CN107556482A - A kind of injectable high intensity chitin based aquagel and its preparation method and application - Google Patents
A kind of injectable high intensity chitin based aquagel and its preparation method and application Download PDFInfo
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Abstract
The present invention provides a kind of preparation method of injectable high intensity chitin based aquagel product, prepare binary or polynary alkynyl-modified hydrophilic polymer A, the chitin derivativ B containing amino is prepared again, the A and B aqueous solution is uniformly mixed, the injection aquagel precursor solution with good fluidity is formed, it is formed in situ the high intensity hydrogel in physiological conditions.The present invention utilizes spontaneous amine alkynes click-reaction, it is chemically crosslinked by forming enamine bonds, original position prepares injection aquagel in physiological conditions, any micromolecule catalyst, initiator or crosslinking agent are not needed, without heating, ultraviolet or radiation etc., it is nontoxic non-stimulated, it is not necessary to any post processing.Using Thermo-sensitive chitosan derivative, in situ being physical crosslinking rapid shaping, further crosslinking prepares injectable high intensity hydrogel in the original location again.Such hydrogel good biocompatibility, there is pH sensitiveness and biodegradable, can be widely applied to biomedicine field.
Description
Technical field
The invention belongs to bio-medical material and field of tissue engineering technology, and in particular to a kind of injectable intelligence high intensity crust
Plain based aquagel product and its preparation method and application.
Background technology
Macromolecule hydrogel be it is a kind of have three-dimensional net structure and adsorb large quantity of moisture and cross-linked polymeric not soluble in water
Thing, because it is close to extracellular matrix, there is good hydrophily, excellent swelling behavior and biocompatibility etc., in life
Thing medicine and tissue engineering material field are with a wide range of applications.For example, hydrogel can be used for the surface of a wound as medicine equipment
Dressing, bonding, closure and antiseep in operation, stops blooding in operation, and tissue filling, Post operation prevents adhesion, or for biology
The bio-medical fields such as the carrier and tissue engineering material of active material and cell.
According to the gel formats of hydrogel, external gel and internal in-situ gel can be divided into.Internal in-situ gel has can
The advantages of injectivity, using the injection aquagel precursor solution with good fluidity, in-situ cross-linked shape in physiological conditions
Into hydrogel, it cannot be only used for open operation and can also be used for Minimally Invasive Surgery.According to the formation basic theory of hydrogel, chemistry can be divided into
Cross-linked hydrogel and the major class of physical cross-linking hydrogel two.Because chemical crosslinking needs the time, and chemical reactivity material
Genotoxic potential and the irreversibility typically to chemically react limit the application of chemically crosslinked aquagel in vivo.Such as Bioglue
It is a kind of based on bovine albumin solution and situ-gel sealing agent inside glutaraldehyde solution, utilizes the amino of bovine serum albumin(BSA)
With the aldehyde radical cross-linking reaction of glutaraldehyde, the state such as the U.S., European Union, Canada and Australia food and medicine pipe has been obtained abroad
Reason department ratifies, and is widely used in cardiovascular and big vascular surgery seepage place and mechanically seals；But Bioglue residuals penta
Dialdehyde can cause cytotoxicity, or even can trigger nervous tissue degeneration [Chinese patent application prospectus
CN201510960917.0].Physical gel typically utilizes its response to extraneous environmental stimulus (such as temperature, pH value, light)
Reversible crosslinking is formed, particularly Thermo-sensitive Injectable polymer hydrogel is widely studied for medical purpose.It is this kind of temperature sensitive
Property gel liquid is kept in low temperature, can equably contain cell/medicine and without surgical operation be implanted into it is suitable so as to improve patient
Ying Xing, when into internal 37 DEG C, quickly form gel and avoid the losses such as cell and bioactive molecule from promoting damage location
Reparative regeneration, because temperature-responsive is to be easiest to realization and maximally effective response relatively, this kind of intelligent aqueous gel is in biological medicine
There is tempting development prospect with tissue engineering material field.
Because chitin and its derivative have extraordinary biocompatibility, biodegradability and a variety of biologies living
Property, it is suitable for the application of bio-medical material and field of tissue engineering technology.Chitin is with carboxylated reagent in sodium hydroxide-urea body
Reacted in system, carboxy chitin [the Chinese invention patent application prospectus with pH sensitiveness and Thermo-sensitive can be made
CN201310641249.6], chitin reacts with hydroxypropylation reagent in sodium hydroxide-urea system, can be made with temperature
The Hydroxypropyl chitosan [Chinese invention patent application prospectus CN201410170871.8] of quick property, Hou Chunlin etc. is reported
Thermo-sensitive hydroxyl butyl chitosan [Chinese patent application prospectus CN200810033699.6] and Thermo-sensitive hydroxyl amyl group shell gather
The preparation of sugared [Chinese patent application prospectus CN201210220246.0].But Thermo-sensitive chitosan derivative passes through liter
The strength of materials made from warm physical gel is weaker.Therefore, a kind of safety and the new injectable water-setting with higher-strength are developed
Glue seems particularly urgent on clinical treatment.
In recent years, click-reaction is turned into its controllable precise, the advantage such as reaction is efficiently quick, yield is high and selectivity is strong
Prepare the strong instrument of hydrogel.However, most of click-reaction reported needs due to transition-metal catalyst be present
Heat or ultraviolet radioactive, precursor prepare the defects of cumbersome, it is restricted in biological field
[Biomacromolecules, 2016,17：1-3；Advanced Functional Materials, 2014,24：1679-
1686；Chinese patent application prospectus CN201610947867.7].Therefore, develop without catalyst and initiator, use
Preparation and the click-reaction that can efficiently prepare injection aquagel in physiological conditions have important section before easy
Learn meaning and application value.In the recent period, this loyal seminar of Tang reports a kind of spontaneous amine-alkynes (amino-yne) and clicks on polymerization instead
Should, they using ester group activate binary terminal alkyne compound high-efficiency polymerization obtains macromolecule at room temperature with binary aliphatic secondary amine
Amount poly- (β amino acrylates) [Journal of the American Chemical Society, 2017,139：
5437].However, up to the present, also form enamine bonds crosslinking not over spontaneous amine-alkynes (amino-yne) click-reaction
Injectable high intensity hydrogel report.
The content of the invention
For deficiency of the prior art, with reference to the basis of previous work, it is an object of the invention to provide a kind of injectable
High intensity chitin based aquagel and its preparation method and application.The present invention is clicked on anti-using spontaneous amine-alkynes (amino-yne)
Should, it is chemically crosslinked by forming enamine bonds, prepares injection aquagel in physiological conditions, it is not necessary to which any small molecule is urged
Agent or crosslinking agent, good biocompatibility, system have pH sensitiveness and biodegradable, can be widely applied to biological medicine
Field.
Technical scheme is as follows：
A kind of preparation method of injectable high intensity chitin based aquagel product, is prepared using following method：
(1) binary or the water solution A of polynary alkynyl-modified hydrophilic polymer are prepared；
(2) aqueous solution B of the chitin derivativ containing amino is prepared；
(3) A and B are uniformly mixed, forms the injection aquagel precursor solution with good fluidity；
(4) the hydrogel precursor solution that step (3) obtains is formed in situ the high intensity hydrogel in physiological conditions.
Preferably, the hydrophilic polymer in the water solution A is poly- containing two or more ester alkynes terminal groups modifications
Ethylene glycol PEG derivatives or their mixture,
The described polyethylene glycol PEG derivant structure formulas containing two (linear) ester alkynes terminal groups modifications are shown below：
The described polyethylene glycol PEG derivant structure formulas containing three (three arms) ester alkynes terminal groups modifications are shown below：
The described polyethylene glycol PEG derivant structure formulas containing multiple (multi-arm) ester alkynes terminal groups modifications are shown below：
Wherein n, x, y, z are 1-1000 integer, it is therefore preferable to 20-400 integer.
Polyethylene glycol has the hydrophily of height, and does not have immunogenicity, and its biocompatibility has been obtained for FDA
Certification, the medical device product prepared by medical polyethylene glycol (PEG) product can be widely applied for the various surgical operations of human body
In the bonding of middle wound, hemostasis, antiseep and the material such as prevent adhesion.And the PEG of different polymerization degree can be chosen, improves water-setting
Colloidality can be to meet various needs.
Preferably, the water solution A can also be by other hydrophilic polymers, such as dextran (such as dextran
1, weight average molecular weight 1000, dextran 3.5, weight average molecular weight 3500, dextran 5, weight average molecular weight 5000) and, over-expense
Change polyhydroxylated polymer (such as Boltorn H40, Boltorn H30, Boltorn H20, hyperbranched polyglycidyl ether, surpass
Branched polyhydroxy polycarboxylic carboxylic acid amide esters), or dendritic carry out it is polynary it is alkynyl-modified obtain it is polynary alkynyl-modified hydrophilic
Property polymer.
Preferably, the chitin derivativ containing amino may be selected from carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl
In base enclosure glycan, hydroxyl butyl chitosan, carboxymethyl chitin, ethoxyl chitin, Hydroxypropyl chitosan, hydroxyl butyl chitin
Any or their mixture；Wherein carboxymethyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxyl butyl shell gather
The acetyl degree scope of sugar is 0.01~0.4；Carboxymethyl chitin, ethoxyl chitin, Hydroxypropyl chitosan, hydroxyl butyl chitin
Acetyl degree scope be 0.7~0.92, molecular weight ranges are 5kDa~1000kDa.
Preferably, the aqueous solution B is the aqueous solution of the chitin derivativ containing amino with Thermo-sensitive, such as China
A kind of hydroxypropyl crust with temperature sensitivity that application for a patent for invention prospectus CN 201410170871.8 is recorded
Element.It also has Thermo-sensitive after being mixed with solution A, at 37 DEG C of physiological temp can rapid physical in situ be cross-linked into gel, then
Injectable high intensity hydrogel product is made in further crosslinking in situ.
Preferably, the mass concentration scope of hydrophilic polymer is 0.5~30wt% in solution A, polymer in solution B
Mass concentration scope is 0.5~20wt%, and the solution A and the reactive functionality alkynyl of solution B and the mol ratio of amino
It is 0.05~5 to be worth scope.Any buffer solution that the optional pH of the described aqueous solution is 5~11, can optionally certainly as solvent
Phosphate buffer, borate buffer solution, histidine buffering liquid, sodium bicarbonate-carbonate buffer solution, Tris-HCl buffer solutions,
The combination of diethanolamine buffer or above-mentioned buffer solution, preferably phosphate normal saline buffer solution, preferable ph 7.0-8.0.
Preferably, step (1), (2), the configuration temperature of (3) are 2-15 DEG C, by A and B quickly full and uniform mixing, shape
Into the injection aquagel precursor solution with good fluidity.Preferably, step (4) described physiological condition is body temperature,
It is chemically crosslinked by forming enamine bonds, is formed in situ the high intensity hydrogel, the process does not need any small molecule to urge
Agent or small molecule crosslinking agent, it is crosslinked to post-process direct in-situ use.
A kind of injectable high intensity chitin based aquagel, adopts and is prepared with the aforedescribed process.
Above-mentioned injectable high intensity chitin based aquagel be applied to the various operations of human body in bonding, closure, hemostasis,
Antiseep prevents adhesion, and bioactive substance and the carrier of cell and organizational project material for bio-medical field
Material.
Compared with prior art, the present invention has following technique effect：
(1) bi-component Injectable in-situ crosslinking aquogel biomaterial prepared by the present invention, utilizes spontaneous amine-alkynes
(amino-yne) click-reaction, it is chemically crosslinked by forming enamine bonds, can solidification obtains mechanics under physiological condition in vivo
The good hydrogel of performance, it is not necessary to any micromolecule catalyst or crosslinking agent, it is not necessary to heat, illumination, the external condition such as radiation,
Its preparation method is simple and quick, the generation of process no coupling product, good biocompatibility, easily realizes industrialized production.According to we institute
Know, the injectable high intensity hydrogel of enamine bonds crosslinking is also formed not over spontaneous amine-alkynes (amino-yne) click-reaction
Report, therefore there is innovative and extremely important meaning.
(2) hydrogel described in can dissolve in the buffer solution of low pH value, adjustment pH to it is neutral when can form gel again,
With pH sensitivity characteristics, in addition also there is good biocompatibility, biodegradable, in biological medicine or tissue work
Journey is with a wide range of applications in field.
(3) general polymerization thing hydrogel product apply bonding in the various operations of human body, closure, hemostasis, antiseep or
When person prevents adhesion, if gel precursors solution viscosity is too big, can not effectively it penetrate into biological tissue space (such as stitching holes),
It can not be sprawled in tissue surface rapidly, bond, block, stopping blooding or preventing adhesiving effect is bad, for bioactive substance and carefully
The bio-medical fields such as the carrier and tissue engineering material of born of the same parents, also require that hydrogel has syringeability, the present invention can use tool
There is the aqueous solution B of Thermo-sensitive chitosan derivative, configuring precursor solution and mixing, system at low temperature has good fluidity,
With syringeability, gel can be cross-linked into by first rapid physical in situ under body temperature in vivo being injected into, then enter one in the original location
Injectable high intensity hydrogel product is made in step chemical reaction crosslinking.
(4) precursor of subject hydrogel is easily obtained or prepared, before the preparation of hydrogel is by changing two kinds of hydrogels
The quantity of functional group's (ester ethynylene group and amino) and adjusted in the concentration of body, polymer with when polymer molecular weight etc..
Brief description of the drawings
Fig. 1 is precursor line style dibasic acid esters alkynes end group PEG (DA-PEG), precursor carboxymethyl chitosan in the embodiment of the present invention 1
(CM-Chitosan) and made 0.28-2.5% hydrogels FTIR spectrograms；
Fig. 2 be the embodiment of the present invention 1 in hydrogel (a) gel rheometer test (37 DEG C, frequency:1Hz；Strain:1%)
(b) relation of storage modulus and Loss modulus and frequency (strain:1%)；
Fig. 3 is the compression failure stress-strain diagram of hydrogel in the embodiment of the present invention 1；
Fig. 4 is 0.28-2.5%DA-PEG/CM-Chitosan hydrogels and COS-7 cells contactings in the embodiment of the present invention 1
The cell proliferation rate of different time；
Fig. 5 is Thermo-sensitive HP-Chitin gels and two-component TA-PEG/HP-Chitin gels in the embodiment of the present invention 4
Storage modulus (G') is at 37 DEG C with the change curve in reaction time；
Fig. 6 is the pH response photos of hydrogel in the embodiment of the present invention 1；
Fig. 7 is molten to be subcutaneously injected into the precursor mixing of the gained hydrogel of the embodiment of the present invention 1 in female BAl BIc/C mice back
Mouse digital photograph (Fig. 7 a) of the liquid after one week and the hydrogel taken out in corresponding site and the digital photograph of surrounding tissue (figure
7b), hydrogel and its H＆E stained slice figures (Fig. 7 c) of surrounding tissue and in different time points Mice Body, scale is in figure
100μm。
Embodiment
With reference to embodiment and accompanying drawing, the invention will be further described, and its object is to help to be better understood from this
The content of invention, but these specific embodiments are not in any way limit the scope of the present invention.
Hydrophilic polymer A preparation
(1) line style dibasic acid esters alkynes end group PEG (DA-PEG) synthesis
Weigh 10.0 grams of PEG (Mn=2000) it is placed in 1.05 grams of propiolic acids in 250mL round-bottomed flasks, adds 150mL and do
Toluene solution after dry makees solvent, adds 0.57 gram of p-methyl benzenesulfonic acid under magnetic stirring and makees catalyst, oil bath is warming up to
140 DEG C, condensing reflux, whole system is cooled to room temperature after reacting 48h.Product mixed solution sinks by concentration and in ether
Form sediment, the light yellow solid that 9.33 grams are obtained after the crude product being collected into recrystallization is dried is line style dibasic acid esters alkynes end group PEG (DA-
PEG), yield 88.6%.1H NMR (500MHz, CDCl3, δ):4.3 (m, COOCH 2), 3.7 (m, COOCH2CH 2O), 3.6 (m,
PEG main chains OCH 2CH 2O), 3.0 (s, CH≡CCOO).The line style PEG of different molecular weight is chosen as raw material, can equally be closed
Into obtaining different polymerization degree (n：The DA-PEG of line style dibasic acid esters alkynes end group 1-1000).
(2) three arm ester alkynes end group PEG (TA-PEG) synthesis
Weigh 10.0 gram of three arm PEG (Mn=2600) it is placed in 3.9 grams of propiolic acids in 250mL round-bottomed flasks, adds 150mL
Dried toluene solution makees solvent, adds 0.6 gram of p-methyl benzenesulfonic acid under magnetic stirring and makees catalyst, is warming up to 140
DEG C, condensing reflux, whole system is cooled to room temperature after reacting 48h.Product mixed solution, will by concentrating and being precipitated in ether
The crude product recrystallization being collected into obtains 8.3 grams of light yellow solid after drying be three arm ester alkynes end group PEG (TA-PEG), yield
For 78%.1H NMR (500MHz, CDCl3, δ):4.3 (m, COOCH 2), 3.7 (m, COOCH2CH 2), 3.6 (m, PEG main chains
OCH 2CH 2O), 2.98 (s, CH≡CCOO).Three arm PEG of different molecular weight are chosen as raw material, can equally synthesize to obtain
Different polymerization degree (n：Three arm ester alkynes end group TA-PEG 1-1000).
(3) synthesis of polynary alkynyl-modified hydrophilic polymer
Hydrophilic polymer of the selection containing polyhydroxy such as dextran (such as dextran 1, weight average molecular weight 1000,
Dextran 3.5, weight average molecular weight 3500, dextran 5, weight average molecular weight 5000), hyperbranched polyhydroxylated polymer (such as
Boltorn H40, Boltorn H30, Boltorn H20, hyperbranched polyglycidyl ether, hyperbranched polyhydroxy polycarboxylic carboxylic acid amide esters),
Or dendritic is dissolved in dry dimethyl sulfoxide (DMSO) (DMSO), appropriate propiolic acid and right is added under magnetic stirring
Toluene sulfonic acide makees catalyst, oil bath temperature reaction, can equally synthesize to obtain polynary alkynyl-modified hydrophilic polymer.
The preparation of chitin derivativ B containing amino
According to the research work [Chinese invention patent application prospectus CN 201410170871.8] of our early stages,
The Hydroxypropyl chitosan of low deacetylation is prepared in sodium hydroxide-urea system using homogeneous method.Weigh 2 grams of first after purification
Shell element is dispersed with stirring in 100 containing the 11wt% sodium hydroxides and 4wt% urea gram aqueous solution freezed in advance, at -20 DEG C
Lower freezing 6h, take out mechanical agitation at room temperature and thawed, repeat 2 chitin water that can be dissolved of freeze-thaw
Solution.11.42 grams of expoxy propane, system mechanical agitation at 2 DEG C are added into obtained chitin solution (100 grams, 2wt%)
2h is reacted, is well mixed reactant, is then warming up to 5 DEG C of reaction 24h, then raises temperature to 15 DEG C of reaction 6h.Finally by system
2 DEG C are cooled to, with 3M hydrochloric acid regulation system pH value to 7, small point of urea and salt in 7 days removing solution of being dialysed with deionized water etc.
Son, freeze-drying obtain white sponge Hydroxypropyl chitosan (HP-Chitin), yield 87%.1H NMR spectras calculate
The acetyl degree for going out product is 0.89, substitution value 0.88.It is M to measure its viscosity average molecular weigh with Ubbelohde viscometerη=410kDa.Should
The HP-Chitin solution of Homogeneous synthesis has temperature sensitivity, and rheological results show the transformation row of reversible sol-gel
For the gel transition temperature of the wherein HP-Chitin solution that concentration is 2wt% is 19.2 DEG C.
According to the research work [Chinese invention patent application prospectus CN201310641249.6] of our early stages, adopt
The carboxymethyl chitin of low deacetylation is prepared in sodium hydroxide-urea system with homogeneous method, hydrogen spectrum NMR spectra is calculated
The acetyl degree of product is 0.87, degree of substitution by carboxymethyl 0.19.Similar, using homogeneous method in sodium hydroxide-urea system
The ethoxyl chitin and hydroxyl butyl chitin of low deacetylation are prepared, these contain the acetyl degree of the chitin derivativ of amino
For scope in 0.7-0.92, its molecular weight ranges is 5kDa~1000kDa.Chitosan derivatives containing amino can be with outsourcing：Carboxylic
Methyl chitosan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxyl butyl chitosan, their acetyl degree scope be 0.01~
0.4.Molecular weight ranges are 5kDa~1000kDa.Such as carboxymethyl chitosan (CM-Chitosan), weight average molecular weight 74kDa,
It is 0.04 that hydrogen spectrum NMR spectra, which measures acetyl degree, degree of substitution by carboxymethyl 0.96.
Embodiment 1：
Spontaneous amine alkynes click-reaction prepares DA-PEG/CM-Chitosan hydrogels.
By DA-PEG (PEG molecular weight is 2000), (CM-Chitosan, weight average molecular weight are with carboxymethyl chitosan
74kDa, deacetylation 0.96, degree of substitution by carboxymethyl 0.96) it is dissolved in respectively in 0.15M pH 7.4 PBS, match somebody with somebody
Put DA-PEG and 3wt% that concentration is 20wt% CM-Chitosan solution.Take 0.137g DA-PEG solution, 1g CM-
Chitosan solution and 0.04g PBSs are carried out after uniformly mixing half a minute, are placed on quiet in 37 DEG C of water bath with thermostatic control
Set to 0 .5-6h, you can the mol ratio for obtaining alkynyl/amino is that 0.28, CM-Chitosan concentration is 2.5wt% hydrogels, is not flowed
It is dynamic, it is designated as 0.28-2.5%DA-PEG/CM-Chitosan hydrogels.
Fig. 1 is 0.28-2.5%DA-PEG/CM-Chitosan hydrogels and its corresponding precursor DA-PEG and CM-
Chitosan infrared spectrum, understand that the alkynyl C ≡ C peaks of DA-PEG in the hydrogel are very weak from the figure, show alkynyl base
This reaction, illustrate that there occurs cross-linking reaction for the system.
The concentration and different alkynyls/amino mol ratio of different carboxymethyl chitosans are chosen, can equally be prepared different
DA-PEG/CM-Chitosan hydrogels, 0.42-2%, 0.28-2%, 0.14-2%, 0.28-1.5%, 0.11- are designated as respectively
2%, wherein before data be alkynyl/amino mol ratio, data below are the mass concentration of final carboxymethyl chitosan.Figure
2a is the gel rheometer test of 0.42-2%DA-PEG/CM-Chitosan hydrogels, illustrates that the mixed aqueous solution can be fast in body temperature
Fast (7 minutes) gel, has good syringeability and internal in-situ Rapid gelation characteristic.Fig. 2 b are the storage of this 6 kinds of hydrogels
Energy modulus and Loss modulus, wherein highest storage modulus value is 4.8kPa, illustrates the mechanical property of this kind of hydrogel and can lead to
The concentration of mole when polymer carboxymethyl's chitosan of alkynyl/amino is overregulated to improve.
Fig. 3 is the compression failure stress-strain diagram of wherein 5 kinds hydrogels, and 0.28-2.5%DA- is understood from the figure
The fracture strength of PEG/CM-Chitosan hydrogels is up to 65kPa, while corresponding breaking strain reaches 58%, shows the water
Gel has preferable mechanical strength.
Fig. 4 is that 0.28-2.5%DA-PEG/CM-Chitosan hydrogels directly contact different time with COS-7 cells
Cell proliferation rate, understand not influence significantly added with hydrogel cell proliferation from the figure, or even the cell after 48h is cultivated
Proliferation rate exceedes control group, shows that prepared hydrogel has excellent cell compatibility.
Embodiment 2：
Spontaneous amine alkynes click-reaction prepares DA-PEG/CM-Chitosan hydrogels.
By DA-PEG (PEG molecular weight be 6000) and carboxymethyl chitosan (CM-Chitosan, molecular weight 74kDa,
Deacetylation is 0.96, degree of substitution by carboxymethyl 0.96) it is dissolved in respectively in 0.15M pH 7.4 PBS, configuration concentration
For 25wt% DA-PEG and 2wt% CM-Chitosan solution.Take 0.15g DA-PEG solution, 1g CM-Chitosan
Solution and 0.18g PBSs are carried out after uniformly mixing half a minute, are placed on 8h in 37 DEG C of water bath with thermostatic control, you can obtain
The mol ratio of alkynyl/amino is the 0.2-1.5%DA-PEG/CM-Chitosan that 0.2, CM-Chitosan concentration is 1.5wt%
Hydrogel.This example illustrates that the molecular weight for changing PEG and concentration do not influence the preparation of injection aquagel.By changing precursor
The molar feed ratio (0.05~5) of alkynyl/amino of solution, and reaction time (0.5~12h), all can obtain a series of DA-
PEG/CM-Chitosan hydrogels.In addition, change concentration (0.5~20wt%), molecular weight and the deacetylation of precursor B solution,
A series of DA-PEG/CM-Chitosan hydrogels can equally be obtained.
Embodiment 3：
Spontaneous amine alkynes click-reaction prepares TA-PEG/CM-Chitosan hydrogels.
Three arm ester alkynes end group TA-PEG (PEG molecular weight is 2600) (CM-Chitosan, are divided with carboxymethyl chitosan
Son amount is 74kDa, deacetylation 0.96, degree of substitution by carboxymethyl 0.96) 0.15M pH 7.4 PBS is dissolved in respectively
In, configuration concentration is 20wt% TA-PEG and 3wt% CM-Chitosan solution.Take 0.39g TA-PEG solution, 1g
CM-Chitosan solution and 0.11gPBS buffer solutions are carried out after uniformly mixing half a minute, are placed in 37 DEG C of water bath with thermostatic control
8h, you can the mol ratio for obtaining alkynyl/amino is the 0.9-2%TA-PEG/CM- that 0.9, CM-Chitosan concentration is 2wt%
Chitosan hydrogels.This example explanation replaces line style dibasic acid esters alkynes end group PEG not influence using three arm ester alkynes end group TA-PEG
The preparation of injection aquagel.Change PEG molecular weight from oligomer to 50,000, or change PEG concentration in 0.5-30wt% scopes
It is interior, the hydrogel of two-component injectable can be made.
Embodiment 4：
Spontaneous amine alkynes click-reaction prepares TA-PEG/HP-Chitin hydrogels.
By three arm ester alkynes end group TA-PEG (PEG molecular weight is 2600) and the Hydroxypropyl chitosan HP- with Thermo-sensitive
Chitin is dissolved in the 0.15M pH 7.4 of 4 DEG C of low temperature PBS respectively, configuration concentration be 20wt% TA-PEG and
3wt% HP-Chitin solution.Take 0.064g TA-PEG solution, 1g HP-Chitin solution and 0.12g PBSs
2-15 DEG C is carried out after uniformly mixing half a minute at low temperature, then is placed on 10h in 37 DEG C of water bath with thermostatic control, you can obtains alkynes
The mol ratio of base/amino is the 1.2-2.5%TA-PEG/HP-Chitin hydrogels that 1.2, HP-Chitin concentration is 2.5wt%.
Because the HP-Chitin solution has temperature sensitivity when concentration is more than 1wt%, rise temperature can form physical gel.
When adding TA-PEG solution, amino and alkynyl in system are realized to enter on the basis of physical gel by forming enamine bonds
Row chemical crosslinking, so as to strengthen the mechanical property of the physical gel.Fig. 5 be 2.5wt%HP-Chitin thermo-responsive hydro gels and
The storage modulus (G') of 1.2-2.5%TA-PEG/HP-Chitin two-component gel solutions changes with time song at 37 DEG C
Line, from the figure it was found from, comprising physics and chemical crosslinking TA-PEG/HP-Chitin hydrogels G' values than only containing physics friendship
The HP-Chitin hydrogels of connection are big 2.5 times, and the chemical crosslinking for showing to carry out on the basis of physical gel can greatly improve water
The mechanical property of gel.Pass through the molar feed ratio (0.05~5) for the alkynyl/amino for changing precursor solution, and reaction time
(0.5~12h), you can obtain a series of TA-PEG/HP-Chitin hydrogels.In addition, change the concentration (0.5 of precursor solution B
~20wt%), molecular weight and deacetylation, can equally obtain a series of TA-PEG/HP-Chitin hydrogels.
The PEG of alkynes end group modification, or adjustment CM- are replaced using other polynary alkynyl-modified hydrophilic polymers
Chitosan acetyl degree scope, molecular weight can make the hydrogel of two-component injectable.Equally, ammonia is contained using other
The chitin derivativ of base such as hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxyl butyl chitosan, carboxymethyl chitin, ethoxy
Chitin hydroxyl butyl chitin, instead of carboxymethyl chitosan or the Hydroxypropyl chitosan of Thermo-sensitive, can make two-component
The hydrogel of injectable.
Performance test
1, the pH sensitivity testses of hydrogel
By 1 gram of 0.28-2.5%DA-PEG/CM-Chitosan hydrogel made from embodiment 1 (represent alkynyl/amino=
0.28, CM-Chitosan concentration is 2.5wt%) place at room temperature, it is vortexed 5 minutes after adding 100 μ L 6M HCl, hydrogel
Fast liquefying into flowable liquid, add 100 μ L 6M NaOH recall to neutrality can plastic immediately, the reversible colloidal sol-
Gel process can be repeated 5 times the above.Fig. 6 is the pH response photos of the hydrogel, and hydrogel made from explanation has pH quick
Perception.
2, Study on biocompatibility inside hydrogel
DA-PEG solution and CM-Chitosan solution are sterilized by 0.22 μm of membrane filtration respectively, by 200 μ L's
0.28-2.5%DA-PEG/CM-Chitosan hydrogel precursors mixed solution is injected into BALB/C female mices by 26G syringe needles
The subcutaneous location of back.A mouse is implemented into euthanasia after injecting 1 week, observes hydrogel formational situation and shape in Mice Body
Looks；Remaining mouse continues to raise, and three mouse are implemented in 2 weeks, 3 weeks and 4 weeks after the injection of hydrogel precursor solution respectively
Euthanasia, hydrogel and its surrounding tissue are taken out, immerse in 4wt% paraformaldehyde solution and fix, sample is embedded in stone
In wax, the thin slice that thickness is about 4 microns is cut into；Sections stained with hematoxylin-eosin (HE) reagent dyeing, under inverted light microscope
Observe inflammatory reaction.
Euthanasia is sentenced after one week in the back that hydrogel precursor solution is subcutaneously injected into mouse, in injection site shape
Into obvious projection as shown in Figure 7a.Hydrogel and its surrounding tissue are taken out (see Fig. 7 b), it is observed that in Mice Body
The transparent aquagel of formation, hydrogel remains in that complete pattern after one week.This illustrates that hydrogel precursor solution can be
Click chemistry reaction, in-situ preparation hydrogel occur in vivo.Hydrogel will be taken out respectively after 1 week, 2 weeks, 3 weeks and 4 weeks, its week again
The H＆E coloration results for enclosing tissue as shown in Figure 7 c, occur more concentrating in hydrogel after injecting 1 week with organization edge intersection
Inflammatory cell infiltration, show as acute inflammatory reaction, and inflammatory cell starts, to hydrogel interior shifting, not cause systematicness
Inflammatory reaction；Inflammatory cell gradually disappears after 2 weeks, only a little inflammatory cell infiltration, predominantly neutrophil leucocyte, has no obvious
Macrophage；Surrounding tissue has recovered normal at the 4th week, without obvious inflammatory reaction.Whole process do not occur as changed colour, suppurate or
The adverse reactions such as edema.Therefore, test result indicates that the injection aquagel has a good biocompatibility, it is expected in people
Bonding, closure in the various operations of body, hemostasis, antiseep either prevent adhesion in application or for bioactive substance and
The bio-medical fields such as the carrier and tissue engineering material of cell.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, one of ordinary skill in the art should
Understand, technical scheme can be modified or equivalent substitution, without departing from the essence of technical solution of the present invention
And scope.
Claims (10)
1. a kind of preparation method of injectable high intensity chitin based aquagel product, it is characterised in that use following method system
It is standby to obtain：
(1) binary or the water solution A of polynary alkynyl-modified hydrophilic polymer are prepared；
(2) aqueous solution B of the chitin derivativ containing amino is prepared；
(3) A and B are uniformly mixed, forms the injection aquagel precursor solution with good fluidity；
(4) the hydrogel precursor solution that step (3) obtains is formed in situ the high intensity hydrogel in physiological conditions.
2. preparation method according to claim 1, it is characterised in that hydrophilic polymer in the water solution A be containing
The polyethylene glycol PEG derivatives or their mixture of two or more ester alkynes terminal groups modifications.
3. preparation method according to claim 1, it is characterised in that hydrophilic polymer in the water solution A be containing
The polyethylene glycol PEG derivatives or their mixture of two or three ester alkynes terminal groups modifications, described contains two ester alkynes end groups
Modified polyethylene glycol PEG derivant structure formulas are shown below：
The described polyethylene glycol PEG derivant structure formulas containing three ester alkynes terminal groups modifications are shown below：
Wherein n, x, y, z are 1-1000 integer.
4. preparation method according to claim 3, it is characterised in that the n, x, y, z are 20-400 integer.
5. preparation method according to claim 1, it is characterised in that the chitin derivativ containing amino is carboxylic first
Base enclosure glycan, hydroxyethyl chitosan, hydroxypropyl chitosan, hydroxyl butyl chitosan, carboxymethyl chitin, ethoxyl chitin, hydroxyl
Any of propyl group chitin, hydroxyl butyl chitin or their mixture；Wherein carboxymethyl chitosan, ethoxy shell gather
Sugar, hydroxypropyl chitosan, the acetyl degree scope of hydroxyl butyl chitosan are 0.01~0.4；Carboxymethyl chitin, ethoxy crust
Element, Hydroxypropyl chitosan, hydroxyl butyl chitin acetyl degree scope be 0.7~0.92, molecular weight ranges be 5kDa~
1000kDa。
6. preparation method according to claim 1, it is characterised in that the mass concentration model of hydrophilic polymer in solution A
Enclose for 0.5~30wt%, the mass concentration scope of polymer is 0.5~20wt% in solution B, and the solution A and solution B
In reactive functionality alkynyl and amino molar ratio scope be 0.05~5.
7. preparation method according to claim 1, it is characterised in that the aqueous solution B is to contain ammonia with Thermo-sensitive
The aqueous solution of the chitin derivativ of base.
8. the preparation method according to claim 1 or 7, it is characterised in that step (1), (2), the configuration temperature of (3) are
2-15 DEG C, step (4) described physiological condition is that physical crosslinking in situ is molded under body temperature, then further spontaneous chemical in the original location
Direct in-situ is crosslinked to use.
9. a kind of injectable high intensity chitin based aquagel, it is characterised in that using the side described in claim any one of 1-8
Method is prepared.
10. the application of injectable high intensity chitin based aquagel as claimed in claim 9, it is characterised in that for human body
Bonding, closure, hemostasis, antiseep in various operations prevent adhesion, and the biological active matter for bio-medical field
The carrier and tissue engineering material of matter and cell.
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| CN112074303A (en) * | 2018-05-08 | 2020-12-11 | 亚州大学校产学协力团 | Injection preparation composition used as filler or drug carrier through click chemistry reaction |
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| CN112533986B (en) * | 2018-07-31 | 2023-07-11 | 国立大学法人东京大学 | Polymer gel with spongy porous structure |
| CN112533986A (en) * | 2018-07-31 | 2021-03-19 | 国立大学法人东京大学 | Polymer gel with spongy porous structure |
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| CN114656652A (en) * | 2020-12-23 | 2022-06-24 | 上海其胜生物制剂有限公司 | Preparation method of low-swelling temperature-sensitive injectable chitosan-based hydrogel |
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