comparative examples 1 and 2 show that the thermal stability (sterilization retention of 70% -80%) of the gel obtained without temperature-variable crosslinking is inferior to that of example 1. Comparative example 3 crosslinking was performed at a higher salt concentration with a sterilization retention of 48.3% and a possible reason for the higher salt concentration affecting the crosslinking reaction capacity of the condensing agent, resulting in a significant decrease in effective crosslinking. The molar concentration of HBCS-1 in comparative example 4 was high, and significant phase separation occurred after the wet heat sterilization, affecting the uniformity of the product. In comparative example 5, non-hydrophobically modified chitosan (i.e., water-soluble carboxymethyl chitosan) was used, and the sterilization stability of the gel was significantly lower than that of example 1, and the gel had a higher degree of granulation and a poorer viscosity.
Test example 2
This example is a comparison of the tensile ductility of the above examples and comparative examples.
The testing method comprises the following steps: the sterilized sample was removed from a 2.25mL prefilled syringe at a rate of 60mm/min, and the height at which the extruded gel broke was used as an indicator for evaluating its tensile ductility, with a greater value for the break height indicating a better tensile ductility. No test was performed because of phase separation after sterilization of comparative example 4.
Table 2 comparison of tensile ductility effects
| Group of | Fracture height (cm) |
| Example 1 | 13.2 |
| Example 2 | 14.5 |
| Example 3 | 9.5 |
| Comparative example 1 | 8.0 |
| Comparative example 2 | 7.0 |
| Comparative example 3 | 8.3 |
| Comparative example 5 | 3.0 |
As can be seen from table 2, the fracture heights of the samples in examples 1,2 and 3 are 13.2cm, 14.5cm and 9.5cm (see fig. 1), which are significantly higher than those of the samples in comparative example, so that the crosslinked hydrogel product of the present invention can achieve better tensile ductility while maintaining mechanical strength, reduce discomfort caused by discontinuous injection, and improve the use experience of patients.
The foregoing is only a preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art, who is within the scope of the present invention, should make equivalent substitutions or modifications according to the technical scheme of the present invention and the inventive concept thereof, and should be covered by the scope of the present invention.
Claims (9)
1. A method for preparing a crosslinked hydrogel, comprising the steps of:
uniformly mixing a carboxyl high molecular compound, a hydrophobically modified chitosan derivative and a condensing agent in a salt solution to obtain a reaction material, carrying out a crosslinking reaction on the reaction material under a variable temperature condition, and carrying out damp-heat sterilization after the crosslinking reaction is finished to obtain crosslinked hydrogel;
the ratio of the mol number of carboxyl in the carboxyl high molecular compound to the mol number of amino in the hydrophobically modified chitosan derivative to the mol number of the condensing agent is 1:0.015-0.5:0.05-0.4;
the temperature change condition is that the reaction is continued at a second crosslinking temperature after the reaction at the first crosslinking temperature, so that the first crosslinking temperature is T1, the second crosslinking temperature is T2, and the temperature difference between the first crosslinking temperature and the second crosslinking temperature is T, and then T= |T1-T2| and T is more than 5 are satisfied.
2. The method for producing a crosslinked hydrogel according to claim 1, wherein the first crosslinking temperature is 0 to 15 ℃, the reaction time is 12 to 30 hours, and the second crosslinking temperature is 16 to 50 ℃, the reaction time is 24 to 72 hours;
or the first crosslinking temperature is 16-50 ℃, the reaction time is 1-24 h, and the second crosslinking temperature is 0-15 ℃ and the reaction time is 24-72 h.
3. The method for preparing a crosslinked hydrogel according to claim 1, wherein the carboxylic polymer is one or more selected from the group consisting of sodium hyaluronate, carboxymethyl chitosan, polyglutamic acid, carboxymethyl chitin, N-succinyl chitosan, sodium alginate, chondroitin sulfate, silk fibroin, succinyl silk fibroin, gelatin, and succinyl gelatin.
4. The method for preparing a crosslinked hydrogel according to claim 1, wherein the hydrophobically modified chitosan derivative is a product of substitution reaction of chitosan and an epoxy compound, and the total substitution degree of hydroxyl groups and amino groups in the chitosan molecular structure is 0.3-2; the epoxy compound is selected from 1, 2-epoxybutane and/or 1, 2-epoxypentane.
5. The method for producing a crosslinked hydrogel according to claim 1, wherein the salt solution contains one or more of a chloride salt, a hydrogen phosphate dibasic salt and a dihydrogen phosphate monobasic salt.
The condensing agent comprises a coupling agent used alone or in combination with a coupling agent/activating agent; the coupling agent is selected from EDC or DMTMM; the coupling agent/activator is one or more selected from EDC/NHS, EDC/HOBt and EDC/sulfoNHS.
6. The method for producing a crosslinked hydrogel according to claim 1, wherein the osmotic pressure of the reaction mass is 150-800mOsmol/kg.
7. The method according to claim 1, further comprising a purification treatment, in particular, a dialysis treatment to remove impurities and a homogenization treatment, before the wet heat sterilization.
8. The method for producing a crosslinked hydrogel according to claim 1, wherein the conditions of wet heat sterilization are at a temperature of 110 to 130 ℃ and a sterilization time of 8 to 20min.
9. The method for producing a crosslinked hydrogel according to any one of claims 1 to 8, wherein the crosslinked hydrogel has a pH of 6.0 to 8.0 and an osmotic pressure of 200 to 400mOsmol/kg.
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