CN108721695A - A kind of preparation method of syringeability composite hydrogel cell carrier holder - Google Patents
A kind of preparation method of syringeability composite hydrogel cell carrier holder Download PDFInfo
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- CN108721695A CN108721695A CN201810531990.XA CN201810531990A CN108721695A CN 108721695 A CN108721695 A CN 108721695A CN 201810531990 A CN201810531990 A CN 201810531990A CN 108721695 A CN108721695 A CN 108721695A
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- polysaccharide
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- hydrazides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/20—Polysaccharides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/52—Hydrogels or hydrocolloids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/80—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special chemical form
- A61L2300/802—Additives, excipients, e.g. cyclodextrins, fatty acids, surfactants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2400/00—Materials characterised by their function or physical properties
- A61L2400/06—Flowable or injectable implant compositions
Abstract
The invention discloses a kind of preparation methods of syringeability composite hydrogel cell carrier holder, furans and hydrazides polysaccharide and furans and aldehyde radical polysaccharide are separately designed by modified-reaction first, then the complex polysaccharide nano-carrier of the pH sensitivities of bioactie agent is loaded by double emulsion methods, eventually by gel precursor solution, the compound realization gelatinizing-in-situ of nano-carrier solution, cell suspending liquid, the preparation of the syringeability composite hydrogel cell carrier holder of final obtained load cells.It is introduced into the cyclodextrin nano particle of the pH sensitivities containing bioactie agent in holder of the present invention to promote the growth of cell in medium, and holder performance meets the basic demand of injectable type, there is larger Social benefit and economic benefit.
Description
Technical field
The present invention relates to for the syringeability holder in organizational project, and in particular to a kind of syringeability composite hydrogel
The preparation method of cell carrier holder.
Background technology
There is new choosing in appearance with organizational project and regeneration medicine technology and gradual perfection, histoorgan reparation
It selects.Process using tissue engineering technique tissue is usually, by the histocyte of in-vitro separation amplification and growth factor or biology
Active material is compound, is then introduced into certain holder, then the method repair deficiency by operation or minimally invasive injection tissue.In addition to kind
Outside daughter cell and active factors, timbering material plays the role of the quality of repair tissue vital.Timbering material is except tool
Have outside good mechanical and physical performance, it is often more important that holder need to be provided suitable for the regenerated microenvironment of tissue tissue；Currently, having
The holder of various structures including porous support, fibrous framework, hydrogel and microcarrier is used for the research of tissue repair
And application.
Different types of holder generates histiocytic function different influences.Histocyte belongs to that anchor dependent form thin
Born of the same parents, they need the surface for being attached on these materials that could grow, typically exhibit out paving in porous support and micrometer fibers holder
The flat sample form of exhibition.However, histocyte in nano fiber scaffold and hydrogel scaffold then at round or ellipse form,
This with it in natural tissues matrix more closely, thus be more advantageous to and maintain histiocytic normal phenotype.Some researches show that,
The rounded or oval stem cell of growth conditions is more likely to break up to histocyte.In addition, the aqueous solution of hydrogel scaffold
Environment is more advantageous to protection cell and the drug such as polypeptide, protein, oligonucleotide and DNA etc. of easy in inactivation, is also beneficial to
Transport nutrition and cell secretory product etc..Due to hydrogel can keep flow regime under certain condition and in external physical or
It forms the bulk material with definite shape and intensity under chemical stimulation, therefore intelligent can prepare injection-type using this
Holder plays it in the advantage for repairing complex-shaped defect and minimally-invasive treatment etc..However, hydrogel also has mechanical strength
It is low, disinfection it is relatively difficult the shortcomings of.
Invention content
In order to overcome shortcoming and deficiency existing in the prior art, it is multiple that the purpose of the present invention is to provide a kind of syringeabilities
The preparation method of Heshui gel cell carrier holder.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of preparation method of syringeability composite hydrogel cell carrier holder, includes the following steps：
(1) furans polysaccharide is prepared：Polysaccharide containing carboxylate radical is dissolved in ethanesulfonic acid buffer, carboxylic fully is added after dissolving
Base activator, is stirred at room temperature；Furylamine is added dropwise again, after being reacted under room temperature environment, it is more that furans is made in dialysis freeze-drying
Sugar；
(2) furans and hydrazides polysaccharide are prepared：Above-mentioned furans polysaccharide is dissolved in ethanesulfonic acid buffer, fully after dissolving
Carboxyl activator is added, is stirred at room temperature；Adipic dihydrazide is added, after being reacted under room temperature environment, furans is made in dialysis freeze-drying
With hydrazides polysaccharide；
Prepare furans and aldehyde radical polysaccharide：Above-mentioned furans polysaccharide is soluble in water, periodic acid fully is added dropwise after dissolving
Sodium water solution after being protected from light, is added excess ethylene glycol and terminates reaction, furans and aldehyde radical polysaccharide are obtained after dialysis freeze-drying；
(3) double emulsion methods prepare the composite cyclodextrin nano-carrier of the pH sensitivities of load bioactie agent：The pH is quick
The preparation of the composite cyclodextrin nano-carrier of sense refers to patent CN201710224454.0；Growth factor is made into PBS solution, institute
The composite cyclodextrin nano-carrier for stating pH sensitivities is made into PBS solution, and low temperature shakes after the two mixing, and it is extra to centrifuge removing
Growth factor obtains the composite cyclodextrin nano-carrier of the pH sensitivities of the load bioactie agent；
(4) furans of step (1) and hydrazides polysaccharide and furans and aldehyde radical polysaccharide are made into aqueous solution respectively；It will
The carrier obtained of step (3) is mixed with above-mentioned furans and hydrazides polysaccharide solution, and carrier is made and furans and hydrazides are more
Sugared mixed liquor；Above-mentioned carrier is mixed with furans and hydrazides polysaccharide mixed liquor and furans and aldehyde radical polysaccharide solution,
Crosslinking agent is added, is uniformly mixed and stands, obtain the syringeability composite hydrogel cell carrier holder.
Further, in the step (1), polysaccharide is Sodium Hyaluronate or heparin, and the mass volume ratio of polysaccharide is 0.1-
5%；The carboxyl activator is 4- (4,6- dimethoxy-triazines) -4- methyl morpholine hydrochlorides or water-soluble carbodiimides
(N- (3-dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride, i.e. DMTMM or EDC, carboxylic
The molar ratio of base activator and carboxylate radical is 10:1-1:2, the time being stirred at room temperature is 10-120min；The furylamine and carboxylic
The molar ratio of acid group is 2:1-1:2, react 2-48h under room temperature environment.Preferably, polysaccharide is hyaluronic acid, the matter of polysaccharide
Amount volume ratio is 0.2-2%；The carboxyl activator is DMTMM, and the molar ratio of carboxyl activator and carboxylate radical is 5:1-1:1,
The time being stirred at room temperature is 20-60min；The molar ratio of the furylamine and carboxylate radical is 2:1-1:1, it is anti-under room temperature environment
Answer 4-24h.Further, the mass volume ratio of the polysaccharide is 0.5-1%；The molar ratio of carboxyl activator and carboxylate radical is
2:1, the time being stirred at room temperature is 30-40min；The molar ratio of the furylamine and carboxylate radical is 1:1；It is anti-under room temperature environment
Answer 8-12h.
In the step (2), furans and hydrazides polysaccharide are prepared：Polysaccharide is Sodium Hyaluronate or heparin, the quality of polysaccharide
Volume ratio is 0.1-5%；The carboxyl activator is 4- (4,6- dimethoxy-triazines) -4- methyl morpholine hydrochlorides or water solubility
Carbodiimides (N- (3-dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride, i.e.,
The molar ratio of DMTMM or EDC, carboxyl activator and carboxylate radical is 10:1-1:2, the time being stirred at room temperature is 10-120min；Institute
The molar ratio for stating adipic dihydrazide and carboxylate radical is 2:1-1:2, react 2-48h under room temperature environment；Prepare furans and aldehyde radical
Polysaccharide:The mass volume ratio of furans polysaccharide is 0.1-5%；A concentration of 0.1-2M of sodium metaperiodate aqueous solution, furans polysaccharide
Volume ratio with sodium metaperiodate aqueous solution is 1:1-100:1.
Preferably, furans and hydrazides polysaccharide are prepared:Polysaccharide is hyaluronic acid, and the mass volume ratio of polysaccharide is 0.2-
2%；The carboxyl activator is DMTMM, and the molar ratio of carboxyl activator and carboxylate radical is 5:1-1:1, the time being stirred at room temperature
For 20-60min；The adipic dihydrazide and the molar ratio of carboxylate radical are 2:1-1:1, react 4-24h under room temperature environment；Prepare furan
Muttering and aldehyde radical polysaccharide:The mass volume ratio of furans polysaccharide is 0.2-2%；The concentration 0.2-1M of sodium metaperiodate aqueous solution；
The volume ratio of furans polysaccharide and sodium metaperiodate aqueous solution is 1:1-50:1；The time being protected from light is 1-3h.
Further, furans and hydrazides polysaccharide are prepared:The mass volume ratio of the polysaccharide is 0.5-1%；Carboxyl is lived
Agent and the molar ratio of carboxylate radical are 2:1, the time being stirred at room temperature is 30-40min；Mole of the adipic dihydrazide and carboxylate radical
Than being 1:1；8-12h is reacted under room temperature environment；Prepare furans and aldehyde radical polysaccharide:The mass volume ratio of furans polysaccharide is
0.2-0.5%；The volume ratio of the concentration 0.5M of sodium metaperiodate aqueous solution, furans polysaccharide and sodium metaperiodate aqueous solution is 40:1；
The time being protected from light is 2h.
Further, in the step (4), the aqueous solution of furans and hydrazides polysaccharide and furans and aldehyde radical polysaccharide
Mass volume ratio be 0.5-15%；Furans and aldehyde radical polysaccharide solution can be mixed to prepare cell and furan with suspension cell
Muttering and aldehyde radical polysaccharide mixed liquor, a concentration of 1,000,000-1,000 ten thousand/mL of suspension cell；A concentration of 0.1-2mg/ of carrier
ML, carrier and furans and hydrazides polysaccharide mixed liquor：Cell is 1 with the volume ratio of furans and aldehyde radical polysaccharide mixed liquor:1；
Crosslinking agent is bismaleimide PEG, and the molar ratio of crosslinking agent and carboxylate radical is 2:1-1:10.Preferably, the furans
Mass volume ratio with hydrazides polysaccharide and furans and the aqueous solution of aldehyde radical polysaccharide is 1-10%；The suspension cell is dense
Degree is 2,000,000-500 ten thousand/mL；The concentration 0.2-1mg/mL of carrier；The molar ratio of crosslinking agent and carboxylate radical is 1:1-1:5.More
Further, the mass volume ratio of the furans and hydrazides polysaccharide and the aqueous solution of furans and aldehyde radical polysaccharide is 2-
5%；A concentration of 3,000,000-400 ten thousand/mL of suspension cell；The concentration 1mg/mL of carrier；Mole of crosslinking agent and carboxylate radical
Than being 1:1-1:2.
The present invention separately designs furans and hydrazides polysaccharide and furans and aldehyde radical polysaccharide by modified-reaction first
Isogel precursor solution；Then the complex polysaccharide nano-carrier of the pH sensitivities of bioactie agent is loaded by double emulsion methods；
It is finally realized and has been loaded by the compound and gelatinizing-in-situ of gel precursor solution, nano-carrier solution and cell suspending liquid again
The preparation of the syringeability composite hydrogel cell carrier holder of cell.
The present invention by DA reaction designings syringeability composite hydrogel cell carrier holder, introduce containing bioactivity because
The cyclodextrin nano particle of the pH sensitivities of son is to promote the growth of cell in medium, holder performance to meet wanting substantially for injectable type
It asks, there is larger Social benefit and economic benefit.
The method of the present invention operating procedure is simple, and reaction condition is mild.Timbering material mechanical strength prepared by the method for the present invention
Height, and there is good biocompatibility.
Description of the drawings
Fig. 1 is the upgrowth situation in hydrogel scaffold for the cell in embodiment 4 (d, e, f) and comparative example (a, b, c)：
Wherein a, d are cell growth first day；B, e are cell growth third day；C, f are cell growth the 5th day；
Fig. 2 is the cell in embodiment 4 (composite hydrogel) and comparative example (pure hydrogel) in water-setting
Active comparative analysis in glue；
Fig. 3 is DNA content analysis of the cell in hydrogel in embodiment 4 and comparative example.
Specific implementation mode
The present invention is described further with reference to the accompanying drawings and examples.
The preparation method of the syringeability composite hydrogel cell carrier holder of the present embodiment, steps are as follows：
(1) synthesis of functionalization polysaccharide
By a certain amount of polysaccharide containing carboxylate radical, (Sodium Hyaluronate or heparin, optimal is hyaluronic acid.Polysaccharide concentration is 0.1-
5% (W/V) is further 0.2-2%, optimal for 0.5-1%), it is dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5)；Carboxyl activator (4- (4,6- dimethoxy-triazines) -4- methyl morpholine hydrochlorides (DMTMM) fully are added after dissolving
Or water-soluble carbodiimides (N- (3-dimethylaminopropyl)-N '-ethylcarbodiimide
Hydrochloride, EDC), preferably DMTMM.The molar ratio of carboxyl activator and carboxylate radical is 10:1-1:2, further
It is 5:1-1:1, optimal is 2:1)；A period of time is stirred at room temperature, and (10-120min is further 20-60min, optimal
For 30-40min) after；Furylamine is added dropwise in syringe, and (molar ratio of furylamine and carboxylate radical is 2:1-1:2, further
It is 2:1-1:1, optimal is 1:1)；Reaction a period of time is stirred at room temperature, and (2-48h is further 4-24h, and optimal is 8-
After 12h), furans polysaccharide is made in dialysis freeze-drying.
Adipic dihydrazide is modified onto above-mentioned furans polysaccharide with same method, preferred polysaccharide concentration, activator class
The proportioning of type and activator and carboxylic acid, soak time, the ratio of amino carboxyl and reaction time are identical as above-mentioned reaction,
Furans and hydrazides polysaccharide are obtained after dialysis freeze-drying.
By a certain amount of above-mentioned furans polysaccharide, (a concentration of 0.1-5% (W/V), is further 0.2-2%, optimal
For 0.2-0.5%) it is soluble in water, sodium metaperiodate aqueous solution is slowly fully added dropwise after dissolving, and (a concentration of 0.1-2M is further
0.2-1M, it is optimal for 0.5M.The volume ratio of polysaccharide solution and sodium periodate solution is 1:1-100:1, it is further 1:1-
50:1, optimal is 40:1)；After being protected from light a period of time (0.5-4h is further 1-3h, optimal for 2h), it is added
Excessive ethylene glycol terminates reaction, and furans and aldehyde radical polysaccharide are obtained after dialysis freeze-drying.
(2) preparation of the composite cyclodextrin nano-carrier of the pH sensitivities of load bioactie agent
The preparation of the composite cyclodextrin nano-carrier of pH sensitivities refers to the embodiment 1 of patent 201710224454.0.?
To after the composite cyclodextrin nano-carrier of above-mentioned pH sensitivities, growth factor is made into 300ng/mL PBS solutions, pH is sensitive to be answered
Cyclization dextrin nano-carrier is made into 1mg/mL PBS solutions, and (4 DEG C) concussion loadings of low temperature for 24 hours, it is more to centrifuge removing after mixing
Remaining growth factor obtains the composite cyclodextrin nano-carrier of the pH sensitivities of load bioactie agent.
(3) preparation of the syringeability composite hydrogel cell carrier holder of load cells
Respectively by the polysaccharide derivates (furans and hydrazides polysaccharide and furans and aldehyde radical polysaccharide) of above-mentioned steps (1)
It is made into aqueous solution, preferred polysaccharide concentration is 0.5-15% (W/V), is further 1-10%, optimal for 2-5%.It will step
Suddenly the carrier with furans and hydrazides polysaccharide of (2) mix, and a concentration of 0.1-2mg/mL of preferred vector is further 0.2-1mg/
ML, it is optimal for 1mg/mL, ultrasonic disperse.Cell after suspension is mixed into furans and aldehyde radical polysaccharide, preferred cell is dense
Degree is 1,000,000-1,000 ten thousand/mL, is further 2,000,000-500 ten thousand/mL, optimal for 3,000,000-400 ten thousand/mL.It will
Above two mixed solution is again with 1:1 volume ratio mixing, is added bismaleimide PEG as crosslinking agent (crosslinking agent and carboxylic
The molar ratio of sour structural unit is 2:1-1:10；Further is 1:1-1:5；Optimal is 1:1-1:2), it is uniformly mixed postposition
Obtain the syringeability composite hydrogel cell carrier holder of load cells for a period of time at 37 DEG C.
Experimental method：
(1) gel time is tested：
The variation of hydrogel monomer mobility, the timing definition hydrogel to lose flowability from solution are observed by observation
Gel time.
(2) cell observation：
The hydrogel of embedded cell is placed in 24 well culture plates, in 37 DEG C, 5%CO2Incubator culture specific time
After take out, with MTT (3- (4,5- dimethylthiazole) -2,5- diphenyltetrazolium bromide bromides, tetrazolium bromide) dye after observation be put in it is aobvious
Its pattern of micro- microscopic observation.
(3) measurement of cell activity：
Cell activity is quantitative by MTT, is as follows：100 μ L 5mg/mL MTT/PBS solution are added to each hole
In, continuation discards culture medium after incubator culture 4h, and 1mL DMSO are added, and takes out 100 μ L, with microplate reader measure its
Absorbance at 570nm.
(4) measurement of DNA content：
Sample is taken out from culture plate, is placed in 2mL centrifuge tubes.After multigelation, it is molten that 1mL papains are added
Liquid in 65 DEG C of water-baths overnight takes out, spare.Use Quant-iTTM Kit detects DNA content.
Embodiment 1
Sodium Hyaluronate (0.5g, 1.25mmol carboxylate radical) is dissolved in (100mM, pH=in 150ml ethanesulfonic acid buffers
5.5) in, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides are added
(DMTMM) (1.25mmol, 0.35g), after 30min is stirred at room temperature, furylamine (0.625mmol, 55 μ is added dropwise in syringe
L), reaction dialysis freeze-drying afterwards for 24 hours is stirred at room temperature.Said derivative 0.5g is taken to be dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5) DMTMM (0.7g, 2.5mmol), is added after dissolving, adipic dihydrazide (ADH, 1.39g, 8mmol) is added, stirs at room temperature
Mixing reaction, dialysis freeze-drying obtains furans and hydrazides hyaluronic acid afterwards for 24 hours.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the height of a concentration of 0.5M is slowly added dropwise
Acid iodide sodium water solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, it is solid to obtain white flock after dialysis freeze-drying
Body obtains furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor
Compound concentration is 2% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (2mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
It is even, it is placed in 4h at 37 DEG C and obtains the syringeability composite hydrogel cell carrier holder of load cells.
Embodiment 2
Sodium Hyaluronate (0.5g, 1.25mmol carboxylate radical) is dissolved in (100mM, pH=in 150ml ethanesulfonic acid buffers
5.5) in, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides are added
(DMTMM) (1.25mmol, 0.35g), after 30min is stirred at room temperature, furylamine (0.625mmol, 55 μ is added dropwise in syringe
L), reaction dialysis freeze-drying afterwards for 24 hours is stirred at room temperature.Said derivative 0.5g is taken to be dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5) DMTMM (0.7g, 2.5mmol), is added after dissolving, adipic dihydrazide (ADH, 1.39g, 8mmol) is added, stirs at room temperature
Mixing reaction, dialysis freeze-drying obtains furans and hydrazides hyaluronic acid afterwards for 24 hours.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the height of a concentration of 0.5M is slowly added dropwise
Acid iodide sodium water solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, it is solid to obtain white flock after dialysis freeze-drying
Body obtains furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor.
Compound concentration is 2% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (2mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
Even, bismaleimide PEG is added, and as crosslinking agent, (molar ratio of the structural unit of crosslinking agent and carboxylic acid is 1:10) 37, are placed in
1h obtains the syringeability composite hydrogel cell carrier holder of load cells at DEG C.
Embodiment 3
Sodium Hyaluronate (0.5g, 1.25mmol carboxylate radical) is dissolved in (100mM, pH=in 150ml ethanesulfonic acid buffers
5.5) in, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides are added
(DMTMM) (1.25mmol, 0.35g), after 30min is stirred at room temperature, furylamine (0.625mmol, 55 μ is added dropwise in syringe
L), reaction dialysis freeze-drying afterwards for 24 hours is stirred at room temperature.Said derivative 0.5g is taken to be dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5) DMTMM (0.7g, 2.5mmol), is added after dissolving, adipic dihydrazide (ADH, 1.39g, 8mmol) is added, stirs at room temperature
Mixing reaction, dialysis freeze-drying obtains furans and hydrazides hyaluronic acid afterwards for 24 hours.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the height of a concentration of 0.5M is slowly added dropwise
Acid iodide sodium water solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, it is solid to obtain white flock after dialysis freeze-drying
Body obtains furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor.
Compound concentration is 2% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (1mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
Even, bismaleimide PEG is added, and as crosslinking agent, (molar ratio of the structural unit of crosslinking agent and carboxylic acid is 1:5) 37, are placed in
1h obtains the syringeability composite hydrogel cell carrier holder of load cells at DEG C.
Embodiment 4
Sodium Hyaluronate (0.5g, 1.25mmol carboxylate radical) is dissolved in (100mM, pH=in 150ml ethanesulfonic acid buffers
5.5) in, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides are added
(DMTMM) (1.25mmol, 0.35g), after 30min is stirred at room temperature, furylamine (0.625mmol, 55 μ is added dropwise in syringe
L), reaction dialysis freeze-drying afterwards for 24 hours is stirred at room temperature.Said derivative 0.5g is taken to be dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5) DMTMM (0.7g, 2.5mmol), is added after dissolving, adipic dihydrazide (ADH, 1.39g, 8mmol) is added, stirs at room temperature
Mixing reaction, dialysis freeze-drying obtains furans and hydrazides hyaluronic acid afterwards for 24 hours.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the height of a concentration of 0.5M is slowly added dropwise
Acid iodide sodium water solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, it is solid to obtain white flock after dialysis freeze-drying
Body obtains furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor.
Compound concentration is 2% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (1mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
It is even, it stirs evenly, bismaleimide PEG is added, and as crosslinking agent, (molar ratio of the structural unit of crosslinking agent and carboxylic acid is 1:
1) it, is placed in 1h at 37 DEG C and obtains the syringeability composite hydrogel cell carrier holder of load cells.
Embodiment 5
Sodium Hyaluronate (1g, 2.5mmol carboxylate radical) is dissolved in 150ml ethanesulfonic acid buffers (100mM, pH=5.5)
In, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides (DMTMM) are added
(10mmol), after 30min is stirred at room temperature, furylamine (2mmol) is added dropwise in syringe, and reaction is stirred at room temperature and dialyses afterwards for 24 hours
Freeze-drying.It takes said derivative 1g to be dissolved in 150ml ethanesulfonic acid buffers (100mM, pH=5.5), DMTMM is added after dissolving
Adipic dihydrazide (ADH, 10mmol) is added in (5mmol), and reaction is stirred at room temperature, and dialysis freeze-drying obtains furans and acyl afterwards for 24 hours
Hydrazine hyaluronic acid.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the high iodine of a concentration of 1M is slowly added dropwise
Acid sodium aqueous solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, white fluffy solid is obtained after dialysis freeze-drying
Obtain furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor.
Compound concentration is 10% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (2mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
Even, bismaleimide PEG is added, and as crosslinking agent, (molar ratio of the structural unit of crosslinking agent and carboxylic acid is 1:10) 37, are placed in
1h obtains the syringeability composite hydrogel cell carrier holder of load cells at DEG C.
Embodiment 6
Sodium Hyaluronate (1g, 2.5mmol carboxylate radical) is dissolved in 150ml ethanesulfonic acid buffers (100mM, pH=5.5)
In, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides (DMTMM) are added
(4mmol), after 30min is stirred at room temperature, furylamine (4mmol) is added dropwise in syringe, and reaction is stirred at room temperature, and dialysis is frozen afterwards for 24 hours
It is dry.It takes said derivative 1g to be dissolved in 150ml ethanesulfonic acid buffers (100mM, pH=5.5), DMTMM is added after dissolving
Adipic dihydrazide (ADH, 2mmol) is added in (2mmol), and reaction is stirred at room temperature, and dialysis freeze-drying obtains furans and hydrazides afterwards for 24 hours
Hyaluronic acid.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the high iodine of a concentration of 1M is slowly added dropwise
Acid sodium aqueous solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, white fluffy solid is obtained after dialysis freeze-drying
Obtain furans and aldehyde radical hyaluronic acid.
Growth factor is made into 300ng/mL PBS solutions, the composite cyclodextrin nano-carrier of pH sensitivities is made into 1mg/mL
PBS solution, (4 DEG C) concussion loadings of low temperature for 24 hours, centrifuge and remove extra growth factor, obtain loading biological work after mixing
The composite cyclodextrin nano-carrier of the pH sensitivities of sex factor.
Compound concentration is 10% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), above-mentioned complex polysaccharide nanometer is carried
Body is mixed into furans and hydrazides polysaccharide derivates solution (2mg/mL), ultrasonic disperse；This two kinds of solution mix in equal volume, and stirring is equal
Even, bismaleimide PEG is added, and as crosslinking agent, (molar ratio of the structural unit of crosslinking agent and carboxylic acid is 1:10) 37, are placed in
1h obtains the syringeability composite hydrogel cell carrier holder of load cells at DEG C.
Comparative example 1
Sodium Hyaluronate (0.5g, 1.25mmol carboxylate radical) is dissolved in (100mM, pH=in 150ml ethanesulfonic acid buffers
5.5) in, fully after dissolving, carboxyl activator 4- (4,6- dimethoxy-triazine) -4- methyl morpholine hydrochlorides are added
(DMTMM) (1.25mmol, 0.35g), after 30min is stirred at room temperature, furylamine (0.625mmol, 55 μ is added dropwise in syringe
L), reaction dialysis freeze-drying afterwards for 24 hours is stirred at room temperature.Said derivative 0.5g is taken to be dissolved in (100mM, pH in 150ml ethanesulfonic acid buffers
=5.5) DMTMM (0.7g, 2.5mmol), is added after dissolving, adipic dihydrazide (ADH, 1.39g, 8mmol) is added, stirs at room temperature
Mixing reaction, dialysis freeze-drying obtains furans and hydrazides hyaluronic acid afterwards for 24 hours.
The hyaluronic acid of furans is further aoxidized with sodium metaperiodate, furans and aldehyde radical hyalomitome are obtained
Acid.It is as follows：The hyaluronic acid of 1g furans is dissolved in 200ml deionized waters, and the height of a concentration of 0.5M is slowly added dropwise
Acid iodide sodium water solution 5ml after being protected from light 2h, is added 1ml ethylene glycol and terminates reaction 1h, it is solid to obtain white flock after dialysis freeze-drying
Body obtains furans and aldehyde radical hyaluronic acid
Compound concentration is 2% furans and hydrazides hyaluronic acid and furans and aldehyde radical hyaluronic acid solution respectively,
Cell after suspending will be mixed into furans and aldehyde radical polysaccharide derivates (4,000,000/mL), this two kinds of solution mix in equal volume
It closes, stirs evenly, be placed in 4h at 37 DEG C and obtain the syringeability composite hydrogel cell carrier holder of load cells.
The specific performance comparative analysis of embodiment 1-4 is as follows：
Gel time in embodiment 1 is 4h, and the gel time in embodiment 2 is 50min, when gel in embodiment 3
Between be 30min, the gel time in embodiment 4 is 10min, and gel time is fewer, and the syringeability of the holder of preparation is better.
Fig. 1 is the upgrowth situation in hydrogel for the cell in embodiment 4 (d, e, f) and comparative example (a, b, c), from figure
In can be seen that cell ovalisation in hydrogel, keep the due phenotype of cell；With the extension of time, thin in hydrogel
Born of the same parents' number gradually increases, and the cell number in embodiment 4 is obviously in extra comparative example.
Fig. 2 is the cell in embodiment 4 (composite hydrogel) and comparative example (pure hydrogel) in water-setting
Activity in glue, as can be seen from Figure with the extension of time, the cell activity in hydrogel gradually increases, in embodiment 4
Cell number is higher than in comparative example.
From DNA content analysis of the cell in the embodiment 4 and comparative example of Fig. 3 in hydrogel, it can be seen that with the time
Extension, the cell DNA content in hydrogel gradually increases, and the cell number in embodiment 4 is significantly higher than in comparative example.
Claims (10)
1. a kind of preparation method of syringeability composite hydrogel cell carrier holder, which is characterized in that the method includes such as
Lower step：
(1) furans polysaccharide is prepared：Polysaccharide containing carboxylate radical is dissolved in ethanesulfonic acid buffer, carboxyl fully, which is added, after dissolving lives
Agent is stirred at room temperature；Furylamine is added, after being reacted under room temperature environment, furans polysaccharide is made in dialysis freeze-drying；
(2) furans and hydrazides polysaccharide are prepared：Above-mentioned furans polysaccharide is dissolved in ethanesulfonic acid buffer, is fully added after dissolving
Carboxyl activator is stirred at room temperature；Adipic dihydrazide is added, after being reacted under room temperature environment, furans and acyl is made in dialysis freeze-drying
Hydrazine polysaccharide；
Prepare furans and aldehyde radical polysaccharide：Above-mentioned furans polysaccharide is soluble in water, it is water-soluble fully to increase sodium iodate after dissolving
Liquid after being protected from light, is added excess ethylene glycol and terminates reaction, furans and aldehyde radical polysaccharide are obtained after dialysis freeze-drying；
(3) double emulsion methods prepare the composite cyclodextrin nano-carrier of the pH sensitivities of load bioactie agent：The pH sensitivities
The preparation of composite cyclodextrin nano-carrier refers to patent CN201710224454.0；Growth factor is made into PBS solution, the pH
Sensitive composite cyclodextrin nano-carrier is made into PBS solution, and low temperature shakes after the two mixing, centrifuges and removes extra growth
The factor obtains the composite cyclodextrin nano-carrier of the pH sensitivities of the load bioactie agent；
(4) furans of step (1) and hydrazides polysaccharide and furans and aldehyde radical polysaccharide are made into aqueous solution respectively；By step
(3) carrier obtained is mixed with above-mentioned furans and hydrazides polysaccharide solution, and carrier is made and furans and hydrazides polysaccharide are mixed
Close liquid；Above-mentioned carrier is mixed with furans and hydrazides polysaccharide mixed liquor and furans and aldehyde radical polysaccharide solution, is added
Crosslinking agent is uniformly mixed and stands, obtains the syringeability composite hydrogel cell carrier holder.
2. a kind of preparation method of syringeability composite hydrogel cell carrier holder as described in claim 1, feature exist
In in the step (1), polysaccharide is Sodium Hyaluronate or heparin, and the mass volume ratio of polysaccharide is 0.1-5%；The carboxyl is lived
Agent is 4- (4,6- dimethoxy-triazines) -4- methyl morpholine hydrochlorides or water-soluble carbodiimides (N- (3-
Dimethylaminopropyl)-N '-ethylcarbodiimide hydrochloride, i.e. DMTMM or EDC, activated carboxylic
The molar ratio of agent and carboxylate radical is 10:1-1:2, the time being stirred at room temperature is 10-120min；The furylamine and carboxylate radical
Molar ratio is 2:1-1:2, react 2-48h under room temperature environment.
3. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 2, feature exist
In in the step (1), polysaccharide is hyaluronic acid, and the mass volume ratio of polysaccharide is 0.2-2%；The carboxyl activator is
The molar ratio of DMTMM, carboxyl activator and carboxylate radical is 5:1-1:1, the time being stirred at room temperature is 20-60min；The furans first
The molar ratio of amine and carboxylate radical is 2:1-1:1, react 4-24h under room temperature environment.
4. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 3, feature exist
In in the step (1), the mass volume ratio of the polysaccharide is 0.5-1%；The molar ratio of carboxyl activator and carboxylate radical is 2:
1, the time being stirred at room temperature is 30-40min；The molar ratio of the furylamine and carboxylate radical is 1:1；It is reacted under room temperature environment
8-12h。
5. a kind of preparation side of syringeability composite hydrogel cell carrier holder as described in claim 1-4 is one of arbitrary
Method, which is characterized in that in the step (2), prepare furans and hydrazides polysaccharide：Polysaccharide is Sodium Hyaluronate or heparin, polysaccharide
Mass volume ratio be 0.1-5%；The carboxyl activator be 4- (4,6- dimethoxy-triazines) -4- methyl morpholine hydrochlorides or
Water-soluble carbodiimides (N- (3-dimethylaminopropyl)-N '-ethylcarbodiimide
The molar ratio of hydrochloride, i.e. DMTMM or EDC, carboxyl activator and carboxylate radical is 10:1-1:2, be stirred at room temperature when
Between be 10-120min；The adipic dihydrazide and the molar ratio of carboxylate radical are 2:1-1:2, react 2-48h under room temperature environment；
Prepare furans and aldehyde radical polysaccharide:The mass volume ratio of furans polysaccharide is 0.1-5%；Sodium metaperiodate aqueous solution it is dense
It is 0.1-2M to spend, and the volume ratio of furans polysaccharide and sodium metaperiodate aqueous solution is 1:1-100:1.
6. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 5, feature exist
In in the step (2), preparing furans and hydrazides polysaccharide:Polysaccharide is hyaluronic acid, and the mass volume ratio of polysaccharide is 0.2-
2%；The carboxyl activator is DMTMM, and the molar ratio of carboxyl activator and carboxylate radical is 5:1-1:1, the time being stirred at room temperature
For 20-60min；The adipic dihydrazide and the molar ratio of carboxylate radical are 2:1-1:1, react 4-24h under room temperature environment；
Prepare furans and aldehyde radical polysaccharide:The mass volume ratio of furans polysaccharide is 0.2-2%；Sodium metaperiodate aqueous solution it is dense
Spend 0.2-1M；The volume ratio of furans polysaccharide and sodium metaperiodate aqueous solution is 1:1-50:1；The time being protected from light is 1-3h.
7. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 6, feature exist
In in the step (2), preparing furans and hydrazides polysaccharide:The mass volume ratio of the polysaccharide is 0.5-1%；Activated carboxylic
The molar ratio of agent and carboxylate radical is 2:1, the time being stirred at room temperature is 30-40min；The molar ratio of the adipic dihydrazide and carboxylate radical
It is 1:1；8-12h is reacted under room temperature environment；
Prepare furans and aldehyde radical polysaccharide:The mass volume ratio of furans polysaccharide is 0.2-0.5%；Sodium metaperiodate aqueous solution
The volume ratio of concentration 0.5M, furans polysaccharide and sodium metaperiodate aqueous solution is 40:1；The time being protected from light is 2h.
8. a kind of preparation method of syringeability composite hydrogel cell carrier holder as described in claim 1, feature exist
In in the step (4), furans and the mass volume ratio of hydrazides polysaccharide and the aqueous solution of furans and aldehyde radical polysaccharide are equal
For 0.5-15%；Furans and aldehyde radical polysaccharide solution can be mixed to prepare cell with suspension cell and furans and aldehyde radical are more
Sugared mixed liquor, a concentration of 1,000,000-1,000 ten thousand/mL of suspension cell；A concentration of 0.1-2mg/mL of carrier, carrier and furans
Change and hydrazides polysaccharide mixed liquor：Cell is 1 with the volume ratio of furans and aldehyde radical polysaccharide mixed liquor:1；Crosslinking agent comes for span
The molar ratio of acid imide PEG, crosslinking agent and carboxylate radical is 2:1-1:10.
9. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 8, feature exist
In, in the step (4), the quality volume of the furans and hydrazides polysaccharide and the aqueous solution of furans and aldehyde radical polysaccharide
Than being 1-10%；A concentration of 2,000,000-500 ten thousand/mL of suspension cell；The concentration 0.2-1mg/mL of carrier；Crosslinking agent with
The molar ratio of carboxylate radical is 1:1-1:5.
10. a kind of preparation method of syringeability composite hydrogel cell carrier holder as claimed in claim 9, feature exist
In, in the step (4), the quality volume of the furans and hydrazides polysaccharide and the aqueous solution of furans and aldehyde radical polysaccharide
Than being 2-5%；A concentration of 3,000,000-400 ten thousand/mL of suspension cell；The concentration 1mg/mL of carrier；Crosslinking agent and carboxylic acid
The molar ratio of root is 1:1-1:2.
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| CN112159534A (en) * | 2020-09-28 | 2021-01-01 | 吾奇生物医疗科技(镇江)有限公司 | Hyaluronic acid hydrogel and preparation method and application thereof |
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