CN104892962A - Preparation method and application of sulfhydryl/disulfide bond controllable self-crosslinked hyaluronic acid hydrogel - Google Patents
Preparation method and application of sulfhydryl/disulfide bond controllable self-crosslinked hyaluronic acid hydrogel Download PDFInfo
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Abstract
The invention discloses a preparation method and an application of sulfhydryl/disulfide bond controllable self-crosslinked hyaluronic acid hydrogel and belongs to the field of biological materials. The hydrogel is prepared from a sulfhydryl-hyaluronic acid compound with free sulfhydryl as a raw material; by adjusting the sulfhydryl density (10-60%), the gel temperature (4-37 DEG C) and the molecular weight (0.1M, 0.3M and 1M) of hyaluronic acid, and using the oxidation-reduction transformation characteristics between sulfhydryl and disulfide bonds to control the forming time, degradation time and the mechanical properties of the gel, a controllable and injectable type intelligent hydrogel with a good three-dimensional network crosslinked structure is constructed. Meanwhile, the hydrogel of the type is single in component, avoids the introduction of exogenous toxic substances before and after gel forming, and has good biocompatibility and degradation performance. Based on the research discovery, the controllable self-crosslinked high-polymer polymer can be applied to minimally invasive repairing of tissue engineering in-situ damages and construction of an intelligent adjustable three-dimensional cell culture scaffold.
Description
Technical field
The invention belongs to technical field of biological material, be specifically related to controlled self-crosslinking hyaluronic acid gel of a kind of sulfydryl/disulfide linkage and its preparation method and application.
Technical background
Organizational engineering (tissue engineering), also someone is called " regenerative medicine ", refers to and utilizes biologically active substance, by the method for vitro culture or structure, reproduces or repair the technology of organ and tissue.This Objective Concept national science foundation of the US council proposed in 1987, within 1988, by its formal definition be: the philosophy and technique of application life science and engineering science, on weave construction under the mammiferous normal and pathology two states of correct understanding and emic basis, research and develop a new branch of science of the biosubstitute for repairing, safeguarding, promote the function and morphology after the various tissue of human body or organ damage.
Bio-medical material (Biomedical Materials), also can referred to as biomaterial (Biomaterials), is for diagnosing, treating, repair or replace pathological tissues, organ to organism or promote the novel High-tech Material of its function.And one of gordian technique of organizational engineering has good biocompatibility and can by the cytoskeleton of human body degraded and absorbed with biomaterial preparation.Gel state is the intermediateness of solid and liquid, and hydrogel refers to and hydrophilic crosslinked Space network of polymer that is swelling and that can keep large quantity of moisture and don't can dissolve can occur in water.Hydrogel is the desirable biomaterial of a class, by simple modification, similar with natural extracellular matrix, gratifying physics and chemistry character can be obtained, good permeability is shown for oxygen, nutritive substance, cell metabolite and water-soluble metal ion simultaneously.The hydrophilic macromolecule preparing hydrogel is by sources divided into natural polymer and synthesis polymer.Natural hydrogel comprises collagen, gelatin, scleroproein, polysaccharide etc., and synthetic hydrogel comprises synthesis class polypeptide, PEG and derivative, PMMA and derivative thereof, PLGA and derivative thereof etc.
Hyaluronic acid (Hyaluronic acid, HA) is the one in glycosaminoglycan, has good water-soluble, biocompatibility and degradation property, in the secretion promoting extracellular matrix and proteoglycan forming process, has played important effect; But fragile physical and mechanical properties and degradation speed too fast in vivo limit its application prospect.In order to obtain the natural polymer hydrogel with desirable physical and mechanical properties and biodegradation rate, the mode of chemically crosslinked is widely used in preparation process.The higher functional group of the chemically reactives such as carboxyl, hydroxyl, amino is often applied in chemical crosslink reaction, conventional chemical cross-linking agent is generally containing bifunctional, such as diamines, two hydrazines, dialdehyde, glycol etc., but these linking agents have cytotoxicity usually, the biocompatibility of hydrogel material will be affected if residual.Need to study a kind of novel chemically crosslinked macromolecule hydrogel, avoid in crosslinking reaction, adding additional chemical material and the cytotoxicity brought.Meanwhile, modern medicine requires that biomaterial in use can have plastic and controllability, realizes the result for the treatment of of Wicresoft.
Summary of the invention
For the problems referred to above, the invention provides the controlled self-crosslinking hyaluronic acid gel of a kind of sulfydryl/disulfide linkage.Described hydrogel physical properties is controlled, has good cell compatibility and biodegradability, can be used for organizational project original position injury repairing or the three-dimensional cell support for building controlled absorbed.
The present invention is achieved through the following technical solutions:
The controlled self-crosslinking hyaluronic acid gel of a kind of sulfydryl/disulfide linkage, there is the sulfydryl-hyaluronic acid compound of free sulfhydryl group for raw material, by material dissolution, then stable disulfide linkage is formed by oxidizing reaction between sulfydryl, thus build the crosslinked progress in Intelligent Hydrogel of three dimensional chemical, by regulating sulfydryl-hyaluronic acid high molecular polymer surface sulfydryl density, gelling temp and hyaluronic molecular weight, utilize the redox conversion characteristic between sulfydryl and disulfide linkage, thus control the formation time of gel, degradation time and mechanical characteristic, build the controllable intelligent type hydrogel with good three-dimensional network crosslinking structure.Alternately, the reaction conditions that described three dimensional chemical is crosslinked is pH value 7.4 ~ 8.0.Under reductive agent effect, the disulfide linkage in described hydrogel can rupture, the rapid avalanche of gel structure, is degraded to the hyaluronic acid monomolecular substance that biocompatibility is good.
Alternately, the sulfydryl-hyaluronic acid compound with free sulfhydryl group take hyaluronic acid as raw material, obtained by half Guang ammonia modification.
Alternately, described hyaluronic molecular weight is: 0.1MDa ~ 1.0MDa.
Alternately, half Guang ammonia percentage of grafting in described sulfydryl-hyaluronic acid compound is; 10% ~ 60%;
Alternately, described sulfydryl-hyaluronic acid compound concentration is 1 ~ 5%; Can be further 1.0% or 2.0% or 3.0%.
By adjusting hyaluronic molecular weight (0.1 MDa, 0.3 MDa, 1 MDa), the percentage of grafting (11.28%, 12.41%, 14.69%, 29.13%, 33.54%, 37.47%, 51.47%, 55.44%, 60.56%) of half Guang ammonia, the concentration (1.0 wt %, 2.0 wt% or 3.0%) of sulfydryl-hyaluronic acid compound, the temperature (4 DEG C, 37 DEG C) of gel environment, the time (from 6 minutes to more than 2 hours) that hydrogel is formed accurately can be controlled.
Alternately, the disulfide linkage in described hydrogel under the reductibility small molecules existent condition such as dithiothreitol (DTT) (DTT) or gsh (GSH), can make the disulfide linkage between hyaluronic acid disconnect, thus makes hydrogel generation reductive cleavage.Further, the micromolecular concentration in system of described reductibility is 0.1 ~ 10mM.By adjusting hyaluronic molecular weight, the percentage of grafting of half Guang ammonia, the micromolecular concentration of reductibility, the degree (from several minutes by several hours) of hydrogel reductive cleavage can be controlled.
Alternately, described hydrogel is specific shape by mould molding.
Alternately, described hydrogel is injectable, by controlling gel time or the envrionment temperature of material, can make product before the injection in fluid state, forming gel state after injection.Described hydrogel has injectable, can gel in-situ, gelation process not produce or residual containing advantages such as cytotoxic substances.Described hydrogel can be implanted by the mode of injection, and forms hydrogel at site of tissue damage, realizes the in-situ immobilization of tissue injury.
The invention provides a kind of the hydrogel plastic of sulfydryl-hyaluronic acid compound and cracking system based on having free sulfhydryl group, described hydrogel plastic and cracking system comprise the controlled self-crosslinking hyaluronic acid gel of above-mentioned sulfydryl/disulfide linkage and reductibility small molecules.Its gelation time and cracking degree can need to adjust flexibly according to applying, and this system has good cell compatibility, and gelation process does not produce has Cytotoxic material, without the introducing of exogenous material, is conducive to the propagation of cell; Have good biodegradability, the reducing substances that cell itself produces also can accelerate disulfide linkage degraded, and gel degradation process does not produce has Cytotoxic material, is conducive to the reparation of cytostromatic secretion and site of tissue damage.
Present invention also offers a kind of preparation method of hyaluronic acid gel of above-mentioned disulfide bond crosslinking, take hyaluronic acid as raw material, sulfydryl-hyaluronic acid the high molecular polymer (spongy solid) with free sulfhydryl group is obtained by half Guang ammonia modification, dissolved, then form disulfide linkage by oxidizing reaction between sulfydryl, build the hydrogel with three dimensional chemical crosslinking structure.
As optional, the sulfydryl-hyaluronic acid compound after dissolving can be placed in corresponding mould and carry out gel reaction to obtain the hydrogel of specified shape; Also sulfydryl-the hyaluronic acid compound after dissolving can be made injection liquid, after injection, make it at target area gel in-situ.
Alternately, in above-mentioned preparation method, take hyaluronic acid as raw material, sulfydryl-hyaluronic acid the high molecular polymer with free sulfhydryl group is obtained by half Guang ammonia modification, be dissolved in the PBS damping fluid of pH=7.4, then adjust ph to 7.4 ~ 8.0, by forming the reaction of disulfide linkage between sulfydryl, form the hydrogel that three dimensional chemical is crosslinked.
As optional, adopt the pH value adding the NaOH solution of 1M.
Alternately, in above-mentioned preparation method, specifically comprise the following steps:
1) hyaluronic half Guang ammonia modification:
Amino on half Guang ammonia and the carboxyl on hyaluronic acid carry out acid amides reaction under the catalysis of N-succinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl), prepare raw material sulfydryl-hyaluronic acid compound;
2) chemically crosslinked:
Half Guang ammonia modified hyaluronic acid of the preparation in step 1) being dissolved, then by forming the reaction of disulfide linkage between sulfydryl, forming the hydrogel that three dimensional chemical is crosslinked.
Alternately, in above-mentioned preparation method, specifically comprise the following steps:
1) hyaluronic half Guang ammonia modification:
Hyaluronate sodium is at N-succinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate
(EDCHCl) react with half Guang ammonia (CSHHCl) hydrochloride under catalysis, add dithiothreitol (DTT) (DTT) subsequently, after reaction terminates, dialyse in deionized water, lyophilize, obtains half Guang ammonia modified hyaluronic acid;
2) chemically crosslinked:
Half Guang ammonia modified hyaluronic acid of the preparation in step 1) is dissolved in the PBS damping fluid of pH=7.4, then passes through mercapto
Form the reaction of disulfide linkage between base, form the hydrogel that three dimensional chemical is crosslinked.
In the preparation process of half Guang ammonia modified hyaluronic acid, add dithiothreitol (DTT) (DTT) can avoid obtaining pars shape product after part half Guang ammonia modified hyaluronic acid generation disulfide bond crosslinking causes lyophilize in preparation process and cannot dissolve in subsequent step.Dialysis procedure can remove again unnecessary dithiothreitol (DTT) (DTT), avoids its residual effect affecting subsequent step in the product.
Alternately, in above-mentioned preparation method, described step 1) is specially:
Hyaluronate sodium is dissolved in deionized water, first adds N-succinimide (NHS), fully dissolve; Then add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) powder, with the pH=4.75 of the HCl solution adjustment reaction solution of NaOH and 1M of 1M, react 2 hours; Add half Guang ammonia (CSHHCl) HCI solution subsequently, react 24 hours; Finally with the pH=8.5 of the NaOH solution adjustment reaction solution of 1M, add dithiothreitol (DTT) (DTT) solution, react 12 hours.
After reaction terminates, with the pH=3.0-3.5 of the HCl solution adjustment reaction solution of 1M, dialyse 72h in the deionized water of pH=3.0-3.5, lyophilize, obtains half Guang ammonia modified hyaluronic acid.
Alternately, in above-mentioned preparation method, carboxyl in hyaluronate sodium: N-succinimide: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: half Guang ammonia hydrochloric acid salt: the ratio of the amount of substance of dithiothreitol (DTT) is 1:2:2 ~ 4:1 ~ 4:3 ~ 12.By adjusting the ratio of amount of substance of half Guang ammonia, hyaluronic acid, NHS and EDCI, the substitution value of half Guang ammonia can be controlled.Alternately, above-mentioned hyaluronic molecular weight is: 0.1MDa ~ 1.0MDa.
Alternately, above-mentioned steps 2) in sulfydryl-hyaluronic acid compound strength of solution be 1.0%-3.0%.
Present invention also offers a kind of application of hyaluronic acid gel of above-mentioned disulfide bond crosslinking, it is characterized in that, used as Three-dimensional cell culture support or be made into injection aquagel.Further, to can be used as organizational project especially cartilage tissue engineered for described Three-dimensional cell culture support; Described injection aquagel can be used for beauty treatment, prevents wound adhesion, original position injury repairing, organizational project etc.
Present invention also offers a kind of Three-dimensional cell culture system, the hyaluronic acid gel comprising above-mentioned disulfide bond crosslinking and the cell be uniformly distributed in wherein.In this culture system, cell can form in whole three-dimensional rack at hydrogel and is uniformly distributed, overcome the more difficult defect moving to internal stent of cell in existing three-dimensional rack, described hydrogel also has good mechanical property, and when cell is bred in the bracket after certain scale, the reducing substances that cell itself produces or exogenous the reductibility small molecules added can make the disulfide bonds in hydrogel, hydrogel generation cracking, the 3-D solid structure of the similar three-dimensional tissue be made up of cell and extracellular matrix completely can be formed after the hyaluronic acid degradation that cracking produces, and degraded product can be absorbed fast by body, culture system is made to have good biocompatibility, can be used for the dimensional culture of various kinds of cell.By regulating and controlling hyaluronic molecular weight, the parameter such as percentage of grafting, the micromolecular concentration of reductibility of half Guang ammonia can realize the controlled degradation of hydrogel scaffold in culture system.
Present invention also offers a kind of construction process of Three-dimensional cell culture system, comprise the following steps:
(1) sulfydryl-hyaluronic acid compound with free sulfhydryl group is dissolved in substratum, then adds cell suspension, mix, form mixed solution;
(2) by forming the reaction of disulfide linkage between sulfydryl, hyaluronic acid in gained mixed solution is made to form the crosslinked hydrogel of three dimensional chemical and by cell encapsulation in wherein.
Alternately, in the construction process of above-mentioned Three-dimensional cell culture system, step (2) is specially: the mixed solution that step (1) is obtained joins in mould, subsequently at 37 DEG C, 0.5%CO
2carry out plastic process in cell culture incubator, finally the hydrogel of parcel cell is taken off from mould, add in substratum, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator;
Or, directly the mixed solution that step (1) is obtained is expelled in organisms, makes hyaluronic acid that three dimensional chemical occur in organisms and be cross-linked to form hydrogel.
Alternately, the concentration after described sulfydryl-hyaluronic acid compound is dissolved in substratum is 1 ~ 3%, preferably 3%.Base-hyaluronic acid compound molecular weight is 0.1MDa ~ 1.0MDa, is preferably 0.3MDa.
Alternately, cell-seeding-density is 5 × 10
6cells/mL.Described cell can be nascent rabbit cartilage cell (chondrocytes), nascent rabbit bone mescenchymal stem cell (MSCs), l cell (L929 cells) etc.
All features disclosed in this specification sheets, or the step in disclosed all methods or process, except mutually exclusive feature and/or step, all can combine by any way.
Beneficial effect of the present invention:
The raw material hyaluronic acid of hydrogel application of the present invention is natural polymer, there is good water-soluble, water-absorbent, gained hydrogel physical properties is controlled, there is easy gel formats, gelation process does not produce containing cytotoxic material, there is good cell compatibility and biodegradability, can be used for organizational project original position injury repairing or for building three-dimensional cell support.
accompanying drawing illustrates:
Fig. 1 is the syntheti c route figure of hydrogel of the present invention;
Fig. 2 is the mechanical property testing result of each hydrogel material prepared in embodiment 1-9;
Fig. 3 is the degradation characteristic (changes in weight) under the reductive agent DTT condition of different concns, and in figure, the DTT concentration of three components is followed successively by 0.1mM, 1mM, 10Mm from top to bottom;
Fig. 4 (is respectively: 3 days, 7 days, 14 days, 21 days with hydrogel Dual culture result of the present invention from top to bottom for rabbit cartilage cell (chondrocytes) nascent described in embodiment 12; From left to right be respectively: observation under the burnt different multiples condition of copolymerization, tem observation);
Fig. 5 (is respectively: 3 days, 7 days, 14 days, 21 days from top to bottom for the cultivation results of rabbit bone mescenchymal stem cell (MSCs) nascent described in embodiment 13; From left to right be respectively: observation under the burnt different multiples condition of copolymerization, tem observation);
The cultivation results that Fig. 6 is MSC cell described in embodiment 13 (is respectively: 3 days, 7 days, 14 days, 21 days from top to bottom; From left to right be respectively: observation under the burnt different multiples condition of copolymerization, tem observation);
Fig. 7 be wrap up chondrocyte cell in embodiment 16 hydrogel at small white mouse culturing in vivo 14 days (in figure left side), 28 days (in figure right side) laser co-focusing photos afterwards;
Fig. 8 be wrap up chondrocyte cell in embodiment 16 hydrogel at small white mouse culturing in vivo 14 days (in figure left side), 28 days (in figure right side) laser co-focusing photos afterwards.
embodiment:
Embodiment is by the following examples described in further detail foregoing of the present invention again.But this should be interpreted as that the scope of the above-mentioned theme of the present invention is only limitted to following example.Not departing from any amendment made within the spirit and principles in the present invention, and the equivalent replacement made according to ordinary skill knowledge and customary means or improvement, all should be included in protection scope of the present invention.
embodiment 1
(1) 400mg hyaluronate sodium is dissolved in 100mL deionized water, first adds 230mg N-succinimide (NHS), fully dissolve; Then add 385mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) powder, with the pH=4.75 of the HCl solution adjustment reaction solution of NaOH and 1M of 1M, react 2 hours; Add half Guang ammonia (CSHHCl) hydrochloride of 10mL 11.36mg/mL subsequently, react 24 hours; Finally with the pH=8.5 of the NaOH solution adjustment reaction solution of 1M, add dithiothreitol (DTT) (DTT) solution of 10mL46.5mg/ml, react 12 hours.
(2), after reaction terminates, with the pH=3.0-3.5 of the HCl solution adjustment reaction solution of 1M, dialyse 72h in the deionized water of pH=3.0-3.5, lyophilize, obtains half Guang ammonia modified hyaluronic acid.
Wherein, hyaluronate sodium molecular weight is 0.1MDa, carboxyl in hyaluronate sodium: N-succinimide: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: half Guang ammonia hydrochloric acid salt: the ratio of the amount of substance of dithiothreitol (DTT) is 1:2:2:1:3.
(3) 30mg sulfydryl-hyaluronic acid compound is dissolved in the PBS damping fluid of 1mL pH=7.4, first this solution of NaOH solution adjustment pH=7.4 ~ 8.0(adding 1M under ice-water bath condition still can keep good mobility under 4 DEG C of conditions after 2 hours), then solution being poured into diameter is 8.5mm, be highly in the cylindrical die of 3.0mm, finally be placed in the insulation can of 37 DEG C, form disulfide linkage by sulfydryl reaction and prepare hydrogel.Gel time under 37 DEG C of conditions is 34 minutes.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia percentage of grafting is 14.69%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 3.3kPa.
embodiment 2
(1) 400mg hyaluronate sodium is dissolved in 100mL deionized water, first adds 230mg N-succinimide (NHS), fully dissolve; Then add 575mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) powder, with the pH=4.75 of the HCl solution adjustment reaction solution of NaOH and 1M of 1M, react 2 hours; Add half Guang ammonia (CSHHCl) hydrochloride of 10mL 22.72mg/mL subsequently, react 24 hours; Finally with the pH=8.5 of the NaOH solution adjustment reaction solution of 1M, add dithiothreitol (DTT) (DTT) solution of 10mL93.0mg/ml, react 12 hours.
(2), after reaction terminates, with the pH=3.0-3.5 of the HCl solution adjustment reaction solution of 1M, dialyse 72h in the deionized water of pH=3.0-3.5, lyophilize, obtains half Guang ammonia modified hyaluronic acid.
Wherein, hyaluronate sodium molecular weight is 0.1MDa, carboxyl in hyaluronate sodium: N-succinimide: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: half Guang ammonia hydrochloric acid salt: the ratio of the amount of substance of dithiothreitol (DTT) is 1:2:3:2:6.
(3) 30mg sulfydryl-hyaluronic acid compound is dissolved in the PBS damping fluid of 1mL pH=7.4, first under ice-water bath condition, add NaOH solution adjustment pH=7.4 ~ 8.0 of 1M, then solution is poured into diameter by (this solution still can keep good mobility under 4 DEG C of conditions after 2 hours) is 8.5mm, be highly in the cylindrical die of 3.0mm, finally be placed in the insulation can of 37 DEG C, form disulfide linkage by sulfydryl reaction and prepare hydrogel.Gel time under 37 DEG C of conditions is 28 minutes
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia percentage of grafting is 37.47%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 12.4kPa.
embodiment 3
(1) 400mg hyaluronate sodium is dissolved in 100mL deionized water, first adds 230mg N-succinimide (NHS), fully dissolve; Then add 770mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) powder, with the pH=4.75 of the HCl solution adjustment reaction solution of NaOH and 1M of 1M, react 2 hours; Add half Guang ammonia (CSHHCl) hydrochloride of 10mL 45.44mg/mL subsequently, react 24 hours; Finally with the pH=8.5 of the NaOH solution adjustment reaction solution of 1M, add dithiothreitol (DTT) (DTT) solution of 10mL186.0mg/ml, react 12 hours.
(2), after reaction terminates, with the pH=3.0-3.5 of the HCl solution adjustment reaction solution of 1M, dialyse 72h in the deionized water of pH=3.0-3.5, lyophilize, obtains half Guang ammonia modified hyaluronic acid.
Wherein, hyaluronate sodium molecular weight is 0.1MDa, carboxyl in hyaluronate sodium: N-succinimide: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: half Guang ammonia hydrochloric acid salt: the ratio of the amount of substance of dithiothreitol (DTT) is 1:2:4:4:12.
(3) 30mg sulfydryl-hyaluronic acid compound is dissolved in the PBS damping fluid of 1mL pH=7.4, first under ice-water bath condition, add NaOH solution adjustment pH=7.4 ~ 8.0 of 1M, then solution is poured into diameter by (this solution still can keep good mobility under 4 DEG C of conditions after 2 hours) is 8.5mm, be highly in the cylindrical die of 3.0mm, finally be placed in the insulation can of 37 DEG C, form disulfide linkage by sulfydryl reaction and prepare hydrogel.Gel time under 37 DEG C of conditions is 18 minutes
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.1MDa, and half Guang ammonia percentage of grafting is 60.56%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 22.0kPa.
embodiment 4
With reference to method described in embodiment 1, its difference is only: hyaluronate sodium molecular weight is 0.3MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 12.41%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 7.7kPa.
Obtained product still kept good mobility before formation gel under 4 DEG C of conditions after 2 hours, and the gel time under 37 DEG C of conditions is 25 minutes.
embodiment 5
With reference to method described in embodiment 2, its difference is only: hyaluronate sodium molecular weight is 0.3MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 33.54%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 16.2kPa.
Obtained product still kept good mobility before formation gel under 4 DEG C of conditions after 2 hours, and the gel time under 37 DEG C of conditions is 18 minutes.
embodiment 6
With reference to method described in embodiment 2, its difference is only: hyaluronate sodium molecular weight is 0.3MDa
Gained sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 55.44%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 35.2kPa.
Obtained product was still keeping good mobility before formation gel under 4 DEG C of conditions after 2 hours, and the gel time under 37 DEG C of conditions is 11 minutes.
embodiment 7
With reference to method described in embodiment 1, its difference is only: hyaluronate sodium molecular weight is 1.0 MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia percentage of grafting is 11.28%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 9.2kPa.
Obtained product still kept good mobility before formation gel under 4 DEG C of conditions after 2 hours, and the gel time under 37 DEG C of conditions is 25 minutes.
embodiment 8
With reference to method described in embodiment 2, its difference is only: hyaluronate sodium molecular weight is 1.0 MDa.
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia percentage of grafting is 29.13%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 17.7kPa.
Obtained product still kept good mobility before formation gel under 4 DEG C of conditions in 116 minutes, and the gel time under 37 DEG C of conditions is 15 minutes.
embodiment 9
With reference to method described in embodiment 2, its difference is only: hyaluronate sodium molecular weight is 1.0 MDa
Gained sulfydryl-hyaluronic acid compound molecular weight is 1.0MDa, and half Guang ammonia percentage of grafting is 51.47%, sulfydryl-hyaluronic acid compound strength of solution 3.0%, 1% deformation quantity, and under 10Hz frequency detecting condition, storage modulus is 42.8kPa.
Obtained product was still keeping good mobility before formation gel under 4 DEG C of conditions in 105 minutes, and the gel time under 37 DEG C of conditions is 6 minutes.
embodiment 10
(1) 400mg hyaluronate sodium is dissolved in 100mL deionized water, first adds 230mg N-succinimide (NHS), fully dissolve; Then add 385mg 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl) powder, with the pH=4.75 of the HCl solution adjustment reaction solution of NaOH and 1M of 1M, react 2 hours; Add half Guang ammonia (CSHHCl) hydrochloride of 10mL 11.36mg/mL subsequently, react 24 hours.
(2), after reaction terminates, with the pH=3.0-3.5 of the HCl solution adjustment reaction solution of 1M, dialyse 72h in the deionized water of pH=3.0-3.5, lyophilize, obtains half Guang ammonia modified hyaluronic acid.
Wherein, hyaluronate sodium molecular weight is 0.1MDa, carboxyl in hyaluronate sodium: N-succinimide: 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate: half Guang ammonia hydrochloric acid salt: the ratio of the amount of substance of dithiothreitol (DTT) is 1:2:2:1:3.
(3) 30mg sulfydryl-hyaluronic acid compound is dissolved in the PBS damping fluid of 1mL pH=7.4, first under ice-water bath condition, add NaOH solution adjustment pH=7.4 ~ 8.0 of 1M, then solution being poured into diameter is 8.5mm, be highly in the cylindrical die of 3.0mm, finally be placed in the insulation can of 37 DEG C, form disulfide linkage by sulfydryl reaction and prepare hydrogel.
In gained hydrogel over-all properties and embodiment 1, product is basically identical.Just the continuous shape desciccate of generating portion sometimes in step (2), is difficult to dissolve in subsequent step.
embodiment 11
Hydrogel obtained in Example 1 ~ 9 respectively, add reductibility small molecules DTT(to detect the degraded situation that DTT concentration is 0.1mM, 1mM, 10Mm Water Under gel respectively), gel progressively can be degraded along with the prolongation of time, until become solution state.
Result is as shown in Figure 3: hydrogel of the present invention can accelerate degraded in a reducing environment, and degradation rate increases with reductant concentration and accelerates.
In addition, change above-mentioned reductibility small molecules into gsh (GSH) by DTT and also can reach substantially identical degradation effect.
embodiment 12
(1) rabbit cartilage cell (chondrocytes) of coming into being is evenly distributed in culture dish, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.Cover with after culture dish until cell, collecting cell, being prepared into 100 μ L concentration is 5.5 × 10
7the cell suspension of cells/mL.
(2) 33mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum, first NaOH solution adjustment pH=7.4 ~ 8.0 of 1M are added, then 100 μ L chondrocyte cell suspensions of above-mentioned preparation are added, mix that (hydrogel final concentration is 3%, and cell-seeding-density is 5 × 10
6cells/mL), solution is poured in mould, subsequently at 37 DEG C, 0.5%CO
2carry out plastic process in cell culture incubator, finally the hydrogel of parcel cell is taken off from mould, join in α-MEM substratum, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 55.44%.
After the hydrogel of parcel chondrocyte cell cultivates 1 day, 3 days, 7 days, 14 days, 21 days, use opticmicroscope respectively, the growing state of laser co-focusing and scan track electron microscope observation cell.Result is as shown in Figure 4: in Dual culture process, chondrocyte cell remains rounded form; Dual culture, after 7 days, obviously can observe the appearance of paired cell; Dual culture, after 14 days, obviously can observe cell mass and matrix secretion phenomenon; Dual culture, after 21 days, obviously can observe a large amount of secretion of matrix and the cell mass of matrix parcel.Observations shows that chondrocyte cell can be bred normally in co-culture system, and keeps the normal biological functions such as secretion matrix.
embodiment 13
(1) rabbit bone mescenchymal stem cell (MSCs) of coming into being is evenly distributed in culture dish, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.Cover with after culture dish until cell, collecting cell, being prepared into 100 μ L concentration is 5.5 × 10
7the cell suspension of cells/mL.
(2) 33mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum, first NaOH solution adjustment pH=7.4 ~ 8.0 of 1M are added, then add 100 μ L MSCs cell suspensions of above-mentioned preparation, mix that (hydrogel final concentration is 3%, and cell-seeding-density is 5 × 10
6cells/mL), solution is poured in mould, subsequently at 37 DEG C, 0.5%CO
2carry out plastic process in cell culture incubator, finally the hydrogel of parcel cell is taken off from mould, join in α-MEM substratum, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 55.44%.
After the hydrogel of parcel MSCs cell cultivates 1 day, 3 days, 7 days, 14 days, 21 days, use opticmicroscope respectively, the growing state of laser co-focusing and scan track electron microscope observation cell.Result is as shown in Figure 5: in Dual culture process, L929 cell remains rounded form; Dual culture, after 7 days, obviously can observe the appearance of paired cell; Dual culture, after 14 days, obviously can observe cell mass and matrix secretion phenomenon; Dual culture, after 21 days, obviously can observe a large amount of cell masses wrapped up by matrix.Observations shows that L929 cell can be bred normally in co-culture system, and keeps the normal biological functions such as secretion matrix.
embodiment 14
(1) l cell (L929 cells) is evenly distributed in culture dish, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.Cover with after culture dish until cell, collecting cell, being prepared into 100 μ L concentration is 5.5 × 10
7the cell suspension of cells/mL.
(2) 33mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum, first NaOH solution adjustment pH=7.4 ~ 8.0 of 1M are added, then add 100 μ L L929 cell suspensions of above-mentioned preparation, mix that (hydrogel final concentration is 3%, and cell-seeding-density is 5 × 10
6cells/mL), solution is poured in mould, subsequently at 37 DEG C, 0.5%CO
2carry out plastic process in cell culture incubator, finally the hydrogel of parcel cell is taken off from mould, join in α-MEM substratum, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.
Wherein, sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 55.44%.
After the hydrogel of parcel L929 cell cultivates 1 day, 3 days, 7 days, 14 days, 21 days, use opticmicroscope respectively, the growing state of laser co-focusing and scan track electron microscope observation cell.Result is as shown in Figure 6: in Dual culture process, MSCs cell remains rounded form, shows certain multiplication capacity.Observations shows that MSCs cell can be bred normally in co-culture system.
embodiment 15
30mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum by 1, first adds the PBS buffered soln adjustment pH=7.4 of 1M, (hydrogel final concentration is 3%), then with syringe, 0.1mL injection of solution is subcutaneous to BALB/C small white mouse.
2 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 33.54%.
3 hydrogels at small white mouse culturing in vivo after 7 days, 28 days, take out, and observe the degraded situation of hydrogel, and with the growing state of laser co-focusing observation of cell.
Experimental result find, this gel can better keep its gel state in vivo, do not occur significance shrink and swelling.
embodiment 16
1 rabbit cartilage cell (chondrocytes) of coming into being is evenly distributed in culture dish, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator.Cover with after culture dish until cell, collecting cell, being prepared into 100 μ L concentration is 5.5 × 10
7the cell suspension of cells/mL.
33mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum by 2, first the PBS buffered soln adjustment pH=7.4 of 1M is added, then add 100 μ L chondrocyte cell suspensions of above-mentioned preparation, mix that (hydrogel final concentration is 3%, and cell-seeding-density is 5 × 10
6cells/mL), with syringe, 0.1mL injection of solution is subcutaneous to BALB/C small white mouse.
3 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 33.54%.
The hydrogel of 4 parcel chondrocyte cells, takes out, with the growing state (as shown in Figure 7) of laser co-focusing observation of cell after 14 days, 28 days at small white mouse culturing in vivo.
Experimental result finds, can better should keep its gel state in vivo at Cellular gels, does not occur that significance is shunk and swelling.Cell can be good at propagation in gel inside.
embodiment 17
1 rabbit cartilage cell (chondrocytes) of coming into being is evenly distributed in culture dish, 37 DEG C, cultivate in 0.5%CO2 cell culture incubator.Cover with after culture dish until cell, collecting cell, being prepared into 100 μ L concentration is 27.5 × 10
7the cell suspension of cells/mL.
33mg sulfydryl-hyaluronic acid compound is dissolved in 1mL α-MEM substratum by 2, first the PBS buffered soln adjustment pH=7.4 of 1M is added, then add 100 μ L chondrocyte cell suspensions of above-mentioned preparation, mix that (hydrogel final concentration is 3%, and cell-seeding-density is 2.5 × 10
7cells/mL), with syringe, 0.1mL injection of solution is subcutaneous to BALB/C small white mouse.
3 wherein, and sulfydryl-hyaluronic acid compound molecular weight is 0.3MDa, and half Guang ammonia percentage of grafting is 33.54%.
The hydrogel of 4 parcel chondrocyte cells, takes out, with the growing state of laser co-focusing observation of cell after 14 days, 28 days at small white mouse culturing in vivo.(as shown in Figure 8)
Experimental result finds, can better should keep its gel state in vivo at Cellular gels, does not occur that significance is shunk and swelling.Cell can be good at propagation in gel inside.
The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall into protection scope of the present invention.
Claims (10)
1. the controlled self-crosslinking hyaluronic acid gel of sulfydryl/disulfide linkage, it is characterized in that, there is the sulfydryl-hyaluronic acid high molecular polymer of free sulfhydryl group for raw material, by regulating its surperficial sulfydryl density, gelling temp and hyaluronic molecular weight, utilize the redox conversion characteristic between sulfydryl and disulfide linkage, thus the formation time of control gel, degradation time and mechanical characteristic, build the controllable intelligent type hydrogel with good three-dimensional network crosslinking structure.
2. hyaluronic acid gel according to claim 1, is characterized in that, the gel time of described hydrogel can accuracy controlling.
3. hyaluronic acid gel according to claim 1, is characterized in that, described hydrogel can realize controlled degradation under reductibility small molecules existent condition.
4. the preparation method of the controlled self-crosslinking hyaluronic acid derivatives of sulfydryl/disulfide linkage as claimed in claim 1, it is characterized in that, take hyaluronic acid as raw material, sulfydryl-hyaluronic acid the high molecular polymer with free sulfhydryl group is obtained by half Guang ammonia modification, dissolved, then form disulfide linkage by oxidizing reaction between sulfydryl, and form the hydrogel that there is three dimensional chemical and be cross-linked.
5. preparation method according to claim 4, it is characterized in that, take hyaluronic acid as raw material, sulfydryl-hyaluronic acid the high molecular polymer with free sulfhydryl group is obtained by half Guang ammonia modification, be dissolved in the PBS damping fluid of pH=7.4, then adding NaOH solution adjust ph to 7.4 ~ 8.0 of 1M, by forming the reaction of disulfide linkage between sulfydryl, forming the hydrogel that three dimensional chemical is crosslinked.
6. preparation method according to claim 4, is characterized in that, comprises the following steps:
1) hyaluronic half Guang ammonia modification:
Hyaluronate sodium reacts with half Guang ammonia (CSHHCl) hydrochloride under the catalysis of N-succinimide (NHS), 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl), add dithiothreitol (DTT) (DTT) subsequently, after reaction terminates, dialyse in deionized water, lyophilize, obtains half Guang ammonia modified hyaluronic acid;
2) half Guang ammonia modified hyaluronic acid of the preparation in step 1) is dissolved in the PBS damping fluid of pH=7.4, is then formed the reaction of disulfide linkage by oxygenizement between sulfydryl, form the hydrogel that three dimensional chemical is crosslinked.
7. an application for the hyaluronic acid gel of disulfide bond crosslinking according to claim 1, is characterized in that, used as Three-dimensional cell culture support or be made into injection aquagel.
8. a Three-dimensional cell culture strutting system, is characterized in that, comprises according to claim 1 based on the controlled self-crosslinking hyaluronic acid gel of sulfydryl/disulfide linkage and the cell that is uniformly distributed in wherein.
9. a construction process for Three-dimensional cell culture system, is characterized in that, comprises the following steps:
(1) sulfydryl-hyaluronic acid compound with free sulfhydryl group is dissolved in substratum, then adds cell suspension, mix, form mixed solution;
(2) form the reaction of disulfide linkage by oxidation between sulfydryl, make the hyaluronic acid in gained mixed solution form the crosslinked hydrogel of three dimensional chemical and by cell encapsulation in wherein.
10. the construction process of Three-dimensional cell culture system according to claim 9, is characterized in that, step (2) is specially: the mixed solution that step (1) is obtained joins in mould, subsequently at 37 DEG C, 0.5%CO
2carry out plastic process in cell culture incubator, finally the hydrogel of parcel cell is taken off from mould, add in substratum, at 37 DEG C, 0.5%CO
2cultivate in cell culture incubator; Or, directly the mixed solution that step (1) is obtained is expelled in organisms, makes hyaluronic acid that three dimensional chemical occur in organisms and be cross-linked to form hydrogel.
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