CN103524748A - Polyamino acid graft copolymer, preparation method thereof and injectable hydrogel - Google Patents
Polyamino acid graft copolymer, preparation method thereof and injectable hydrogel Download PDFInfo
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Abstract
The present invention provides a kind of polyaminoacid graft copolymer and preparation method thereof, syringeability hydrogel, the polyaminoacid graft copolymer is as shown in the formula (I). Compared with existing natural polymer,The present invention using the poly(L-glutamic acid) with formula (V) structure as main chain,It can simulate native protein and polypeptide to a certain extent,With good biocompatibility and degradability; The graft polymers has the branch of formula (II) structure,It contains phenol structure and adjustable different grafting rate,It can be crosslinked under the action of horseradish peroxidase and hydrogen peroxide,Formation condition is mild,Reaction rate is easy to control,Therefore the higher syringeability hydrogel of mechanical strength can be prepared; And the graft copolymer also includes the branch of formula (III) structure,It can also adjust different grafting rates,To make polyaminoacid graft copolymer shown in formula (I) that there is good water solubility.
Description
Technical field
The invention belongs to biotechnology, bio-medical material and tissue engineering technique field, relate in particular to polyamino acid graft copolymer and preparation method thereof, syringeability hydrogel.
Background technology
Hydrogel is the polymkeric substance with cross-linked network structure that a class can absorb and keep large quantity of moisture fast, in polymer network structure, contain hydrophilic radical or hydrophilic segment, they can be combined with water in water surrounding, and this hydrogel structure can spread hydrophilic small molecules therein.
Syringeability hydrogel is the novel hydrogels system occurring in recent years, it has unique solution-gel conversion characteristic, before becoming gel, is the low viscous aqueous solution, is convenient to injection, and after in injection of solution arrives organism, can original position form fast hydrogel.
Syringeability hydrogel is convenient to wrap medicine carrying thing, Cell and organism bioactive molecule, and can be applicable to repair complex-shaped wound, and realizes minimally-invasive treatment, so ，Qi part drug discharges and original position Repair of tissue defect aspect is with a wide range of applications.
Prior art discloses multiple syringeability hydrogel, as the block copolymer hydrogel of polyoxyethylene glycol and PLLA, when temperature variation, can realize the reversible change of colloidal sol and gel; The segmented copolymer that polyoxyethylene-polyoxytrimethylene polymkeric substance and polyalanine form, under finite concentration, also can form syringeability hydrogel.But, the poor stability of syringeability hydrogel prepared by prior art, the drawbacks limit such as physical strength is low its application in bio-medical field.
Syringeability hydrogel based on enzyme-catalyzed cross-linking is because its formation condition is gentle, speed of reaction is easily controlled, physical strength is relatively high and have the good advantages such as biocompatibility, receives increasing concern.Conventionally, at horseradish peroxidase (HRP) and hydrogen peroxide (H
2o
2) katalysis under, the polymkeric substance that contains phenol or anils can form the cross-linking set of C-C and C-O key, and then original position forms hydrogel.
At present, the hydrogel based on enzyme-catalyzed cross-linking mainly concentrates on natural polysaccharide and protein, as glucose, hyaluronic acid, chitosan, alginate, Mierocrystalline cellulose and gelatin etc.But natural polymer composition is single, performance is difficult to regulation and control.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide polyamino acid graft copolymer and preparation method thereof, syringeability hydrogel, and this polyamino acid graft copolymer percentage of grafting is controlled.
The invention provides a kind of polyamino acid graft copolymer, as shown in the formula (I):
Described-R
1there is formula (II) structure:
Described-R
2there is formula (III) structure:
Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.
Preferably, the scope of described m is 10≤m≤300.
The present invention also provides a kind of preparation method of polyamino acid graft copolymer, comprises the following steps:
Tyrasamine, the poly glycol monomethyl ether with formula (IV) structure, the PLGA with formula V structure are mixed with organic solvent, under the effect of coupling agent, condensation reaction occurs, obtain the polyamino acid graft copolymer shown in formula (I); Described-R
1there is formula (II) structure; Described-R
2there is formula (III) structure;
Wherein, x, y, z, m and n are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000; N=x+y+z.
Preferably, described tyrasamine with the mol ratio of carboxyl of PLGA with formula V structure for (0.1～0.4): 1.
The poly glycol monomethyl ether preferably, with formula (IV) structure is (0.1～0.4) with the mol ratio of carboxyl with the PLGA of formula V structure: 1.
Preferably, described coupling agent is EDC hydrochloride, N, one or more in N '-dicyclohexylcarbodiimide and N-hydroxy-succinamide.
The present invention also provides a kind of syringeability hydrogel, comprises polyamino acid graft copolymer, aqueous solvent, horseradish peroxidase and the hydrogen peroxide shown in formula (I);
Described-R
1there is formula (II) structure:
Described-R
2there is formula (III) structure:
Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.
Preferably, the polyamino acid graft copolymer shown in described formula (I) and aqueous solvent are mixed into the first mixing solutions containing polyamino acid graft copolymer 2～30wt%.
Preferably, described horseradish peroxidase and aqueous solvent are mixed into the second mixing solutions of 0.01mg/mL～2.0mg/mL.
Preferably, described hydrogen peroxide and aqueous solvent are mixed into the 3rd mixing solutions of 0.1mmol/L～200mmol/L.The invention provides a kind of polyamino acid graft copolymer and preparation method thereof, syringeability hydrogel, by tyrasamine, there is the poly glycol monomethyl ether of formula (IV) structure, the PLGA of formula V structure mixes with organic solvent, under the effect of coupling agent, there is condensation reaction, can obtain the polyamino acid graft copolymer shown in formula (I).Compare with existing natural polymer, it is main chain that the PLGA with formula V structure is take in the present invention, and it can simulate natural protein and polypeptide to a certain extent, has good biocompatibility and degradation property; This graftomer has the side chain of formula (II) structure, it contains phenol structure and can regulate different percentage of grafting, can under the effect of horseradish peroxidase and hydrogen peroxide, be cross-linked, formation condition is gentle, speed of reaction is easily controlled, and therefore can prepare the syringeability hydrogel that physical strength is higher; And this graft copolymer also comprises the side chain of formula (III) structure, adjustable different percentage of grafting also, good water-soluble thereby the polyamino acid graft copolymer shown in the formula of making (I) has.
Accompanying drawing explanation
Fig. 1 is the hydrogen nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer shown in the formula (I) preparing in the embodiment of the present invention 5;
Fig. 2 is the hydrogen nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer shown in the formula (I) preparing in the embodiment of the present invention 6;
Fig. 3 is the hydrogen nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer shown in the formula (I) preparing in the embodiment of the present invention 7;
Fig. 4 is the hydrogen nuclear magnetic resonance spectrogram of the polyamino acid graft copolymer shown in the formula (I) preparing in the embodiment of the present invention 8;
Fig. 5 is that the syringeability hydrogel gel time for preparing in the embodiment of the present invention 7 is with horseradish peroxidase concentration curve figure;
Fig. 6 is that the syringeability hydrogel gel time for preparing in the embodiment of the present invention 7 is with horseradish peroxidase concentration curve figure;
Fig. 7 is that the syringeability hydrogel gel time for preparing in the embodiment of the present invention 7 is with horseradish peroxidase concentration curve figure;
Fig. 8 is that the syringeability hydrogel gel time for preparing in the embodiment of the present invention 8 is with horseradish peroxidase concentration curve figure;
Fig. 9 is that the syringeability hydrogel gel time for preparing in the embodiment of the present invention 8 is with concentration of hydrogen peroxide change curve;
Figure 10 is the syringeability hydrogel dynamic mechanical test figure preparing in the embodiment of the present invention 8.
Embodiment
The invention provides a kind of polyamino acid graft copolymer, as shown in the formula (I):
Described-R
1there is formula (II) structure:
Described-R
2there is formula (III) structure:
Wherein, x, y, z and m are the polymerization degree; 3≤x≤400, are preferably 6≤x≤300, more preferably 15≤x≤210; 3≤y≤400, are preferably 6≤y≤300, more preferably 15≤y≤210; 0≤z≤900, are preferably 5≤z≤788, more preferably 10≤z≤570; 30≤x+y+z≤1000, are preferably 50≤x+y+z≤800, more preferably 100≤x+y+z≤600; The scope of described m is preferably 10≤m≤300, more preferably 10≤m≤227.
According to the present invention, described R
1weight be preferably 2%～15% of polyamino acid graft copolymer weight, more preferably 5%～15%; Described R
2weight be preferably 50%～80% of polyamino acid graft copolymer weight, more preferably 60%～70%.
Polyamino acid graftomer of the present invention has the side chain of formula (II) structure, it contains phenol structure and can regulate different percentage of grafting, can under the effect of horseradish peroxidase and hydrogen peroxide, be cross-linked, formation condition is gentle, speed of reaction is easily controlled, and therefore can prepare the syringeability hydrogel that physical strength is higher; And this graft copolymer also comprises the side chain of formula (III) structure, adjustable different percentage of grafting also, good water-soluble thereby the polyamino acid graft copolymer shown in the formula of making (I) has.
The present invention also provides the preparation method of the polyamino acid graft copolymer shown in above-mentioned formula (I), comprises the following steps:
Tyrasamine, the poly glycol monomethyl ether with formula (IV) structure, the PLGA with formula V structure are mixed with organic solvent, under the effect of coupling agent, condensation reaction occurs, obtain the polyamino acid graft copolymer shown in formula (I).
Wherein, m and n are polymkeric substance, and described m is same as above, does not repeat them here; Described n=x+y+z.
The present invention does not have special restriction to the source of all raw materials, for commercially available.
It is main chain that polyamino acid graft copolymer of the present invention be take the PLGA with formula V structure, and it can simulate natural protein and polypeptide to a certain extent, has good biocompatibility and degradation property.The PLGA in the present invention with (V) structure is preferably prepared in accordance with the following methods: take normal hexyl Amine and/or triethylamine as initiator; in the first organic solvent, be there is to ring-opening polymerization in γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride; then carry out benzyl deprotection, obtain having the PLGA of formula V structure.
Described γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride is preferably prepared in accordance with the following methods: γ-benzyl-Pidolidone ester carries out cyclization with two (trichloromethyl) carbonic ethers, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.Described cyclization preferably carries out under anhydrous condition in the second organic solvent, described the second organic solvent is the organic solvent of solubilized γ-benzyl well known to those skilled in the art-Pidolidone ester and two (trichloromethyl) carbonic ethers, there is no special restriction, in the present invention, be preferably tetrahydrofuran (THF); Described γ-benzyl-Pidolidone ester, two (trichloromethyl) carbonic ether with the second organic solvent preferably according to 1g:(0.6～0.8) g:(10～15) ratio of ml mixes; The temperature of described cyclization is preferably 30 ℃～70 ℃, more preferably 40 ℃～60 ℃; The time of described cyclization is preferably 0.5～3h, more preferably 1～3h; This cyclization preferably carries out under the condition of protection of inert gas; Described rare gas element is rare gas element well known to those skilled in the art, there is no special restriction, is preferably nitrogen in the present invention.The present invention does not have special restriction to the source of γ-benzyl-Pidolidone ester, two (trichloromethyl) carbonic ether and the second organic solvent, for commercially available.
After cyclization, preferably carry out purification processes.The method of described purification is method well known to those skilled in the art, the present invention preferably carries out sedimentation with cold sherwood oil or normal hexane, filtration is drained, by solid acetic acid ethyl dissolution, cold water washing repeatedly, spend the night with anhydrous magnesium sulfate drying by organic phase, after filtering, uses ethyl acetate and normal hexane recrystallization, solid vacuum-drying 12～48h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
It is initiator that normal hexyl Amine and/or triethylamine are take in the present invention, and γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride, in the first organic solvent, ring-opening polymerization is occurred to.Wherein, the mol ratio of described initiator and γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride is preferably 1:(50～800), 1:(100～600 more preferably); Described the first organic solvent is the organic solvent that can dissolve normal hexyl Amine, triethylamine and γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride well known to those skilled in the art, there is no special restriction, in the present invention, be preferably one or more in DMF, chloroform and dioxane; The temperature of described ring-opening polymerization is preferably 0 ℃～40 ℃, more preferably 10 ℃～30 ℃; The time of described ring-opening polymerization is preferably 24～72h, more preferably 48～72h; In the present invention, this ring-opening polymerization preferably carries out under the protection of rare gas element, and this rare gas element is preferably nitrogen.
After ring-opening polymerization, preferably carry out purification process.This purification process is preferably carried out sedimentation with ether, and suction filtration is gathered (γ-benzyl-Pidolidone ester).
Gathered (γ-benzyl-Pidolidone ester) and afterwards, carried out benzyl deprotection.The present invention does not have special restriction to the method for benzyl deprotection; can be method of sloughing benzyl or carbobenzoxy-(Cbz) well known to those skilled in the art; the present invention preferably adopts following methods to carry out: after poly-(γ-benzyl-Pidolidone ester) that obtain dissolved with dichloro acetic acid; the mixing solutions that adds Hydrogen bromide and acetic acid; room temperature reaction, obtains having the PLGA of formula V structure.Wherein, the ratio of described poly-(γ-benzyl-Pidolidone ester), dichloro acetic acid and Hydrogen bromide and acetic acid mixing solutions is preferably 1g:(5～15) ml:(1～5) ml; In the mixing solutions of described Hydrogen bromide and acetic acid, hydrobromic volume fraction is preferably 20%～40%; The time of this room temperature reaction is preferably 1～2h; After room temperature reaction, preferably carry out purification processes, reaction solution is carried out in ether to sedimentation, filter, solid is dissolved in to N, in N-dimethyl sulfoxide (DMSO), with the dialysis tubing of corresponding sized molecules amount, in water, dialyse three days, after freeze-drying, obtain having the PLGA of formula V structure.
By thering is PLGA, the tyrasamine of formula V structure, the poly glycol monomethyl ether with formula (IV) structure mixes with organic solvent, under the effect of coupling agent, condensation reaction occurs.The wherein said poly glycol monomethyl ether with formula (IV) structure is preferably (0.1～0.4) with the mol ratio of carboxyl with the PLGA of formula V structure: 1, more preferably (0.12～0.38): 1, then be preferably (0.15～0.35): 1; Described tyrasamine is preferably (0.1～0.4) with the mol ratio of carboxyl with the PLGA of formula V structure: 1, more preferably (0.12～0.38): 1, then be preferably (0.15～0.35): 1; Described coupling agent is preferably (0.25～0.8) with the mol ratio of carboxyl with the PLGA of formula V structure: 1, more preferably (0.3～0.7): 1; In the present invention, preferably with EDC hydrochloride, N, one or more in N '-dicyclohexylcarbodiimide and N-hydroxy-succinamide are coupling agent; Described organic solvent is that well known to those skilled in the art can dissolving has PLGA, the tyrasamine of formula V structure, the poly glycol monomethyl ether with formula (IV) structure and the organic solvent of coupling agent, there is no special restriction, in the present invention, be preferably one or more in DMF, dimethyl sulfoxide (DMSO) and N-Methyl pyrrolidone.
In the present invention, above-mentioned raw materials preferably carries out according to following order of addition(of ingredients): PLGA and the coupling agent with formula V structure are dissolved in organic solvent, room temperature reaction 4～12h, then add tyrasamine and there is formula (IV) structure poly glycol monomethyl ether and carry out condensation reaction.
In the present invention, the temperature of condensation reaction is preferably 20 ℃～60 ℃, more preferably 30 ℃～50 ℃; The time of described condensation reaction is preferably 24～72h, more preferably 36～56h.
After condensation reaction, preferably carry out purification process, reaction solution is dialysed in water with the dialysis tubing of corresponding sized molecules amount, preferably dialyse 2～4 days, after lyophilize, obtain the polyamino acid graft copolymer shown in formula (I).
The present invention by the tyrasamine of different feed ratio, the poly glycol monomethyl ether with formula (IV) structure and the PLGA with formula V structure by condensation reaction, obtain the polyamino acid graftomer shown in the formula (I) of different percentage of grafting, it can be used as the geopolymer gel material with enzyme-catalyzed cross-linking function, there is good water-soluble, regulatable one-tenth gel time and cross-link intensity, in bio-medical field, be with a wide range of applications.
The present invention also provides a kind of syringeability hydrogel, comprises polyamino acid graft copolymer, aqueous solvent, horseradish peroxidase and the hydrogen peroxide shown in formula (I); Wherein, the polyamino acid graft copolymer shown in described formula (I) is same as above, does not repeat them here.
In syringeability hydrogel, polyamino acid graft copolymer and aqueous solvent shown in described formula (I) are mixed to form mixing solutions, and preferably itself and aqueous solvent are mixed into the first mixing solutions of 2～30wt%, and more preferably 5%～28%, then be preferably 8%～25%.
In syringeability hydrogel of the present invention, the form that described horseradish peroxidase all mixes with aqueous solvent with hydrogen peroxide exists.Wherein, described horseradish peroxidase preferably with aqueous solvent is mixed into the second mixing solutions of 0.01mg/mL～2.0mg/mL, 0.02mg/ml～1.5mg/ml more preferably, then be preferably 0.05mg/ml～1mg/ml; Described hydrogen peroxide is preferably mixed into the 3rd mixing solutions of 0.1mmol/L～200mmol/L with aqueous solvent, 0.2mmol/L～150mmol/L more preferably, then be preferably 0.5mmol/L～100mmol/L.
Described aqueous solvent is aqueous solvent well known to those skilled in the art, there is no special restriction, is preferably water, physiological saline, buffered soln, tissue culture medium or body fluid in the present invention.
In the present invention, polyamino acid graft copolymer, horseradish peroxidase and hydrogen peroxide shown in formula (I) are dissolved in respectively in aqueous solvent, obtain the corresponding aqueous solution, then by aqueous solution, can form the cross-linking set of C-C and C-O key, form fast hydrogel, and can be by regulating percentage of grafting, the concentration of polyamino acid graft copolymer shown in formula (I), the concentration of the concentration of horseradish peroxidase and hydrogen peroxide of junket ammonia to regulate forming the physical strength of gel time and gel; Therefore meanwhile, because the poly-propylhomoserin graft copolymer shown in formula (I) contains poly glycol monomethyl ether side chain, have good water-soluble.In sum, hydrogel provided by the invention has advantages of that formation condition gentleness, speed of reaction are easily controlled and physical strength is convenient to regulation and control, and there is biocompatibility and degradability, can be used for biomedical materials field, especially at aspects such as medicine controlled releasing and organizational projects, have broad application prospects.
In order to further illustrate the present invention, below in conjunction with embodiment, polyamino acid graft copolymer provided by the invention and preparation method thereof, syringeability hydrogel are described in detail.
In following examples, reagent used is commercially available.
1.1 poly glycol monomethyl ethers that are 1000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 8ml triethylamine and 16ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
1.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g1.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
1.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
1.4 are dissolved in by the γ-benzyl obtaining in 2g1.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride the N that 20ml has dewatered; in dinethylformamide; add 25 μ L normal hexyl Amines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h, after reaction finishes, uses ether sedimentation; solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
1.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g1.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By nuclear-magnetism, calculate, its molecular weight is 4600.
1.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g1.5, then (EDCHCl(point is in centre to add 0.35g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride, lower same)) and 0.22g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.16g tyrasamine with formula (IV) structure that in 1g1.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
1.7 the polyamino acid graft copolymer shown in the formula obtaining in 1.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, obtain syringeability hydrogel.Adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 1.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 12%, and the percentage of grafting of tyrasamine is 10%, and productive rate is 87%.
Embodiment 2
2.1 poly glycol monomethyl ethers that are 1000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 8ml triethylamine and 16ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
2.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g2.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
2.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
2.4 are dissolved in by the γ-benzyl obtaining in 2g2.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride the N that 20ml has dewatered; in dinethylformamide; add 25 μ L normal hexyl Amines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h, after reaction finishes, uses ether sedimentation; solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
2.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g2.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By nuclear-magnetism, calculate, its molecular weight is 4600.
2.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g2.5, then add 0.48g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.29g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.22g tyrasamine with formula (IV) structure that in 1.24g2.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
2.7 the polyamino acid graft copolymer shown in the formula obtaining in 2.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, obtain syringeability gel.While being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 2.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 15%, and the percentage of grafting of tyrasamine is 16%, and productive rate is 84%.
Embodiment 3
3.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
3.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g3.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
3.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
3.4 are dissolved in by the γ-benzyl obtaining in 2g3.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride the N that 20ml has dewatered; in dinethylformamide; add 6 μ L normal hexyl Amines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h, after reaction finishes, uses ether sedimentation; solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
3.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g3.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By nuclear-magnetism, calculate, its molecular weight is 21000.
3.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g3.5, then add 0.45g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.27g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.2g tyrasamine with formula (IV) structure that in 2.32g3.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
3.7 the polyamino acid graft copolymer shown in the formula obtaining in 3.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 3.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 16%, and the percentage of grafting of tyrasamine is 14%, and productive rate is 89%.
Embodiment 4
4.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
4.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g4.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
4.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
4.4 are dissolved in by the γ-benzyl obtaining in 2g4.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride the N that 20ml has dewatered; in dinethylformamide; add 6 μ L normal hexyl Amines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h, after reaction finishes, uses ether sedimentation; solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
4.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g4.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By nuclear-magnetism, calculate, its molecular weight is 21000.
4.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g4.5, then add 0.60g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.36g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.27g tyrasamine with formula (IV) structure that in 3.1g4.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
4.7 the polyamino acid graft copolymer shown in the formula obtaining in 4.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 4.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 21%, and the percentage of grafting of tyrasamine is 19%, and productive rate is 82%.
Embodiment 5
5.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
5.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g5.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
5.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
5.4 are dissolved in the γ-benzyl obtaining in 2g5.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
5.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g5.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
5.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g5.5, then add 0.45g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.27g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.20g tyrasamine with formula (IV) structure that in 2.32g5.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
5.7 the polyamino acid graft copolymer shown in the formula obtaining in 5.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 5.6, obtain its hydrogen nuclear magnetic resonance spectrogram, as shown in Figure 1; Obtaining result is: the percentage of grafting of poly glycol monomethyl ether is 14%, and the percentage of grafting of tyrasamine is 15%, and productive rate is 79%.
Embodiment 6
6.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
6.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g6.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
6.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
6.4 are dissolved in the γ-benzyl obtaining in 2g6.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
6.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g6.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
6.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g6.5, then add 0.60g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.36g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.27g tyrasamine with formula (IV) structure that in 3.1g6.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
6.7 the polyamino acid graft copolymer shown in the formula obtaining in 6.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 6.6, obtain its hydrogen nuclear magnetic resonance spectrogram, as shown in Figure 2; Obtaining result is: the percentage of grafting of poly glycol monomethyl ether is 21%, and the percentage of grafting of tyrasamine is 20%, and productive rate is 85%.
Embodiment 7
7.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
7.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g7.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
7.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
7.4 are dissolved in the γ-benzyl obtaining in 2g7.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
7.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g7.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
7.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g7.5, then add 0.74g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.45g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.34g tyrasamine with formula (IV) structure that in 3.9g7.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
7.7 the polyamino acid graft copolymer shown in the formula obtaining in 7.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
When polyamino acid graft copolymer mass concentration is 5%, when concentration of hydrogen peroxide is 4.9mmol/L, obtain syringeability hydrogel gel time with horseradish peroxidase concentration curve, as shown in Figure 5.As shown in Figure 5, this polyamino acid graft copolymer can form hydrogel fast, and becomes gel time to regulate and control.
When polyamino acid graft copolymer mass concentration is 10%, when concentration of hydrogen peroxide is 4.9mmol/L, obtain syringeability hydrogel gel time with horseradish peroxidase concentration curve, as shown in Figure 6.As shown in Figure 6, this polyamino acid graft copolymer can form hydrogel fast, and becomes gel time to regulate and control.
When polyamino acid graft copolymer mass concentration is 15%, when concentration of hydrogen peroxide is 4.9mmol/L, obtain syringeability hydrogel gel time with horseradish peroxidase concentration curve, as shown in Figure 7.As shown in Figure 7, this polyamino acid graft copolymer can form hydrogel fast, and becomes gel time to regulate and control.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 7.6, obtain its hydrogen nuclear magnetic resonance spectrogram, as shown in Figure 3; Obtaining result is: the percentage of grafting of poly glycol monomethyl ether is 26%, and the percentage of grafting of tyrasamine is 24%, and productive rate is 86%.
Embodiment 8
8.1 poly glycol monomethyl ethers that are 2000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 4ml triethylamine and 8ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
8.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g8.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
8.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
8.4 are dissolved in the γ-benzyl obtaining in 2g8.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
8.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g8.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
8.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g8.5, then add 0.89g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.53g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.4g tyrasamine with formula (IV) structure that in 4.6g8.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
8.7 the polyamino acid graft copolymer shown in the formula obtaining in 8.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
When polyamino acid graft copolymer mass concentration is 10%, when concentration of hydrogen peroxide is 24.5mmol/L, obtain syringeability hydrogel gel time with horseradish peroxidase concentration curve, as shown in Figure 8.As shown in Figure 8, this polyamino acid graft copolymer can form hydrogel fast, and becomes gel time to regulate and control.
When polyamino acid graft copolymer mass concentration is 10%, when horseradish peroxidase concentration is 0.5mg/ml, obtain syringeability hydrogel gel time with concentration of hydrogen peroxide change curve, as shown in Figure 9.As shown in Figure 9, this polyamino acid graft copolymer can form hydrogel fast, and becomes gel time to regulate and control.
When polyamino acid graft copolymer mass concentration is 10%, horseradish peroxidase concentration is 0.5mg/ml, when concentration of hydrogen peroxide is 24.5mmol/L, utilizes rheometer to measure the three-dimensional storage modulus of injection aquagel situation over time, obtain its dynamic mechanical test figure, as shown in figure 10.As shown in Figure 10, this polyamino acid graft copolymer can form hydrogel fast, and gel-strength can regulate and control.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 8.6, obtain its hydrogen nuclear magnetic resonance spectrogram, as shown in Figure 4; Obtaining result is: the percentage of grafting of poly glycol monomethyl ether is 31%, and the percentage of grafting of tyrasamine is 29%, and productive rate is 76%.
Embodiment 9
9.1 poly glycol monomethyl ethers that are 5000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 1.6ml triethylamine and 3.2ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
9.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g9.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
9.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
9.4 are dissolved in the γ-benzyl obtaining in 2g9.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
9.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g9.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
9.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g9.5, then add 0.6g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.36g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.27g tyrasamine with formula (IV) structure that in 7.75g9.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
9.7 the polyamino acid graft copolymer shown in the formula obtaining in 9.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 9.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 19%, and the percentage of grafting of tyrasamine is 20%, and productive rate is 81%.
10.1 poly glycol monomethyl ethers that are 5000 by 10g number-average molecular weight and 100ml toluene are in 140 ℃ of azeotropic water removings, and then toluene is removed in decompression, adds 50ml methylene dichloride to dissolve and obtains poly glycol monomethyl ether solution.Under 0 ℃, anhydrous condition, in poly glycol monomethyl ether solution, slowly add 1.6ml triethylamine and 3.2ml Methanesulfonyl chloride, 0 ℃ of reaction 2h, rise to stirring at room reaction 24h, after reaction finishes, remove by filter throw out, filtrate is used ether sedimentation, filters and obtains solid, after room temperature vacuum-drying 24h, obtain methylsulphonic acid poly glycol monomethyl ether ester.
10.2 are dissolved in the methylsulphonic acid poly glycol monomethyl ether ester obtaining in 5g10.1 and 5g ammonium chloride in 250ml ammoniacal liquor, room temperature reaction 72h, after reaction finishes, with sodium-chlor saturated reaction liquid, then use dichloromethane extraction reaction product, the sodium chloride aqueous solution washing that the organic phase obtaining is 5% with massfraction, collect organic phase, by anhydrous sodium sulphate, be dried, filter, after filtrate is concentrated, use ether sedimentation, filter, the solid vacuum-drying 24h obtaining, the poly glycol monomethyl ether of (IV) structure that obtains thering is formula.
10.3 add to the dried tetrahydrofuran (THF) of 150ml in dry reaction flask; under nitrogen atmosphere, add 10g γ-benzyl-Pidolidone ester and 6g triphosgene, under nitrogen protection; in 55 ℃ of reaction 2～3h; after reaction solution clarification, stirring at room 30min, then precipitates with cold sherwood oil; filtration is drained; by solid acetic acid ethyl dissolution, cold water washing three times, organic phase is spent the night with anhydrous magnesium sulfate drying.Remove by filter after sal epsom, filtrate is gone in dry reaction flask, use ethyl acetate and normal hexane recrystallization three times, solid vacuum-drying 24h, obtains γ-benzyl-Pidolidone ester-N-carboxylic acid inner-acid anhydride.
10.4 are dissolved in the γ-benzyl obtaining in 2g10.3-Pidolidone ester-N-carboxylic acid inner-acid anhydride in the dioxane that 20ml dewatered; add 4 μ L triethylamines as initiator; under the condition of nitrogen protection; stirring at room reaction 72h; after reaction finishes; use ether sedimentation, solid vacuum-drying 24h, is gathered (γ-benzyl-Pidolidone ester).
10.5 are dissolved in poly-(γ-benzyl-Pidolidone ester) that in 1g10.4, obtain in 10ml dichloro acetic acid; until completely dissolved; adding 3ml volume fraction is 33% hydrobromic acetum; room temperature deprotection reaction 2h, after reaction finishes, uses ether sedimentation; product after suction filtration is with moving into dialysis tubing after dmso solution; dialyse three days, lyophilize, obtains PLGA.By viscosimetry, test, its molecular weight is 110000.
10.6 are dissolved in 50ml dimethyl sulfoxide (DMSO) by the PLGA obtaining in 1g10.5, then add 1.0g1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCl) and 0.55g N-hydroxy-succinamide (NHS) activated carboxyl 24h, add again poly glycol monomethyl ether and the 0.4g tyrasamine with formula (IV) structure that in 11.6g10.2, obtain, after room temperature reaction 24h, reaction solution moves in dialysis tubing, dialyse three days, lyophilize, obtains the polyamino acid graft copolymer shown in formula (I).
10.7 the polyamino acid graft copolymer shown in the formula obtaining in 10.6 (I) is mixed with to mass concentration is that 6%～20% phosphate buffer soln is the first solution; The phosphate buffer soln that hydrogen peroxide is mixed with to 2.45～49mmol/L is the second solution; The phosphate buffer soln that horseradish peroxidase is mixed with to 0.0156～0.5mg/ml is the 3rd solution; 200 μ L the first solution, 50 μ L the second solution, 50 μ L the 3rd solution are fully mixed, adopt tubule inverted type to observe into gel situation, while being inverted with tubule, in 30s, do not flow for gelation.
Utilize nucleus magnetic resonance to analyze the polyamino acid graft copolymer shown in the formula (I) obtaining in 10.6, obtain result and be: the percentage of grafting of poly glycol monomethyl ether is 31%, and the percentage of grafting of tyrasamine is 32%, and productive rate is 87%.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a polyamino acid graft copolymer, as shown in the formula (I):
Described-R
1there is formula (II) structure:
Described-R
2there is formula (III) structure:
Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.
2. polyamino acid graft copolymer according to claim 1, is characterized in that, the scope of described m is 10≤m≤300.
3. a preparation method for polyamino acid graft copolymer, is characterized in that, comprises the following steps:
Tyrasamine, the poly glycol monomethyl ether with formula (IV) structure, the PLGA with formula V structure are mixed with organic solvent, under the effect of coupling agent, condensation reaction occurs, obtain the polyamino acid graft copolymer shown in formula (I); Described-R
1there is formula (II) structure; Described-R
2there is formula (III) structure;
Wherein, x, y, z, m and n are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000; N=x+y+z.
4. preparation method according to claim 3, is characterized in that, described tyrasamine with the mol ratio of carboxyl of PLGA with formula V structure for (0.1～0.4): 1.
5. preparation method according to claim 3, is characterized in that, described in the poly glycol monomethyl ether with formula (IV) structure and the mol ratio of carboxyl with the PLGA of formula V structure be (0.1～0.4): 1.
6. preparation method according to claim 3, is characterized in that, described coupling agent is EDC hydrochloride, N, one or more in N '-dicyclohexylcarbodiimide and N-hydroxy-succinamide.
7. a syringeability hydrogel, is characterized in that, comprises polyamino acid graft copolymer, aqueous solvent, horseradish peroxidase and the hydrogen peroxide shown in formula (I);
Described-R
1there is formula (II) structure:
Described-R
2there is formula (III) structure:
Wherein, x, y, z and m are the polymerization degree, 3≤x≤400; 3≤y≤400; 0≤z≤900; 30≤x+y+z≤1000.
8. syringeability hydrogel according to claim 7, is characterized in that, the polyamino acid graft copolymer shown in described formula (I) and aqueous solvent are mixed into the first mixing solutions of 2～30wt%.
9. syringeability hydrogel according to claim 7, is characterized in that, described horseradish peroxidase and aqueous solvent are mixed into the second mixing solutions of 0.01mg/mL～2.0mg/mL.
10. syringeability hydrogel according to claim 7, is characterized in that, described hydrogen peroxide and aqueous solvent are mixed into the 3rd mixing solutions of 0.1mmol/L～200mmol/L.
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