CN108610491A - Thermo-sensitive graft polymers, the hydrogel that cell can be carried and preparation method and application - Google Patents
Thermo-sensitive graft polymers, the hydrogel that cell can be carried and preparation method and application Download PDFInfo
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Abstract
The invention belongs to intellectual material, high molecular material and biomedical engineering field are related to a kind of Thermo-sensitive graft polymers, the hydrogel that can carry cell and preparation method and application.The present invention is by poly glycol monomethyl ether carboxylated, by ethyl cellulose bromination.Brominated ethyl cellulose is subjected to DCC with the poly glycol monomethyl ether of carboxylated and reacts acquisition graft copolymer EC Br/mPEG.Again by EC Br/mPEG and the active monomer MEO of removing2MA and OEGMA475And PMDETA, CuBr carry out ATRP reactions, obtain graft copolymer.Product is dialysed to transparent, freezing is dissolved in water to form hydrogel solution after being evaporated.Hydrogel is further made injectable and wraps up Cellular gels microballoon.Gained gel micro-ball of the invention adjustable, no toxic biological property with response temperature, has wide application prospect in terms of medical treatment.The synthetic method is simple and practicable, and raw material all can industrialized production.
Description
Technical field
The invention belongs to intellectual material, high molecular material and biomedical engineering field, and in particular to a kind of Thermo-sensitive connects
Graft copolymer, the hydrogel that cell can be carried and preparation method and application.
Background technology
Intelligent organic material obtains extensive concern in recent years, these materials can to environmental stimuli, as temperature, pH value, light,
Salt, sugar etc. respond, and are all widely used in nanosecond science and technology, biomedical sector.It is point that temperature-sensitive hydrogel, which is with water,
The water-soluble high-molecular substance of dispersion media introduces a part of hydrophobic grouping and hydrophilic radical.Wherein hydrophilic radical and hydrone knot
It closes, and the cross-linked polymer of hydrophobic grouping water-swellable forms tridimensional network.Temperature-sensitive hydrogel maximum is characterized in having
Minimum critical inversion temperature (LCST).
Methacrylic acid -2 (2- methoxy ethoxies) ethyl ester (MEO2) and methoxypolyethylene glycol methacrylate MA
(OEGMA475) homologue as polyethylene glycol (PEG), it can be obtained by different rate of charges of the adjusting between them different
LCST, with Human Physiology temperature mutually suitable for (Lutz, J.F.；Hoth,A.Macromolecules2006,39,893.doi:
10.1021/ma0517042).P (MEO simultaneously2MA-co-OEGMA475) also there is good biocompatibility, in biomedicine
There is good application prospect in field.
Invention content
It is an object of the present invention to overcome the deficiencies of the prior art and provide a kind of Thermo-sensitive graft polymers, cell can be carried
Hydrogel and preparation method and application, the temperature-responsive of gained temperature-sensitive hydrogel it is adjustable.
P(MEO2MA-co-OEGMA475) when higher than its LCST, hydrophilic and hydrophobic is changed into hydrophobic by hydrophilic, passes through tune
Save the MEO of different ingredient proportions2MA and OEGMA475, its phase transition temperature can be controlled.Its phase transition temperature and rate of charge
Relationship is about at line style relationship, wherein when molar ratio is
MEO2MA：OEGMA475=92:8, phase transition temperature is 37 DEG C of (Lutz J F.Polymerization of
oligo(ethylene glycol)(meth)acrylates:Toward new generations of smart
biocompatible materials[J].Journal of Polymer Science Part A Polymer
Chemistry,2008,46(11):3459-3470.)。
In order to form gel structure, the present invention introduces poly glycol monomethyl ether (mPEG) on main chain ethyl cellulose (EC)
As permanent hydrophilic group, to maintain its hydrogel structure.Poly glycol monomethyl ether (mPEG) has good hydrophily, one
End is blocked by monomethyl ether, and one end, which is hydroxyl, still reactivity.When so that mPEG being grafted on ethyl cellulose (EC), it will not occur
Chemical crosslinking.
Use ethyl cellulose as main chain, grafting poly glycol monomethyl ether (mPEG) and methacrylic acid -2 in the present invention
(2- methoxy ethoxies) ethyl ester (MEO2MA) with methoxypolyethylene glycol methacrylate (OEGMA475) copolymer, form life
Object nontoxicity temperature-sensitive hydrogel, gained temperature-sensitive hydrogel keeps gel state when its temperature is higher than LCST, when temperature is low
When LCST, become solution.
In order to realize above-mentioned target, the present invention provides following technical solutions：
The present invention is by poly glycol monomethyl ether carboxylic (mPEG) carboxylated, by ethyl cellulose (EC) bromination.By brominated second
Base cellulose (EC-Br) carries out DCC with the poly glycol monomethyl ether (mPEG-SA) of carboxylated and reacts acquisition graft copolymer EC-
Br/mPEG.Again by EC-Br/mPEG and the active monomer MEO of removing2MA and OEGMA475And PMDETA, CuBr carry out ATRP
Reaction, finally obtains graft copolymer EC- (MEO2MA-co-OEGMA475)/mPEG.Product is dialysed to transparent, freezing is evaporated
After be dissolved in water to form hydrogel, by the variation of temperature to realize load cytosis.
The present invention provides the preparation that a kind of formation can carry the Thermo-sensitive graft polymers needed for the temperature-sensitive hydrogel of cell
Method is as follows：
(1) ethyl cellulose is dried, is dried for 24 hours in 60 DEG C of vacuum drying ovens.Ethyl cellulose after drying is dissolved in
In dry methylene chloride and a small amount of triethylamine is added, 2- bromine isobutyryls are slowly added dropwise under ice bath in flask, are stirred to react 30h.
It is washed, is washed with sodium bicarbonate, anhydrous magnesium sulfate drying is filtered, concentration；It is precipitated in anhydrous ether, is purified after filtering, is dry,
Obtain product EC-Br.
(2) monomethyl ether polyethylene glycol is dissolved in solvent toluene, and flows back 12h in 120 DEG C of azeotropic to remove with solvent toluene
Then water steams half solvent, succinic anhydride excess is added after system cooling, in 150 degree of back flow reaction 5h.Product is dissolved
In the sodium bicarbonate solution of 1mol/L, it is cooled to 0~5 DEG C and filters.Filtrate is acidified with 5% hydrochloric acid, then with three chloromethanes
Alkane extracts.It collects organic phase to be washed with deionized, be dried with anhydrous magnesium sulfate, concentrate, precipitated in ice ether, dry sample
Product obtain product mPEG-SA.
(3) products therefrom mPEG-SA in products therefrom EC-Br in (1) and (2) is dissolved in dry solvent A, according to
Suitable DMAP and DCC is added in reaction designing, and wherein rate of charge is acid：Alcohol：DCC：DMAP=4:1:4:2, in 20 DEG C of environment
Lower reaction 48h.Products therefrom is filtered to take into filtrate, dialyse and is lyophilized to obtain product EC-Br/mPEG.
(4) under nitrogen protection, by products therefrom and OEGMA in (3)475Add with MEO2MA, PMDETA, CuBr and solvent B
Enter into single-necked flask to carry out ATRP reactions, system is passed through：Vacuumize-three bouts of applying argon gas after, argon gas atmosphere, 60 DEG C,
It is reacted under the conditions of magnetic agitation, reacts 4~8 hours, dialyse and be lyophilized, obtain Thermo-sensitive graft polymers EC- (MEO2MA-co-
OEGMA475)/mPEG.The recipe ratio that wherein feeds intake is adjusted according to the low critical inversion temperature of required gel (LCST).
The Thermo-sensitive graft polymers EC- (MEO obtained by above-mentioned preparation method2MA-co-OEGMA475The structure of)/mPEG
Formula is as follows：
The present invention also provides a kind of temperature-sensitive hydrogels for carrying cell formed by above-mentioned Thermo-sensitive graft polymers.Institute
The preparation method for stating the temperature-sensitive hydrogel that can carry cell is：Thermo-sensitive graft polymers is dissolved in the water, in predetermined temperature
Lower place 1 hour forms hydrogel completely to determine.(it is MEO with molar ratio2MA：OEGMA475=92:For 8, make a reservation for
Temperature is 37 DEG C).
It, can be micro- to prepare injectable load cell after Thermo-sensitive graft polymers provided by the invention is changed into hydrogel
Ball, the preparation method which carries cell microsphere are：
(1) specific cells are collected first, are cultivated, and specific cultural method is determined according to the type of cell.
(2) secondly by Thermo-sensitive graft polymers and certain distilled water mixed preparing hydrogel solution, hydrogel solution exists
37 DEG C are sterilized by vacuum filter, and hydrogel mixing with cells liquor is mixed to get with cell and its culture solution obtained by step (1).
(3) finally hydrogel cell mixture obtained by step (2) is fitted into syringe, and by hydrogel cell mixture
It instills in sterile mineral oil, is gradually heating to 37 degree.Because the hydrophobic interaction of oil makes drop harden into particle.Use nylon
They are collected by filtration screen cloth, are put into the sterile beaker of phosphate buffered saline (PBS) and shake cleaning, are blotted, obtained with sterile towel
Cellular gels microballoon is carried to injectable.
In the present invention, solvent A is dry tetrahydrofuran, methyl phenyl ethers anisole, toluene, n,N-Dimethylformamide, N, N- diethyl
One or more of formamide or DMAC N,N' dimethyl acetamide.
In the present invention, solvent B is dry tetrahydrofuran, methyl phenyl ethers anisole, n,N-Dimethylformamide, N, N- diethylformamides
Or one or more of DMAC N,N' dimethyl acetamide.
In the present invention, OEGMA475And MEO2MA is the monomer for having been removed polymerization inhibitor.
The advantage of the invention is that：Raw material sources are extensive, methacrylic acid -2 used (2- methoxy ethoxies) ethyl ester
(MEO2MA), methoxypolyethylene glycol methacrylate (OEGMA475), poly glycol monomethyl ether (mPEG), hydroxyethyl cellulose
(EC), solvent, precipitating reagent etc. can industrialized production, synthetic method is simple and practicable.EC- (the MEO of preparation2MA-co-
OEGMA475)/mPEG, can be by adjusting its OEGMA475And MEO2Its temperature-responsive of MA proportion adjustments (is with molar ratio
MEO2MA：OEGMA475=92:For 8, it is predefined as 37 DEG C), and biological nontoxic, cell can be carried.The grafting hydrogel material
The injectable gel microballoon for forming package cell in vitro, under the protection of gel micro-ball, treatment cell is injected into vivo, and
And release can be pinpointed in lesion region (due to local temperature low area caused by blood vessel blockage), the efficiency for the treatment of is improved, is
Treatment provides a kind of thinking, is had a wide range of applications in fields such as medicine controlled releasing, biological intelligence switch, biosensors.
Description of the drawings
Fig. 1 is to prepare Thermo-sensitive graft polymers EC- (MEO2MA-co-OEGMA475The reaction line map of)/mPEG.
Fig. 2 is the nuclear-magnetism figure of EC-Br prepared by embodiment 1；
Fig. 3 is the nuclear-magnetism figure of EC-Br/mPEG prepared by embodiment 1；
Fig. 4 is EC- (MEO prepared by embodiment 12MA-co-OEGMA475The nuclear-magnetism figure of)/mPEG.
Specific implementation mode
The following examples are further illustrations of the invention, it rather than limits the scope of the invention.
Embodiment 1
The preparation of Thermo-sensitive graft polymers, reaction route figure are as shown in Figure 1：
(1) by 2.27g (0.12mmol) ethyl cellulose, 1.46ml (10.5mmol) triethylamine, solvent is 10ml dryings
Dichloromethane is added in dry reaction bottle, and 2.259ml (21mmol) 2- bromines isobutyryl and 5ml dryings two are added in the first funnel
Chloromethanes.It is slowly added dropwise under ice bath.Shading is reacted 30 hours.Product first washs three respectively with saturated sodium bicarbonate and distilled water
Secondary, the drying of organic phase solution anhydrous magnesium sulfate is stood overnight, and is precipitated in n-hexane after filtering, product, vacuum is obtained by filtration
40 degree dry 48 hours, obtain product brominated ethyl cellulose EC-Br, the nuclear-magnetism figure of EC-Br is as shown in Figure 2.
(2) 6g (1.5mmol) monomethyl ether polyethylene glycol and toluene are steamed into half to remove after 12 hours in 140 degree of azeotropic
4.5g (45mmol) succinic anhydrides were added in 150 degree of back flow reactions 5 hours in water.Residue is dissolved in saturated sodium bicarbonate, cold
But 0~5 filter is spent, filtrate is acidified with 5% hydrochloric acid, is purified with chloroform, and anhydrous sodium sulfate drying is added in deionization washing,
Ice ether precipitation is injected, filtering drying obtains carboxylation monomethyl ether polyethylene glycol.
(3) carboxylation monomethyl ether polyethylene glycol obtained by 6g steps (2) is taken, bromination ethyl cellulose obtained by 1.5g steps (1),
0.8g DMAP, 0.6g DCC carry out esterification and carry out esterification at room temperature in anhydrous tetrahydro furan 24 hours, after filtering
Dialysis freeze-drying obtains EC-Br/mPEG, and the nuclear-magnetism figure of EC-Br/mPEG is as shown in Figure 3.
(4) under dry argon gas, by EC-Br/mPEG, 0.15g CuBr, 0.33g PMDETA obtained by 3.19g steps (3),
2.5ml MEO2MA,0.18ml OEGMA475, 60 degree are reacted 3 hours in dry tetrahydrofuran.Dialysis freeze-drying obtains product EC-
(MEO2MA-co-OEGMA475)/mPEG, EC- (MEO2MA-co-OEGMA475The nuclear-magnetism figure of)/mPEG is as shown in Figure 4.
The preparation of the temperature-sensitive hydrogel of cell can be carried：
(5) by step (4) products therefrom EC- (MEO2MA-co-OEGMA475)/mPEG takes 0.5g and matches 0.5ml distilled water
Obtain hydrogel solution.
Injectable carries the preparation of Cellular gels microballoon：
(6) by hydrogel solution obtained by step (5), at 37 degree, culture obtains hydrogel/carefully together with cell after disinfection
Born of the same parents' suspension.Hydrogel cell mixture is fitted into syringe.Mixed solution is instilled in room-temperature sterile mineral oil, is gradually risen
Temperature is to 37 degree.Because the drop that the hydrophobic interaction of oil is hardens into particle, they are collected by filtration with nylon net cloth, is put into
Have and shake cleaning in the sterile beaker of phosphate buffered saline (PBS), blotted with sterile towel, obtains injectable and carry Cellular gels microballoon.
Embodiment 2
The preparation of Thermo-sensitive graft polymers：
(1) by 2.27g (0.12mmol) ethyl cellulose, 1.46ml (10.5mmol) triethylamine, solvent is 10ml dryings
Dichloromethane is added in dry reaction bottle, and 2.259ml (21mmol) 2- bromines isobutyryl and 5ml dryings two are added in the first funnel
Chloromethanes.It is slowly added dropwise under ice bath.Shading is reacted 30 hours.Product first washs three respectively with saturated sodium bicarbonate and distilled water
Secondary, the drying of organic phase solution anhydrous magnesium sulfate is stood overnight, and is precipitated in n-hexane after filtering, product, vacuum is obtained by filtration
40 degree dry 48 hours, obtain product brominated ethyl cellulose.
(2) 6g (1.5mmol) monomethyl ether polyethylene glycol and toluene are steamed into half to remove after 12 hours in 140 degree of azeotropic
4.5g (45mmol) succinic anhydrides were added in 150 degree of back flow reactions 5 hours in water.Residue is dissolved in saturated sodium bicarbonate, cold
But 0~5 filter is spent, filtrate is acidified with 5% hydrochloric acid, is purified with chloroform, and anhydrous sodium sulfate drying is added in deionization washing,
Ice ether precipitation is injected, filtering drying obtains carboxylation monomethyl ether polyethylene glycol.
(3) carboxylation monomethyl ether polyethylene glycol obtained by 2.5g steps (2) is taken, bromination ethyl cellulose obtained by 0.5g steps (1),
0.4g DMAP, 0.3g DCC carry out esterification and carry out esterification at room temperature in anhydrous tetrahydro furan 24 hours, after filtering
Dialysis freeze-drying obtains EC-Br/mPEG.
(4) under dry argon gas, by EC-Br/mPEG, 0.07g CuBr, 0.33g PMDETA obtained by 1.9g steps (3),
2.0ml MEO2MA,0.15ml OEGMA475, 60 degree are reacted 3 hours in dry tetrahydrofuran.Dialysis freeze-drying obtains product EC-
(MEO2MA-co-OEGMA475)/mPEG。
The temperature-sensitive hydrogel of cell can be carried and injectable carries the preparation process of Cellular gels microballoon with embodiment 1
Foregoing description is only the description to present pre-ferred embodiments, is not any restriction to the scope of the invention.Appoint
Any change or modification what those skilled in the art makes according to the technology contents of the disclosure above should all regard
For equivalent effective embodiment, the range of technical solution of the present invention protection is belonged to.
Claims (8)
1. a kind of preparation method of Thermo-sensitive graft polymers, which is characterized in that be as follows：
(1) ethyl cellulose is dried, is dried for 24 hours in 60 DEG C of vacuum drying ovens.Ethyl cellulose after drying is dissolved in drying
In dichloromethane and a small amount of triethylamine is added, 2- bromine isobutyryls are slowly added dropwise under ice bath in flask, are stirred to react 30h, then
It is washed, is washed with sodium bicarbonate, anhydrous magnesium sulfate drying is filtered, concentration；It is precipitated in anhydrous ether again, is purified after filtering, is dry
It is dry, obtain product EC-Br；
(2) monomethyl ether polyethylene glycol is dissolved in solvent toluene, and flows back 12h to remove water, so in 120 DEG C of azeotropic with solvent toluene
After steam half solvent, it is excessive to be added succinic anhydride after system cooling, and in 150 degree of back flow reaction 5h, product is dissolved in
It in the sodium bicarbonate solution of 1mol/L, is cooled to 0~5 DEG C and filters, filtrate is acidified with 5% hydrochloric acid, then uses chloroform
Extraction is collected organic phase and is washed with deionized, dried with anhydrous magnesium sulfate, concentrate, precipitated in ice ether, dries sample,
Obtain product mPEG-SA；
(3) products therefrom mPEG-SA in products therefrom EC-Br in step (1) and step (2) is dissolved in dry solvent A,
Suitable DMAP and DCC is added according to reaction designing, wherein rate of charge is acid：Alcohol：DCC：DMAP=4:1:4:2, at 20 DEG C
48h is reacted under environment, products therefrom is filtered to take into filtrate, dialyse and is lyophilized to obtain product EC-Br/mPEG；
(4) under nitrogen protection, by products therefrom and OEGMA in step (3)475And MEO2MA, PMDETA, CuBr and solvent B add
Enter into single-necked flask to carry out ATRP reactions, system is passed through：Vacuumize-three bouts of applying argon gas after, argon gas atmosphere, 60 DEG C,
It is reacted under the conditions of magnetic agitation, reacts 4~8 hours, dialyse and be lyophilized, obtain product EC- (MEO2MA-co-OEGMA475)/
mPEG；The recipe ratio that wherein feeds intake is adjusted according to the low critical inversion temperature of required gel (LCST).
2. the preparation method of Thermo-sensitive graft polymers according to claim 1, it is characterized in that solvent A is dry tetrahydrochysene furan
Mutter, methyl phenyl ethers anisole, toluene, N,N-dimethylformamide, N, one kind in N- diethylformamides or DMAC N,N' dimethyl acetamide or
It is several.
3. the preparation method of Thermo-sensitive graft polymers according to claim 1, it is characterized in that solvent B is dry tetrahydrochysene furan
It mutters, methyl phenyl ethers anisole, N,N-dimethylformamide, N, one or more of N- diethylformamides or DMAC N,N' dimethyl acetamide.
4. the preparation method of Thermo-sensitive graft polymers according to claim 1, feature OEGMA475And MEO2MA is
Through the monomer for removing polymerization inhibitor.
5. a kind of Thermo-sensitive graft polymers that preparation method according to claim 1 obtains, structure are as follows：
6. the temperature-sensitive hydrogel for carrying cell that a kind of Thermo-sensitive graft polymers by described in claim 5 is formed.
7. a kind of preparation method by the temperature-sensitive hydrogel for carrying cell described in claim 6, it is characterised in that：It will be temperature sensitive
Property graft polymers be dissolved in the water, at a predetermined temperature place one hour to ensure to form hydrogel completely.
8. a kind of temperature-sensitive hydrogel for carrying cell by described in claim 6 answering in injectable carries Cellular gels microballoon
With the preparation process that the injectable carries Cellular gels microballoon is as follows：
(1) specific cells are collected, are cultivated, specific cultural method is determined according to the type of cell；
(2) by temperature sensing polymer and certain distilled water mixed preparing hydrogel solution, hydrogel solution passes through vacuum at 37 DEG C
Filtration sterilization is mixed to get hydrogel cell mixture with cell and its culture solution obtained by step (1)；
(3) hydrogel cell mixture obtained by step (2) is fitted into syringe, and the instillation of hydrogel cell mixture is sterile
In mineral oil, 37 degree are gradually heating to, they are collected by filtration with nylon net cloth, is put into the sterile burning of phosphate buffered saline (PBS)
Cleaning is shaken in cup, is blotted with sterile towel, is obtained injectable and is carried Cellular gels microballoon.
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| WO2020134385A1 (en) * | 2018-12-28 | 2020-07-02 | 华南理工大学 | Temperature-sensitive polymer, synthesis method thereof, and temperature-sensitive injectable hydrogel |
| CN112043863A (en) * | 2020-08-02 | 2020-12-08 | 华南理工大学 | Titanium-based implant capable of selectively expressing 'anti-fouling-cell adhesion promotion-sterilization' functions in response to temperature change and preparation method thereof |
| CN116655995A (en) * | 2023-05-23 | 2023-08-29 | 浙江工业大学 | Preparation method of temperature-pH responsive ethyl cellulose aerogel and application of temperature-pH responsive ethyl cellulose aerogel in oil-water separation |
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Application publication date: 20181002 |