CN103724455B - A kind of preparation method of derivatives of hyaluronic acids and hydrogel thereof - Google Patents
A kind of preparation method of derivatives of hyaluronic acids and hydrogel thereof Download PDFInfo
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Abstract
The invention discloses a kind of derivatives of hyaluronic acids and preparation method thereof, described method by hyaluronic acid or its salt and anhydride hybrid reaction in anhydrous solvent, product through precipitation, separate, obtain the derivatives of hyaluronic acids with cross linkable after purification.The method preparation process is simple and the extent of reaction can be passed through to control response parameter and realize, it is thus achieved that product property stable, can preserve for a long time.The derivatives of hyaluronic acids with cross linkable of gained can prepare hyaluronic acid gel in a mild condition, has good biocompatibility, can be used for bioactive substance carrier material, tissue engineering bracket material and soft tissue filling material.
Description
Technical field
The preparation method that the present invention relates to a kind of derivatives of hyaluronic acids and hydrogel thereof, belongs to the preparation field of biomedical material.
Technical background
Hyaluronic acid is due to unique composition and construction features so that it is have splendid water retention property, simultaneously because have concurrently good biocompatibility, biodegradable and receive much concern in field of tissue engineering technology, also embody higher using value simultaneously.In hyaluronic research and application, generally it is required for it being carried out chemical modification thus realizing in hyaluronan molecule or intermolecular cross-linking, or with other raw material with covalently cross-linked formation composite, to obtain the biomedical material with more preferably mechanical property, biological degradability, the performance such as shapeable and functional.
In existing report, the chemical modification method introducing unsaturated bond in hyaluronan molecule has two classes: one is in aqueous phase system, under cryogenic, dripping the continuous regulation system pH value of methacrylic acid anhydride solution in hyaluronic acid solution is alkalescence, thus obtaining the derivatives of hyaluronic acids of grafting methacrylic acid；Another kind is in anhydrous solvent, hyaluronic acid is made to react with unsaturated acyl chlorides (acryloyl chloride or methacrylic chloride), thus obtaining the derivatives of hyaluronic acids of grafting acrylic or methacrylic acid, for instance: Chinese invention patent, publication number: CN103113495A.
The present invention, with hyaluronic acid or its salt and unsaturated acid anhydride for raw material, reacts in anhydrous solvent, and preparation has the derivatives of hyaluronic acids of cross linkable, overcomes the deficiency of existing method and technology；Ultraviolet radiation or peroxide initiator initiated polymerization can be adopted further, prepare single hyaluronic acid gel or form multiple elements design hydrogel material with other materials containing active sulfydryl.Up to the present, there is no relevant documents and materials report.
Summary of the invention
Inventor is by finding to have the disadvantage that in prior art to comprehensive analysis of prior art
1. when adopting aqueous phase system to react, preparation process is loaded down with trivial details, it is necessary to constantly regulates the pH value in reactant liquor, easily causes the fluctuation of reaction condition and affect the extent of reaction；Simultaneously for the derivatives of hyaluronic acids that the bigger and modified degree of molecular weight is higher, particularly after preserving after a while, its water solublity substantially reduces, even can not be water-soluble；
2. adopting in anhydrous solvent, the method for hyaluronic acid and unsaturated acyl chloride reaction, the reactivity of unsaturated acyl chlorides is high, and the extent of reaction is wayward；Simultaneously as product is identical with the product structure of aqueous phase system, the shortcoming equally also not easily with preservation for a long time, water solublity reduction.
Present invention aims to the deficiencies in the prior art, the preparation method that a kind of derivatives of hyaluronic acids obtaining cross linkable and hydrogel thereof are provided, preparation process is simple and the extent of reaction can be passed through to control response parameter and realize, it is thus achieved that product property stable, can preserve for a long time.
The present invention is achieved through the following technical solutions:
A kind of preparation method of derivatives of hyaluronic acids, by hyaluronic acid or its salt and anhydride hybrid reaction in anhydrous solvent, product through precipitation, separate, obtain the derivatives of hyaluronic acids with cross linkable after purification.
Alternately, anhydrous solvent described in said method is at least one in Methanamide, DMF, oxolane.Hyaluronic acid or its salt being fully dissolved with in this anhydrous solvent is beneficial to the success of modified-reaction and carries out.
Alternately, the anhydride described in said method can be the one in maleic anhydride (also known as maleic anhydride), citraconic anhydride, cis-3-carboxyl glutaconic anhydride (cis-aconitic anhydride), 4-amylene acid anhydride, crotonic anhydride, methacrylic anhydride.Preferential selection ring-type or carboxylic unsaturated acid anhydride, the unsaturated terminal chain group with carboxyl can be introduced on hyaluronic acid or its salinity subchain with ester bond form, do not affect the dissolubility of derivatives of hyaluronic acids, be one of principal character being different from other derivatives of hyaluronic acids.Specifically, when selecting maleic anhydride, citraconic anhydride, cis-3-carboxyl glutaconic anhydride, can with the one in A class group below ester bond form grafting；When selecting 4-amylene acid anhydride, crotonic anhydride, methacrylic anhydride, can with the one in B class group below ester bond form grafting.
A class:、、、、
B class:、、
Wherein, in hyaluronan molecule, introduce the derivatives of hyaluronic acids that A class group obtains, not only make it have cross linkable, and its water solublity will not be impacted, even can be more beneficial for dissolving.
Alternately, said method specifically includes following process:
1) joining in anhydrous solvent by hyaluronic acid or its salt, stirring is to fully dissolving；
2) anhydride is dissolved in identical anhydrous solvent, joins under stirring condition in step 1) gained solution；
3) to step 2) reactant liquor of gained adds molal quantity account for the catalyst that can react hydroxyl moles 0-20% on hyaluronic acid;
4), after reacting 3-30h at 30-100 DEG C, it is added drop-wise in the cryoprecipitate agent in stirring after the cooling of question response liquid, is sufficiently stirred for and makes product precipitate out；
5) separate；
6) purification, it is thus achieved that there is the derivatives of hyaluronic acids of cross linkable.
Alternately, said method step 1) carries out at 30-100 DEG C, and the concentration of hyaluronic acid or its salt is 1-20mg/ml.
Alternately, said method step 2) described in the molal quantity of anhydride be 0.5-50 times that hyaluronic acid can react hydroxyl moles.The mol ratio of raw material is to affect one of principal element of unsaturated terminal chain percent grafting on derivatives of hyaluronic acids product molecule.
Alternately, the catalyst described in said method step 3) is the one in pyridine, PA, 4-(dimethylamino) pyridine (DMAP).
Alternately, separating step described in said method step 5) particularly as follows: by the solid-liquid mixed liquor high speed centrifugation of gained in step 4), will be deposited in precipitant to disperse, centrifugal, and repeat this step 2-4 time.It is centrifuged by repeatedly disperseing, removes Residual reactants and partial impurities.
Alternately, purification step described in said method step 6) is particularly as follows: after being substantially soluble in deionized water by the precipitation of above-mentioned acquisition, regulate pH value of solution to neutral, this solution is transferred to dialysis band again, fully dialyse with deionized water postlyophilization, it is thus achieved that there is the derivatives of hyaluronic acids of cross linkable.By the soluble small molecular impurity removed in product of dialysing.
Alternately, described hyaluronic acid or its salt can be the Natural hyaluronic acid or its salt that extract in animal tissue, it is also possible to be the hyaluronic acid or its salt that adopt microbial fermentation technology to produce.Described hyaluronic acid or its salt can be hyaluronate sodium, potassium hyaluronate, hyaluronic acid ammonium, hyaluronic acid magnesium, calcium hyauronate or other there is the one in the hyaluronic acid inorganic salt of good biocompatibility, it is preferable that hyaluronic acid or hyaluronate sodium.
Alternately, described hyaluronic acid or the molecular weight of its salt are at 30kDa-3000kDa, it is preferable that molecular weight is at 1000kDa-2000kDa.
Alternately, described precipitant can be the one in ethanol, methanol, isopropanol, acetone.
Alternately, described dialysis time is usually 1-5d, and period, every 4-8h changed a dialysis solution
Present invention also offers a kind of derivatives of hyaluronic acids, described derivatives of hyaluronic acids adopts said method to be prepared from.
The preparation method that present invention also offers a kind of hydrogel, the method is that derivatives of hyaluronic acids of the present invention is dissolved in physiological water medium, through peroxide initiator cause or when light trigger exists through ultraviolet radiation cause self-polymerization reaction or with other copolyreaction containing the material of active mercapto groups, it is thus achieved that the hyaluronic acid gel of single component or the hyaluronic acid multiple elements design hydrogel with other material covalent cross-linking containing active mercapto groups.
Alternately, in the preparation method of above-mentioned hydrogel, it is 10-100mg/mL that described derivatives of hyaluronic acids is dissolved in the derivatives of hyaluronic acids solution concentration of physiological water medium gained.
Alternately, in the preparation method of above-mentioned hydrogel, described physiological water medium can be the one in deionized water, normal saline, phosphate buffered saline (PBS) or other isosmotic solution.
Alternately, in the preparation method of above-mentioned hydrogel, described light trigger can be 2-hydroxyl-4 '-(hydroxy ethoxy)-2-methyl phenyl ketone (I2959), 1-hydroxycyclohexylphenyl acetone (I184), one in 2,2-dimethoxy-phenylf 1-Phenylethanone .s (I651).
Alternately, in the preparation method of above-mentioned hydrogel, described photoinitiator concentration is 0.08-1.5wt%.
Alternately, in the preparation method of above-mentioned hydrogel, described ultraviolet radiation is employing wavelength is 320-480nm, and light intensity is 10-50mW/cm2Ultraviolet radiation 15-900s.
Alternately, in the preparation method of above-mentioned hydrogel, described peroxide initiator can be the one in Ammonium persulfate., hydrogen peroxide, potassium peroxydisulfate, sodium peroxydisulfate.
Alternately, in the preparation method of above-mentioned hydrogel, described peroxide initiator concentration is 0.1-2.0wt%.
Alternately, in the preparation method of above-mentioned hydrogel, the described material containing active mercapto groups is cysteamine or its salt, cysteine or its ester, dithiothreitol dithio etc. containing at least one in the small molecule material of active sulfydryl or by least one in the natural macromolecular materials such as the modified chondroitin sulfate of the above-mentioned small molecule material containing active sulfydryl, dermatan sulfate, chitosan, chitin, heparin, alginic acid or its salt.
Alternately, in the preparation method of above-mentioned hydrogel, in described hyaluronic acid multiple elements design hydrogel, derivatives of hyaluronic acids and the described weight ratio containing active mercapto groups material are 100:1-1:100.
Present invention also offers a kind of hydrogel, described hydrogel is adopted and is prepared from the aforedescribed process.Described hydrogel under proper condition can plastic rapidly, it is adaptable to in-situ injection molding, and plastic mechanical property is better, has the advantages such as more excellent potential source biomolecule performance concurrently simultaneously.
Present invention also offers the application of a kind of above-mentioned derivatives of hyaluronic acids and hydrogel thereof: use it for the initiation material of soft tissue filling material or for bioactive substances such as packaging medicine, somatomedin, enzyme, carrier as transmission or slow release or the timbering material as organizational project, can be used for the reparation of tissue and reconstruction after embedding various adult cell, stem cell.
All features disclosed in this specification, or the step in disclosed all methods or process, except mutually exclusive feature and/or step, all can combine by any way.
Beneficial effects of the present invention:
1, the derivatives of hyaluronic acids preparation method with cross linkable of the present invention is simple to operate, and modified-reaction degree is controlled, and adopts derivatives of hyaluronic acids prepared by optimum condition to overcome existing method to prepare the defect of like product.The product prepared by the method can keep good water solublity, stable performance, it is easy to preserve for a long time for a long time.
2, the derivatives of hyaluronic acids with cross linkable of the present invention can prepare hyaluronic acid gel in a mild condition, has good biocompatibility, can be used for bioactive substance carrier material, tissue engineering bracket material and soft tissue filling material.
Accompanying drawing explanation
Fig. 1 be in the embodiment of the present invention 1 preparation hyaluronic acid-butene dioic acid ester nuclear magnetic resonance, NMR (1HNMR) collection of illustrative plates；
Fig. 2 is dynamic mechanical analysis (DMA) result of the hyaluronic acid-butene dioic acid ester hydrogel of preparation in example 3 of the present invention；
Fig. 3 be embodiment 7 adopts tradition hyaluronic acid-methacrylate of preparing of aqueous phase system nuclear magnetic resonance, NMR (1HNMR) collection of illustrative plates；
Fig. 4 is the process chart of the preparation method of derivatives of hyaluronic acids of the present invention, and in figure, dotted portion is optional step；
Specific implementation method:
The preparation technology flow process of the present invention is (as shown in Figure 4): the dissolving of hyaluronic acid or its salt, the interpolation of anhydride solution and catalyst, stirring reaction postprecipitation in a heated condition, by centrifugation, clean after dissolve, adjust pH fully dialysing, lyophilization acquisition derivatives of hyaluronic acids.
Wherein, hyaluronic acid or its salt to be dissolved in anhydrous solvent, and add anhydride solution and catalyst under agitation；After reacting the regular hour under certain temperature conditions, question response liquid cools down and is added drop-wise in cryoprecipitate agent and makes it fully precipitate out, and repeatedly cleans, is centrifuged；The precipitation of acquisition is dissolved in deionized water and regulates pH to neutrality, deionized water is fully dialysed, then lyophilization obtains the derivatives of hyaluronic acids with cross linkable.
Several most preferred embodiments for the present invention set forth below, it should be understood that these embodiments only for illustration purposes, are never limited in protection scope of the present invention.
Embodiment 1
Prepare hyaluronic acid-butene dioic acid ester derivant according to the following steps:
(1) weighing 0.2g hyaluronic acid (about 0.527mmol, the molecular weight of its construction unit is 379.32g/mol, as follows) and join in 100mL dry formamide, at 50 DEG C, stirring is to fully dissolving；
(2) weighing 0.517g maleic anhydride (about 5.27mmol, molecular weight is 98.06g/mol) and be dissolved in dry formamide, join above-mentioned (1) solution under stirring condition, the molal quantity of maleic anhydride is 10 times that can react hydroxyl moles on hyaluronic acid；
(3) without catalyst；
(4) at 50 DEG C, react 5h, after question response liquid is cooled to room temperature, in the cold dehydrated alcohol of 500mL being added drop-wise in stirring, make product precipitate out；
(5) by above-mentioned solid-liquid mixed liquor high speed centrifugation, will be deposited in dehydrated alcohol to disperse, centrifugal, and this step 2-4 time repeatedly；
(6) precipitation of above-mentioned acquisition is substantially soluble in deionized water, regulates pH value of solution to neutral, then this solution is transferred to dialysis band, postlyophilization that deionized water is fully dialysed, it is thus achieved that have the hyaluronic acid-butene dioic acid ester derivant of cross linkable.
Adopt1Hyaluronic acid-butene dioic acid the ester derivant of gained is detected by HNMR, and result is as shown in Figure 1.Contrast unmodified hyaluronic acid nuclear magnetic spectrum it appeared that the chemical shift of unsaturated bond both sides hydrogen in butene dioic acid ester is 6.0ppm and 6.7ppm, by showing that with the area integral ratio calculation of hyaluronan molecule methyl hydrogen the percent grafting of this hyaluronic acid-butene dioic acid ester derivant is about 27%.
Embodiment 2
Prepare hyaluronic acid-4-pentenoate derivant according to the following steps:
(1) weighing 0.4g hyaluronic acid (about 1.06mmol) and join in 100mL anhydrous tetrahydro furan, at 60 DEG C, stirring is to fully dissolving；
(2) weighing 0.961g4-amylene acid anhydride (about 5.27mmol, molecular weight is 182.22g/mol) and be dissolved in anhydrous tetrahydro furan, join above-mentioned (1) solution under stirring condition, the molal quantity of 4-amylene acid anhydride is 5 times that can react hydroxyl moles on hyaluronic acid；
(3) in above-mentioned reactant liquor, 0.1gDMAP catalyst is added；
(4) at 60 DEG C, react 10h, after question response liquid is cooled to room temperature, in the cold anhydrous isopropyl alcohol of 800mL being added drop-wise in stirring, make product precipitate out；
(5) by above-mentioned solid-liquid mixed liquor high speed centrifugation, will be deposited in anhydrous isopropyl alcohol to disperse, centrifugal, and this step 2-4 time repeatedly；
(6) precipitation of above-mentioned acquisition is substantially soluble in deionized water, regulates pH value of solution to neutral, then this solution is transferred to dialysis band, postlyophilization that deionized water is fully dialysed, it is thus achieved that have the hyaluronic acid-4-pentenoate derivant of cross linkable.
Embodiment 3
Prepare the hyaluronic acid-butene dioic acid ester hydrogel of single component according to the following steps:
Seal the hyaluronic acid-butene dioic acid ester derivant of preservation after weighing 20mg lyophilization, be dissolved in 1.0mL and contain 0.1%2-hydroxyl-4 ' aqueous solution of-(hydroxy ethoxy)-2-methyl phenyl ketone (I2959) initiator；Take appropriate volume by this solution after particular mold, be 365nm, light intensity at wavelength be 30mW/cm2Ultraviolet radiation 120s initiated polymerization, formed hyaluronic acid gel.
Single component hyaluronic acid-butene dioic acid ester the hydrogel adopting DMA gained detects, and dynamic force frequency is 1.0Hz and 10Hz, and testing result is as shown in Figure 2.It can be seen that the storage modulus average of this hydrogel is more than 0.2MPa.
Embodiment 4
Prepare the hyaluronic acid-4-pentenoate hydrogel of single component according to the following steps:
Seal the hyaluronic acid-4-pentenoate derivant of preservation after weighing 40mg lyophilization, be dissolved in 1.0mL deionized water；Now join 10% ammonium persulfate solution, take 10mL before use and join above-mentioned derivatives of hyaluronic acids solution, take appropriate volume after being sufficiently mixed and in particular mold, stand reaction to be polymerized complete, form hyaluronic acid gel.
Embodiment 5
Prepare the hyaluronic acid-chondroitin sulfate hydrogel of binary compound according to the following steps:
Seal the hyaluronic acid-butene dioic acid ester derivant of preservation after weighing 20mg lyophilization, be dissolved in 1.0mL and contain 0.2%2-hydroxyl-4 ' aqueous solution of-(hydroxy ethoxy)-2-methyl phenyl ketone (I2959) initiator；Weigh the 20mg chondroitin sulfate derivatives (as adopted EDC/NHS reaction system, the chondroitin sulfate derivatives obtained after being reacted with Mercaptamine by chondroitin sulfate) containing active sulfydryl, be dissolved in 1.0mL deionized water；Take appropriate above two solution and be placed on particular mold by different volumes than after mixing, be 365nm, light intensity at wavelength be 30mW/cm2Ultraviolet radiation 60s initiated polymerization, formed hyaluronic acid-chondroitin sulfate binary composite aquogel.
Embodiment 6 dissolubility contrast experiment
Adopt derivatives of hyaluronic acids preparation method of the present invention to prepare respectively hyaluronic acid-butene dioic acid ester derivant (being designated as sample 1) that percent grafting is about 50% and hyaluronic acid-methacrylate derivative (being designated as sample 2) that percent grafting is about 50%, hyaluronic acid-the methacrylate derivative (being designated as sample 3) adopting percent grafting prepared by traditional water solution system to be about 50%, the method adopting hyaluronic acid and unsaturated acyl chloride reaction prepares hyaluronic acid-methacrylate derivative (being designated as sample 4) that percent grafting is about 50%, take above-mentioned four kinds of samples and carry out dissolubility contrast:
Cryodesiccated hyaluronic acid-butene dioic acid ester derivant under agitation, soluble in water formed 20mg/ml transparent uniform solution；Long-term preservation does not affect its dissolubility；
Hyaluronic acid-methacrylate derivative (sample 2-4) that after lyophilization, short-term (2-3d) preserves, under agitation, the solid calculated according to 20mg/ml concentration is difficult to the transparent uniform solution of completely soluble formation；Preserve one week or after the longer time, its dissolubility reduces further.Wherein the dissolubility of sample 2 and holding time are all slightly better than sample 3 and sample 4.
Higher for molecular weight and that percent grafting is higher derivatives of hyaluronic acids, adopts the phenomenon that product water dissolubility prepared by traditional method declines to become apparent from.
Embodiment 7 percent grafting contrast experiment
Several factors that the percent grafting that method of the present invention prepares derivatives of hyaluronic acids is limited mainly by following raw material and process are adopted to affect: the water content of anhydrous solvent, anhydride and hyaluronic acid can react the molar ratio of hydroxyl, reaction temperature and response time etc..Adopt and add desiccant or use the method for molecular sieve can obtain dry anhydrous solvent；Hyaluronic acid raw material for different molecular weight, its initial action concentration can control within the scope of 0.2-2mg/mL, anhydride and hyaluronic mol ratio can be controlled in 1.0-15 times, react 4-6h and can obtain the derivatives of hyaluronic acids that percent grafting is 5-70% under 40-70 DEG C of condition.
The percent grafting that tradition aqueous phase system prepares derivatives of hyaluronic acids is adopted to be limited mainly by the impact of following factor: the raw material ratio of reaction, reaction temperature, response time and course of reaction need to regulate the pH value etc. of solution system always.Owing to anhydride is prone to hydrolysis, therefore adopt and need during aqueous phase system reaction use large excess of anhydride starting material and control relatively low reaction temperature continuous stirring reaction more than 12h, simultaneously need to constantly regulate pH value between 8-9 to promote that reaction carries out；This preparation method consumption excess raw material, course of reaction are wayward, so that the percent grafting of derivatives of hyaluronic acids easily fluctuates.The methacrylic anhydride adopting mol ratio to be 15 times and hyaluronic acid successive reaction 24h in 4 DEG C of aqueous phase systems, the percent grafting of the derivatives of hyaluronic acids obtained when comparatively ideal control reaction pH is about 20%.Methacrylic anhydride and the hyaluronic acid successive reaction 6h in 4 DEG C of aqueous phase systems adopting mol ratio to be 10 times, it is thus achieved that product carry out1HNMR analyzes, and result is as it is shown on figure 3, its percent grafting is about 5.04%.
Being found by contrast experiment, when the factors such as the raw material ratio of reaction, reaction temperature, response time are identical, the method for the invention is compared with tradition Aqueous phase, and percent grafting is considerably higher, and reaction is easier to control.
Embodiment 8
In embodiment 1 and embodiment 2, adopt hyaluronate (as hyaluronate sodium, potassium hyaluronate, hyaluronic acid ammonium, hyaluronic acid magnesium, calcium hyauronate or other there is the hyaluronic acid inorganic salt of good biocompatibility) replace hyaluronic acid to be successfully prepared derivatives of hyaluronic acids equally, adopt commercially available hyaluronic acid or its salt or adopt the Natural hyaluronic acid or its salt that extract in animal tissue, also or adopt hyaluronic acid that microbial fermentation technology produces or its salt all can successfully prepare derivatives of hyaluronic acids of the present invention.Molecular weight hyaluronic acid within the scope of 30kDa-3000kDa or its salt all can successfully react.
In embodiment 1 and embodiment 2, DMF is adopted also to be successfully prepared derivatives of hyaluronic acids of the present invention as anhydrous solvent.
In embodiment 1 and embodiment 2, it is respectively adopted citraconic anhydride, cis-3-carboxyl glutaconic anhydride (cis-aconitic anhydride), 4-amylene acid anhydride, crotonic anhydride as anhydride starting material, is successfully prepared corresponding derivatives of hyaluronic acids.Products therefrom passes through1HNMR is analyzed to identify errorless.
Temperature described in the step (1) of embodiment 2 is 30 DEG C or 100 DEG C, and gained hyaluronic acid concentration is 1mg/ml or 20mg/ml；In step (2), the molal quantity of anhydride is 0.5 or 50 times that can react hydroxyl moles on hyaluronic acid；Step (3) adopt pyridine or PA as catalyst, catalyst molal quantity accounts for the ratio that can react hydroxyl moles on hyaluronic acid 10% or 20%, step (4) and (5) adopt methanol or acetone as precipitant, in step (4), reaction temperature is 30 DEG C or 100 DEG C, and the response time is 3h or 30h；All it is successfully prepared derivatives of hyaluronic acids of the present invention.
Embodiment 9
Embodiment 3 and embodiment 5 adopt various derivatives of hyaluronic acids of the present invention be all successfully prepared for hyaluronic acid gel or the hyaluronic acid multiple elements design hydrogel of corresponding single component.
Described aqueous solution is changed into normal saline or phosphate buffered saline (PBS) by embodiment 5；Described light trigger is changed into 1-hydroxycyclohexylphenyl acetone (I184) or 2,2-dimethoxy-phenylf 1-Phenylethanone. (I651)；Making described derivatives of hyaluronic acids solution concentration is 10mg/mL or 100mg/mL, and described light trigger mass percentage concentration in reaction system is 0.08-1.5wt%；The wavelength adopted during the radiation of described ultraviolet is 320nm or 480nm, and light intensity is 10mW/cm2Or 50mW/cm2Ultraviolet light, radiated time is 15 seconds or 900 seconds；Chondroitin sulfate derivatives is changed into cysteamine or its salt, cysteine or its ester, dithiothreitol dithio or passes through at least one in the modified dermatan sulfate of the small molecule material containing active sulfydryl, chitosan, chitin, heparin, alginic acid or its salt, successfully prepare a series of hyaluronic acid and the material covalent cross-linking binary containing active mercapto groups, the even more polynary composite aquogel of ternary, gained composite aquogel1HNMR is analyzed to identify.
Embodiment 10
Take a kind of derivatives of hyaluronic acids of preparation in embodiment of the present invention and be dissolved in deionized water, normal saline, phosphate buffered saline (PBS) or other isosmotic solution, be made into 10mg/mL or 100mg/mL solution；Add hydrogen peroxide, Ammonium persulfate., potassium peroxydisulfate or sodium peroxydisulfate as peroxide initiator, making described peroxide initiator mass percentage concentration in reaction system is 0.1%-2.0%, take appropriate volume after being sufficiently mixed in particular mold, to stand reaction to be polymerized complete, form the hyaluronic acid gel of single component.
In above-mentioned steps, before adding peroxide initiator, add cysteamine or its salt, cysteine or its ester, dithiothreitol dithio or pass through at least one in the natural macromolecular materials such as the modified chondroitin sulfate of the small molecule material containing active sulfydryl, dermatan sulfate, chitosan, chitin, heparin, alginic acid or its salt, successfully prepare a series of hyaluronic acid and the material covalent cross-linking binary containing active mercapto groups, the even more polynary composite aquogel of ternary, gained composite aquogel1HNMR is analyzed to identify.
The foregoing is only the preferred embodiments of the present invention, be merely illustrative for the purpose of the present invention, and nonrestrictive；Those of ordinary skill in the art understand, and it can be carried out many changes in the spirit and scope that the present invention limits, amendment, and even equivalence is changed, but falls within protection scope of the present invention.
Claims (12)
1. the preparation method of a derivatives of hyaluronic acids, it is characterized in that, by hyaluronic acid or its salt and anhydride hybrid reaction in anhydrous solvent, product is through precipitation, separate, the derivatives of hyaluronic acids with cross linkable is obtained after purification, described hyaluronate is hyaluronate sodium, potassium hyaluronate, hyaluronic acid ammonium, hyaluronic acid magnesium, calcium hyauronate or other there is the one in the hyaluronic acid inorganic salt of good biocompatibility, described anhydride is ring-type unsaturated acid anhydride or carboxylic unsaturated acid anhydride or 4-amylene acid anhydride, crotonic anhydride, one in methacrylic anhydride.
2. the preparation method of derivatives of hyaluronic acids according to claim 1, it is characterised in that described anhydrous solvent is at least one in Methanamide, DMF, oxolane.
3. the preparation method of derivatives of hyaluronic acids according to claim 1, it is characterized in that, described anhydride is the one in maleic anhydride (also known as maleic anhydride), citraconic anhydride, cis-3-carboxyl glutaconic anhydride (cis-aconitic anhydride), 4-amylene acid anhydride, crotonic anhydride, methacrylic anhydride.
4. the preparation method of derivatives of hyaluronic acids according to claim 1, it is characterised in that specifically include following process:
1) hyaluronic acid or its salt are joined in anhydrous solvent, and stirring is dissolved to abundant at 30-100 DEG C；
2) being dissolved in by anhydride in identical anhydrous solvent, join in step 1) gained solution under stirring condition, the molal quantity of anhydride is 0.5-50 times that can react hydroxyl moles on hyaluronic acid；
3) to step 2) reactant liquor of gained adds molal quantity account for the catalyst that can react hydroxyl moles 0-20% on hyaluronic acid, described catalyst is the one in pyridine, PA, 4-(dimethylamino) pyridine (DMAP)；
4) after reacting 3-30h at 30-100 DEG C, being added drop-wise in the cryoprecipitate agent in stirring, be sufficiently stirred for and make product precipitate out after the cooling of question response liquid, described precipitant is the one in ethanol, methanol, isopropanol, acetone；
5) by the solid-liquid mixed liquor high speed centrifugation of gained in step 4), will be deposited in precipitant to disperse, centrifugal, and repeat this step 2-4 time；
6), after the precipitation of above-mentioned acquisition being substantially soluble in deionized water, regulate pH value of solution to neutral, then this solution is transferred to dialysis band, postlyophilization of fully dialysing with deionized water, it is thus achieved that have the derivatives of hyaluronic acids of cross linkable.
5. a derivatives of hyaluronic acids, it is characterised in that described derivatives of hyaluronic acids adopts any one of method in claim 1-4 to be prepared from, and introduces the unsaturated terminal chain group with carboxyl with ester bond form on hyaluronic acid or its salinity subchain.
6. the preparation method of a hydrogel, it is characterized in that, derivatives of hyaluronic acids described in claim 5 is dissolved in physiological water medium, through peroxide initiator cause or when light trigger exists through ultraviolet radiation cause self-polymerization reaction or with other copolyreaction containing the material of active mercapto groups, it is thus achieved that the hyaluronic acid gel of single component or the hyaluronic acid multiple elements design hydrogel with other material covalent cross-linking containing active mercapto groups.
7. the preparation method of hydrogel according to claim 6, it is characterised in that it is 10-100mg/mL that described derivatives of hyaluronic acids is dissolved in the derivatives of hyaluronic acids solution concentration of physiological water medium gained.
8. the preparation method of hydrogel according to claim 6, it is characterized in that, the described material containing active mercapto groups is the small molecule material containing active sulfydryl or by least one in the modified chondroitin sulfate of the above-mentioned small molecule material containing active sulfydryl, dermatan sulfate, chitosan, chitin, heparin, alginic acid or its salt.
9. the preparation method of hydrogel according to claim 8, it is characterised in that the described small molecule material containing active sulfydryl is at least one in cysteamine or its salt, cysteine or its ester, dithiothreitol dithio.
10. a hydrogel, it is characterised in that described hydrogel adopts the method described in claim 6 to be prepared from.
11. the application of a hydrogel according to claim 10, it is characterized in that, use it for the initiation material of soft tissue filling material or for wrapping biological active matter, carrier as transmission or slow release or the timbering material as organizational project, for the reparation organized and reconstruction after embedding various adult cell, stem cell.
12. the application of a hydrogel according to claim 11, it is characterised in that described bioactive substance is medicine or somatomedin or enzyme.
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