CN102627767A - Potential of hydrogen (pH) response random copolymer based on poly-beta amino ester and preparation method and application thereof - Google Patents

Potential of hydrogen (pH) response random copolymer based on poly-beta amino ester and preparation method and application thereof Download PDF

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CN102627767A
CN102627767A CN2012100878750A CN201210087875A CN102627767A CN 102627767 A CN102627767 A CN 102627767A CN 2012100878750 A CN2012100878750 A CN 2012100878750A CN 201210087875 A CN201210087875 A CN 201210087875A CN 102627767 A CN102627767 A CN 102627767A
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macromonomer
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CN102627767B (en
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章莉娟
张灿阳
杨友强
赵斌
孙尧
郭新东
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South China University of Technology SCUT
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Abstract

The invention discloses a potential of hydrogen (pH) response random copolymer based on poly-beta amino ester and a preparation method and application thereof. Firstly, polylactic acid with two ends sealed with hydroxide radicals is synthesized through a solution polymerization method, propylene serves as a macro-monomer after acidification, and a pH response amphiphilic copolymer is prepared through the Michael gradual addition reaction. The proportion of functional groups in the polymer molecule can be regulated. The synthetic process is simple, and the production rate is high. By means of a polymeric micelle obtained through a dialysis method and with a hydrophobic group (polylactic acid) and a pH response group (poly-beta amino ester) forming a kernel and a hydrophilic group forming a casing, water slight-solubility anti-cancer drugs can be coated into the kernel of the micelle, and the drugs can be slowly released in low quantity under neutral or weak alkaline conditions and can be quickly and controllably released under weakly acidic conditions.

Description

Based on gathering-pH of β amino ester response random copolymers and method for making and application
Technical field
The present invention relates to biological medicine and use the macromolecule polymer material field, be specifically related to a kind of based on gather- βPH response random copolymers and the method for making and the application of amino ester.
Background technology
Cancer becomes the main killer of harm humans life and health day by day, and chemotherapy is most important efficacious therapy means except that operation.But there is following two large problems: the one, the toxic side effect of medicine; The 2nd, the resistance of tumour (cancer cells).Though a lot of cancer therapy drugs can be treated cancer on pharmacology; Yet, because the hydrophobic or poorly water-soluble (like Zorubicin, NSC 94600, taxol etc.) of medicine influences result of treatment clinically;, thereby still face huge challenge for the application of this kind anti-cancer drugs thing.
The nano core-shell structure micella that is formed by amphipathic multipolymer is made the carrier of slightly water-soluble cancer therapy drug, can control the release of medicine.Have lot of advantages, can improve the apparent water-soluble of medicine like it; Provide passive and active target ability; Avoid reticuloendothelial system (RES) in the body engulf or by tissue absorption such as liver spleens; Thereby help prolonging micella in blood cycling time and in the accumulation at tumor tissues position; Improve the activity of medicine, thereby effectively improve drug effect, reduce spinoff at privileged site.In addition; Polymer micelle has lower micelle-forming concentration, bigger solubilising space, Stability Analysis of Structures; And can to poorly water soluble drugs tangible solubilizing effect be arranged through effect packaging medicines such as chemistry, physics and static according to polymer hydrophobic segmental different properties.
The cancer therapy drug medicine-carried system of a success must have higher pharmaceutical pack carrying capacity, longer steady state in blood of human body circulation environment, and good target function, toxic side effect is little, characteristics such as good biocompatibility.PH responsive polymer micella is owing to can keep dense structure under the normal pH environment of human body; Reduce the seepage of medicine; And at weakly acidic condition (tumour cell) thus swelling taking place down or dissociates promotes the medicine snap-out release, thereby be considered to the ideal carrier of insoluble cancer therapy drug targeted therapy.The pH value of healthy tissues is generally 7.4.The inner anoxic condition of solid tumor makes tumour cell anaerobic glycolysis produce lactic acid, and the shortage of inside tumor vascular system can not fully be discharged the lactic acid of generation, causes solid tumor inside to have different sour environments, like the weak acid environment pH in the tenuigenin E7.06 about, the acidity in endosome and the lysosome is stronger, and generally about 5.0, lysosomal pH value is 4.0-5.0 to the pH value of early stage endosome.The sour environment of tumour can be used as the quick medicament release that signal is used to trigger carrier micelle, promotes cell endocytic micella and organoid target thereof.
When pH changed, the ionization state of the weakly alkaline polymkeric substance of basic groups such as band amido, pyridine or imidazolyl or the slightly acidic polymkeric substance of band carboxylic group changed, and causes its solvability in water to change.Weakly alkaline polymer poly (N commonly used; N-diethylammonium amido ethyl-methyl propenoate) [poly (N; N-diethylaminoethyl methacrylate); PDEA], gather (4-vinylpridine) [poly (4-vinylpyridine), PVP], polyhistidyl [poly (L-histidine), Phis] and gather ( β-amido ester) [poly ( β-amino esters)] etc. water insoluble under alkalescence or neutrallty condition; And under acidic conditions, positively charged takes place to change mutually because basic group takes place protonated; Cause that its micellar structure changes; Thereby realize that medicine-carried system keeps stable under home, and release efficiency is preferably arranged in that weak acid pH environment is next, so can be used as the carrier of insoluble cancer therapy drug targeted therapy.
Langer [J. Am. Chem. Soc. 2000,122,10761] synthesized a series of novel degradables and gathered- βAmino ester (PAE) is used for the transmission of DNA, and PAE issues unboiled water at acid or alkaline condition and separates and produce 1, and 4-butyleneglycol and amino acid are compared with PEI, and PAE and hydrolysate thereof are nontoxic basically.The microballoon of PAE also is used for water soluble medicament-entrapping; Keep medicine stablizing under extracellular and tenuigenin pH environment; And under endosome and lysosome pH environment, discharge, medicine gets into tenuigenin and then performance drug effect [Angewandte Chemie-International Edition, 2001 again from endosome or lysosome; 40,1707].In the process of preparation PAE microballoon, add PEG, form the PAE nanoparticle that PEG modifies, the bag that is used for taxol carries and discharges, and can obviously alleviate the prominent phenomenon of releasing, and improving release efficiency Journal of Controlled Release, 2003,86,223].Patented claim KR2009056478-A has announced a kind of degradable polymer of being made up of urethane derivative main chain and pH responsiveness side chain; It can pass through the carrier of the form of micella or gel as components such as medicine, polypeptide, nucleic acid, protein, is used for tumour, inflammation and other treatment of diseases.LEE [Journal of Controlled Release; 2007,123,109] synthetic bi-block copolymer MPEG-PAE bag carries Zorubicin DOX; Extracorporeal releasing experiment shows; This material is the good carrier of insoluble cancer therapy drug targeted therapy tumour, and to release phenomenon serious but medicine is prominent, and controlled-release effect is undesirable.Patented claim CN102295737-A has announced the preparation method of multiple response property amphiphilic polyurethane materials such as a kind of pH of having, temperature and light.Patented claim WO2010018286-A1, ES2333087-A1 have announced that a kind of branch-shape polymer and linear polyhistidyl that is connected by polyoxyethylene glycol passes through the micella that electrostatic interaction forms, and it is used for the carrier of medicine and diagnostic reagent.Through changing environment PH the electrically charged character of polyhistidyl is changed, the bag of regulation and control MICELLAR STRUCTURE realization pH response carries and discharges.Patented claim US2005070721-A1, US7229973-B2 have announced a kind of mixed micelle by polylactic acid-polyglycol and the preparation of polyethylene glycol-Histidine, and have shown that this micella has the tumor-targeting function of pH response.Patented claim CN102268137-A has announced the preparation method of a kind of star POLYACTIC ACID-polymethyl acrylic acid dimethylaminoethyl segmented copolymer, and points out to wrap a year hydrophobicity chemotherapeutics, is used for treatment for cancer.
Report from existing research how effectively to improve drug loading and alleviation or eliminate the prominent phenomenon of releasing; How to improve medicine-carried system to the response particularity of environment pH variation and the efficient of sustained release, be still current two topmost difficult problems that face.Common micelle medicine carrying system; Drug-loaded layer and pH response layer have certain space length; Make that the such process of micellar pH response-micella swelling-drug release is slow, accurate inadequately and sensitive to the response that environment pH changes, and if increase too much pH response group; Can make its medicine carrying ability drop again, and prominent release phenomenon and also can aggravate.
Summary of the invention
For the bag of the stability that effectively improves the micelle medicine carrying delivery system, pH response sensitivity, medicine carries efficient and sustained release performance, the present invention design synthetic a kind of based on gather- βThe pH response linear random copolymer of amino ester and its production and application, its self-assembled micelle is used for the targeted drug delivery system of slightly water-soluble cancer therapy drug.
General plotting of the present invention is following: hydrophobic material PLA and pH responsive materials PAE are designed to the random copolymerization block, form the bi-block copolymer material with hydrophilic block again.The amphipathic nature polyalcohol molecular structure of this pH response/hydrophobic grouping random copolymerization can increase pH response region in its self-assembled micelle; Improve drug loading; Make pH response-micella swelling-drug release be rendered as a synchronous process; Thereby improve micellar pH response sensitivity and release efficiency, improve the controlled release properties of micelle medicine carrying system, also can effectively alleviate prominent releasing simultaneously.Hydrophilic block forms the micella shell, helps micella stability, strengthens anti-albumen and thrombocyte adsorptive power, prolongs micella cycling time in vivo.Behind the carrier micelle drug administration by injection, in normal human's environment, because that hydrophobic material PLA and pH response block PAE is all is hydrophobic, the micella kernel is tight, Stability Analysis of Structures, and release amount of medicine is few.Along with pH changes, arrive under the solutions of weak acidity, PAE is protonated, and micelle surface becomes positively charged, and helps through cytolemma, gets into tumour cell, and the protonated micella generation swelling that makes of PAE, and drug release rate is accelerated.Can adapt to different release requests through the content of pH response block in the telomerized polymer material.
The present invention realizes through following technical scheme: earlier synthetic two ends envelope hydroxyl macromonomer, again with the monomer acrylated, utilize progressively addition reaction of Michael subsequently, and promptly obtain pH response amphipathic nature polyalcohol material.The self-assembly in the aqueous solution of this polymer molecule is a nano-micelle, and effectively bag carries poorly water soluble drugs.
The object of the invention realizes through following technical scheme:
A kind of based on gather- βThe pH response random copolymers of amino ester, structural formula does
Figure 2012100878750100002DEST_PATH_IMAGE001
x=2~10，y=1~6，n=113。
The number-average molecular weight of said multipolymer is 10752 ~ 14536 g/mol.
Described a kind of based on gather- βThe molecular formula of the pH response linear random copolymer of amino ester is: MPEG- b-(PLA- Co-PAE).
Described based on gather- βThe preparation method of the pH response random copolymers of amino ester may further comprise the steps:
(1) preparation macromonomer A: under protection of inert gas and anhydrous condition,, react 12 ~ 36 h down at 80 ~ 120 ℃ with monomer, catalyst system A, initiator and solvent, remove solvent after, deposition, filtration, drying obtain macromonomer A;
(2) preparation macromonomer B: macromonomer A is dissolved in the solvent; Add reductive agent; Be cooled to 0 ℃; Add acylating agent then, keep 0 ℃ to rise to behind reaction 2 h down and react 12 ~ 36 h under the room temperature again, then the macromonomer B after deposition, filtration, dry terminal hydroxyl acrylated;
(3) preparation macromonomer D: hydrophilic macromonomer C is dissolved in the solvent; Add reductive agent; Be cooled to 0 ℃; Add acylating agent then, keep 0 ℃ to rise to behind reaction 2 h down and react 12 ~ 36 h under the room temperature again, then the macromonomer D after deposition, filtration, dry terminal hydroxyl acrylated;
(4) preparation based on gather- βThe pH response linear random copolymer of amino ester: the macromonomer B that under protection of inert gas and anhydrous condition, respectively macromonomer D, diacrylate, diamines and step (2) is obtained is dissolved in the solvent; The mixing back is reacted 60 ~ 80 h down at 50 ~ 80 ℃, and deposition, filtration, oven dry obtain pH response linear random copolymer;
The parts by weight of reactant are following in the said step (1):
84.7 ~ 98.04 parts of monomers
0.33 ~ 2.6 part of catalyst system A
1.62 ~ 12.7 parts of initiators;
The parts by weight of reactant are following in the said step (2):
61 ~ 91.32 parts of macromonomer A
4.8 ~ 17 parts of reductive agents
4.13 ~ 23.68 parts of acylating agents;
The parts by weight of reactant are following in the said step (3):
90.9 ~ 96.2 parts of hydrophilic macromonomer C
1.9 ~ 5.45 parts of reductive agents
1.9 ~ 3.35 parts of acylating agents;
The parts by weight of reactant are following in the said step (4):
20.36 ~ 41.81 parts of macromonomer D
9.8 ~ 25.57 parts of diacrylates
12.96 ~ 31.45 parts of diamines
16.72 ~ 42.25 parts of macromonomer B.
Said monomer is D, the L-rac-Lactide;
Said diamines is 1,3-pair-4-piperidines propane;
Said diacrylate is a 1,6 hexanediol diacrylate;
Said hydrophilic macromonomer C is a mono methoxy polyethylene glycol, and its structural formula is following:
Figure 444256DEST_PATH_IMAGE002
N=113 wherein.
Catalyst system A is the toluene solution of stannous octoate described in the step (1);
The said initiator of step (1) is 1, the 4-butyleneglycol;
The said solvent of step (1) is toluene or methyl-phenoxide; The amount of solvent is enough to the reactant of dissolving step (1)
Step (2) and (3) said reductive agent are triethylamine;
Step (2) and (3) said acylating agent are acrylate chloride;
Step (2) and (3) said solvent are methylene dichloride;
The said solvent of step (4) is a chloroform.
The said solvent that removes of step (1) is that reaction product is reduced pressure rotary evaporation except that desolvating;
Deposition described in the step (1) is that the product that removes behind the solvent is dissolved in chloroform or the methylene dichloride, solution is joined in the mixing solutions of 0 ℃ of cold methanol being equivalent to 10 times of its volumes and water again, and cold methanol and water volume ratio are 1:1;
Step (2) and (3) said deposition are that reacted solution is joined in the 0 ℃ of cold normal hexane that is equivalent to 10 times of its volumes; Preferably reacted solution is added hydrochloric acid, gained lower floor solution joins in the 0 ℃ of cold normal hexane that is equivalent to 10 times of lower floor's liquor capacities.
The said deposition of step (4) is that reacted solution is joined in the 0 ℃ of cold diethyl ether that is equivalent to 10 times of its volumes.
Described based on gathering-micelle medicine carrying system of the poorly water soluble drugs that the pH of β amino ester response random copolymers makes.
The preparation method of the micelle medicine carrying system of described poorly water soluble drugs, be with based on gather- βThe pH response random copolymers and the poorly water soluble drugs of amino ester are dissolved in the organic solvent; Stir behind 6 h with the pH damping fluid 24 h postlyophilizations of dialysing; Obtain, said organic solvent is N or DMSO 99.8MIN. or acetone, and said pH damping fluid is the buffered soln of pH value 7 ~ 9.
The present invention also provides a kind of micelle medicine carrying system of described poorly water soluble drugs to carry and APPLICATION IN TRANSMISSION at the bag of slightly water-soluble cancer therapy drug.
Said poorly water soluble drugs is the medicine that solubleness is less than or equal to 1 g in 1 L water.
The said catalyst system A of step (1) is a stannous octoate, and its consumption is 0.33% ~ 3% of a hydrophobic monomer quality, is preferably 1% ~ 1.5%;
The said initiator of step (1) is 1, the 4-butyleneglycol, and its consumption is 1.67% ~ 15% of a hydrophobic monomer quality, is preferably 5% ~ 8%;
The said reductive agent of step (2) is a triethylamine, and its consumption is 1.2 ~ 4 times of macromonomer A mole number, is preferably 2 ~ 2.8 times;
The said acidylate of step (2) is an acrylate chloride, and its consumption is 1.5 ~ 10 times of macromonomer A mole number, is preferably 3 ~ 4 times;
The said reductive agent of step (2) is a triethylamine, and its consumption is 1 ~ 3 times of macromonomer E mole number;
The said acidylate of step (2) is an acrylate chloride, and its consumption is 1.1 ~ 2.2 times of macromonomer E mole number;
The ratio of the said two kinds of diacrylates of step (3) (A-PLA-A:HDD) is 1/3 ~ 1/6, is preferably 1/4 ~ 1/5;
Temperature of reaction in the said step (1) is preferably 95 ~ 105 ℃, and the reaction times is 20 ~ 24 h;
Said step rises to room temperature in (2), and the reaction times is preferably 20 ~ 24 h;
Temperature of reaction in the said step (3) is preferably 55 ~ 65 ℃, and the reaction times is 70 ~ 75 h.
Described a kind of based on gather- βThe micelle medicine carrying system of the pH response linear random copolymer of amino ester; Can be used for the drug administration by injection mode; Regulate and control medicine through tumour cell and normal cell environment pH difference and in normal meta-alkalescence or neutral environment, slowly perhaps do not discharge, in weakly acidic tumor environment, faster discharge.
The invention has the beneficial effects as follows: the present invention design synthetic a kind of based on gather- βThe pH response linear random copolymer and the self-assembled micelle drug-loading system thereof of amino ester; Hydrophobic grouping and pH response group are designed to the random copolymerization form; Both can keep than high drug load; Can effectively regulate pH responding range in its self-assembled micelle again, make the not only variation of response environment pH value rapidly of micella, and effectively alleviate prominent releasing and control drug release.The long hydrophilic block of micella shell can improve micella shell density, strengthens wetting ability, anti-albumen and the thrombocyte adsorptive power of micelle surface, prolongs micellar cycling time, thereby improves the controlled release properties of micelle medicine carrying system.This micelle medicine carrying system Stability Analysis of Structures in home, the micella kernel is tight, and the cumulative release amount is few, thus the protection medicine.In weakly acidic condition, the micella swelling, drug release rate is accelerated, and because micelle surface has positive electricity, helps it and get into tumour cell through cytolemma.The rate of release that can come regulating medicine through the content of pH response group and hydrophobic grouping in the telomerized polymer molecular material satisfies the release request of different pharmaceutical.The critical aggregate concentration of this polymer molecule is lower, is merely 1.2 ~ 9.5 mg/L, thereby the stability of its carrier micelle is high.
Description of drawings
Fig. 1 is the building-up reactions formula of macromonomer A.
Fig. 2 is the building-up reactions formula of macromonomer A and hydrophilic macromonomer C acrylated.
Fig. 3 is the reaction formula that obtains the linear copolymerized polymer of pH response amphiphilic.
Fig. 4 is the gel permeation chromatography figure (GPC) of HO-PLA-OA among the embodiment 1, and moving phase is THF (THF).
Fig. 5 be HO-PLA-OH among the embodiment 1 proton nmr spectra ( 1H NMR), solvent is deuterochloroform (d-CDCl 3).
Fig. 6 be MPEG-A among the embodiment 1 proton nmr spectra ( 1H NMR), solvent is deuterochloroform (d-CDCl 3).
Fig. 7 is the 1H NMR spectrum of A-PLA-A among the embodiment 1, and solvent is d-CDCl 3
Fig. 8 is MPEG-among the embodiment 1 b-(PLA- Co-PAE) GPC elution curve.
Fig. 9 is MPEG-among the embodiment 1 b-(PLA- Co-PAE) 1H NMR spectrum, solvent is d-CDCl 3
Figure 10 is MPEG-among the embodiment 7 b-(PLA- Co-PAE) micelle-forming concentration test curve.
Figure 11 is that embodiment 8 empty micellar particle diameters are with the pH variation diagram.
Figure 12 is that embodiment 8 empty micellar current potentials are with the pH variation diagram.
Figure 13 is ESEM (SEM) figure of carrier micelle among the embodiment 9.
Figure 14 is MPEG-among the embodiment 10 b-(PLA- Co-PAE) (case study on implementation 1 product) carry Zorubicin micellar release in vitro curve.
Figure 15 is for implementing 11 empty micellas to the toxicity graphic representation to HepG2.
Figure 16 is carrier micelle in the case study on implementation 11 and the free Zorubicin toxicity graphic representation after to HepG2 effect 24 h.
Figure 17 is carrier micelle in the case study on implementation 11 and the free Zorubicin toxicity graphic representation after to HepG2 effect 48 h.
Embodiment
Content for a better understanding of the present invention is described further below in conjunction with the practical implementation instance.The abbreviation contrast of uses such as monomer, catalyzer, initiator is as follows in an embodiment:
Title Abbreviation Title Abbreviation
Rac-lactide LA Triethylamine TEA
1, the 4-butyleneglycol BDO Acrylate chloride AC
1,6 hexanediol diacrylate HDD Stannous octoate Sn(Oct) 2
1,3-pair-4-piperidines propane TDP Mono methoxy polyethylene glycol MPEG
Embodiment 1
(1) (A:B:C=100:5:1, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (20 g, 93.9 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.3 mg, 1.41 parts) and initiator 1,4-butyleneglycol (1 g, 4.69 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 100 ℃ of oil baths, react 24 h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h.Can know that by Fig. 5 pre-product is synthetic.Can know by Fig. 4 M n=1998, PDI=1.19.
(2) acrylated of HO-PLA-OH (AB:D:F=10:1:1.36, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (5 g, 80.9 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (50 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.5 g, 8.09 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.68 g with ice-cooled; 11.01 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 24 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.Can know that by Fig. 7 pre-product is synthetic.
(3) acrylated of MPEG (hydrophilic macromonomer C:D:F=25:10:7, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, (2.5 g, n=45) are dissolved in the solution that forms 10 % in the methylene dichloride (25 mL) by 93.63 parts with MPEG; Place single necked round bottom flask, slowly drip anhydrous TEA (0.1 g, 3.75 parts) with syringe, with ice-cooled to 0 ℃; Under agitation dropwise drip AC (0.07 g, 2.62 parts), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 24 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of volumes) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.Can know that by Fig. 6 pre-product is synthetic.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=20:25:9.05:11.57, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (2.5 g, 38.1 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (0.905 g, 13.79 parts), TDP (1.157 g, 17.63 parts); A-PLA-A (2 g, 30.48 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 65 ℃, magnetic agitation is reacted 70 h.The solution that obtains slowly joins in 0 ℃ of ether of 300 mL (being equivalent to 10 times of volumes) deposition three times, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtains pale yellow powder.Right Fig. 8 learns and obtains pre-product.Can know by Fig. 9 M n=10952, PDI=1.39.Obtain the x=7 of product, y=1, n=113.
The reaction formula of pH response amphipathic nature polyalcohol molecule of the present invention is shown in Fig. 1-3.
Embodiment 2
(1) (A:B:C=100:15:3, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (10 g, 91.32 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.15 mg, 1.37 parts) and initiator 1,4-butyleneglycol (0.8 g, 7.31 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 95 ℃ of oil baths, react 30 h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h. M n=1020，PDI=1.22。
(2) acrylated of HO-PLA-OH (AB:D:F=25:7:9, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (2.5 g, 61 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.7 g, 17 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.9 g with ice-cooled; 22 parts), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 20 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(3) acrylated of MPEG (macromonomer C:D:F=50:3:2, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, MPEG (2.5 g, 91.9 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.13 g, 4.78 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.09 g with ice-cooled; 3.32 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 20 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=100:90:113:139, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (0.9 g, 20.36 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (1.13 g, 25.57 parts), TDP (1.39 g, 31.45 parts); A-PLA-A (1g, 22.62 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 70 ℃, magnetic agitation is reacted 70 h.Product usefulness, the solution that obtains slowly join and precipitate deposition in three times in 0 ℃ of ether of 300 mL, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtain pale yellow powder. M n=11374，PDI=1.25。Obtain the x=5 of product, y=3, n=113.
Embodiment 3
(1) (A:B:C=300:5:1, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (30 g, 98.04 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.1 mg, 0.33 part) and initiator 1,4-butyleneglycol (0.5 g, 1.63 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 105 ℃ of oil baths, react 20 h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h. M n=2960，PDI=1.32。
(2) acrylated of HO-PLA-OH (AB:D:F=75:4:3.4, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (7.5 g, 91.02 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (75 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.4 g, 4.85 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.34 g with ice-cooled; 4.13 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 20 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(3) acrylated of MPEG (macromonomer C:D:F=50:1:1, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, MPEG (2.5 g, 96.2 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.05 g, 1.9 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.05 g with ice-cooled; 1.9 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 20 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=150:125:34:46, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (2.5 g, 35.21 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (0.68 g, 9.8 parts), TDP (0.92 g, 12.96 parts); A-PLA-A (3g, 42.25 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 55 ℃, magnetic agitation is reacted 80 h.Product usefulness, the solution that obtains slowly join and precipitate deposition in three times in 0 ℃ of ether of 300 mL, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtain pale yellow powder. M n=13540，PDI=1.36。Obtain the x=10 of product, y=1, n=113.
Embodiment 4
(1) (A:B:C=300:5:3, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (30 g, 97.4 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.3 mg, 0.98 part) and initiator 1,4-butyleneglycol (0.5 g, 1.62 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 100 ℃ of oil baths, react 24 h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h. M n=2500，PDI=1.27。
(2) acrylated of HO-PLA-OH (AB:D:F=25:4:9, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (2.5 g, 65.78 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.4 g, 10.53 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.9 g with ice-cooled; 23.68 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 24 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(3) acrylated of MPEG (macromonomer C:D:F=50:1:2, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, MPEG (2.5 g, 91.9 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.13g, 4.78 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.09 g with ice-cooled; 3.32 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 24h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=250:250:113:139, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (2.5 g, 33.24 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (1.13 g, 15.03 parts), TDP (1.39 g, 18.49 parts); A-PLA-A (2.5g, 33.24 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 60 ℃, magnetic agitation is reacted 75 h.Product usefulness, the solution that obtains slowly join and precipitate deposition in three times in 0 ℃ of ether of 300 mL, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtain pale yellow powder. M n=12552，PDI=1.38。Obtain the x=4 of product, y=2, n=113.
Embodiment 5
(1) (A:B:C=100:15:3, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (10 g, 84.7 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.3 g, 2.6 parts) and initiator 1,4-butyleneglycol (1.5 g, 12.7 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 80 ℃ of oil baths, react 36h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h. M n=992，PDI=1.35。
(2) acrylated of HO-PLA-OH (AB:D:F=25:7:9, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (2.5 g, 61 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.7 g, 17 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.9 g with ice-cooled; 22 parts), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 36 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(3) acrylated of MPEG (macromonomer C:D:F=50:3:2, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, MPEG (2.5 g, 90.9 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.15g, 5.45 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.1 g with ice-cooled; 3.35 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 36 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=100:90:113:139, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (0.9 g, 20.36 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (1.13 g, 25.57 parts), TDP (1.39 g, 31.45 parts); A-PLA-A (1 g, 22.62 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 80 ℃, magnetic agitation is reacted 60 h.Product usefulness, the solution that obtains slowly join and precipitate deposition in three times in 0 ℃ of ether of 300 mL, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtain pale yellow powder. M n=10752，PDI=1.04。Obtain the x=4 of product, y=3, n=113.
Embodiment 6
(1) (A:B:C=100:5:1, A represent the monomer rac-Lactide to synthetic HO-PLA-OH, and B represents initiator 1, and 4-butyleneglycol, C are represented catalyst S n (Oct) 2, ratio is mass percent, down together).Stirrer and monomer rac-Lactide (20 g, 94.33 parts) are added in the 100 mL reaction flasks of band stirrer, bleed after the sealing-ventilate three times, under argon shield, add solvent toluene (40 mL) and catalyst S n (Oct) with syringe successively 2(0.2 g, 0.95 part) and initiator 1,4-butyleneglycol (1 g, 4.72 parts).Behind liquid nitrogen freezing, vacuumize-heat up-inflate three times, under argon shield, in 120 ℃ of oil baths, react 12 h.After reaction finishes; The solution that obtains is removed toluene through the decompression rotary evaporation, with chloroform polymer dissolution is obtained solution again, slowly joins 300 mL (being equivalent to 10 times of liquor capacities) refrigerated methanol (1:1; V/v) deposition in; Behind deionized water wash 2 times,, obtain the HO-PLA-OH product at 45 ℃ of following vacuum-drying 48 h. M n=1204，PDI=1.20。
(2) acrylated of HO-PLA-OH (AB:D:F=10:1:1.36, AB represents HO-PLA-OH, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, HO-PLA-OH (5 g, 80.9 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (50 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.5 g, 8.09 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.68 g with ice-cooled; 11.01 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 12 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(3) acrylated of MPEG (macromonomer C:D:F=25:10:7, C represents MPEG, D represents reductive agent TEA, F represents acylating agent AC, down with).Get baking exsiccant 50 mL reaction flasks, MPEG (2.5 g, 93.63 parts) is dissolved in the solution that forms 10 % in the methylene dichloride (25 mL), place single necked round bottom flask; Slowly drip anhydrous TEA (0.1 g, 3.75 parts) with syringe, to 0 ℃, under agitation dropwise drip AC (0.07 g with ice-cooled; 2.62 part), at 0 ℃ of following reaction 2 h, rise to room temperature after; Continue reaction 12 h, reacted solution adds Hydrogen chloride, and gained lower floor solution joins in 0 ℃ of normal hexane of 300 mL (being equivalent to 10 times of lower floor's liquor capacities) and precipitates; Filter, at 45 ℃ of following vacuum-drying 48 h, obtain white powder then.
(4) synthetic segmented copolymer MPEG- b-(PLA- Co-PAE) (ABF:EF:G:H=20:25:9.05:11.57, ABF represent the product A-PLA-A of step (2), and EF represents the product MPEG-A of step (3), and G represents diacrylate HDD, and H represents diamines TDP, down together).MPEG-A (2.5 g, 41.81 parts) is added in the 100 mL single port reaction flasks, freezing after the sealing-as to vacuumize-the Tong argon gas, three times, under argon shield, add anhydrous chloroform (10 mL) dissolving, freezing-as to vacuumize-the Tong argon gas, three times.Respectively with certain amount of H DD (1.13 g, 18.9 parts), TDP (1.35 g, 22.57 parts); A-PLA-A (1 g, 16.72 parts) is dissolved in the anhydrous chloroform (10 mL), mixes then, and mixed solution is joined in the reaction flask with syringe; Under 50 ℃, magnetic agitation is reacted 80 h.Product usefulness, the solution that obtains slowly join in 0 ℃ of ether of 300 mL and precipitate three times, after the filtration, at 35 ℃ of following vacuum-drying 48 h, obtain pale yellow powder. M n=14536，PDI=1.53。Obtain the x=2 of product, y=6, n=113.
Embodiment 7
The fluorescent probe method is measured embodiment 1 product MPEG- b-(PLA- Co-PAE) micelle-forming concentration.
(1) preparation pyrene solution: pyrene is mixed with 6*10 with acetone -5The solution of M.
(2) join sample solution: take by weighing 10 mg MPEG- b-(PLA- Co-PAE) be dissolved in 5 mL acetone, dropwise join in the 100 mL deionized waters, obtain 0.1 mg/mL solution behind the volatilization acetone, be diluted to a series of concentration (0.0001 ~ 0.1 mg/ml) subsequently.Get 20 10 mL volumetric flasks, every adds 0.1 mL pyrene solution, and the polymers soln that adds above-mentioned different concns then respectively is made into sample liquid.The concentration of pyrene is 6*10 in the sample liquid -7M.
(3) fluorescence spectrum test: as emission wavelength, specimen liquid is got at the excitation spectrum of 300-350nm with 373 nm I 337/ I 336Ratio is seen Figure 10 to concentration logarithm logC mapping, and curve break is the micelle-forming concentration value.Record MPEG- b-(PLA- Co-PAE) micelle-forming concentration is 1.5 mg/L.
Embodiment 8
Preparation MPEG- b-(PLA- Co-PAE) blank micella, and characterize the situation that its particle diameter and current potential change with pH.Adopt dialysis method to prepare blank micella.Accurately take by weighing 40 mg MPEG- b-(PLA- Co-PAE) be dissolved in the 50 mL Ns, stir 2h under the room temperature, change dialysis tubing (MWCO3500 ~ 4000) over to, with 1 L deionized water, 48 h that dialyse, preceding 12 h, per 2 h change the medium of once dialysing, and back 36 h do not have 6 h to change the medium of once dialysing.With the micellar solution via hole diameter is the filtering head filtration postlyophilization of 0.8 μ m.Blank micella is added in the pre-assigned different pH buffered soln successively, place 0.5 h.Adopt dynamic light scattering method to survey MPEG- b-(PLA- Co-PAE) particle diameter and current potential be with the variation of pH value.See Figure 11 and Figure 12.
Embodiment 9
Adopt dialysis method to prepare carrier micelle.Accurately take by weighing 20 mg Zorubicins, 40 mg MPEG-b-(PLA-co-PAE) are dissolved in the 50 mL Ns, drip TEA (0.02 mL) after stirring 2 h under the room temperature; Change dialysis tubing (MWCO3500 ~ 4000) over to; With 1 L deionized water, 48 h that dialyse, preceding 12 h, per 2 h change the medium of once dialysing; Back 36 h do not have 6 h to change the medium of once dialysing.With the micellar solution via hole diameter is the filtering head filtration postlyophilization of 0.8 μ m.It is spherical adopting SEM to observe its pattern, sees Figure 13.
Embodiment 10
Extracorporeal releasing experiment: accurately take by weighing 3 mg carrier micelle powder, place dialysis tubing (MWCO3500 ~ 4000), (pH 5.0 to add 3 mL PBS buffered soln then respectively; 6.5 with 7.4); Seal dialysis tubing, change medicament dissolution instrument over to, add 40 mL PBS damping fluids.Design temperature is at 37 ℃, and stirring velocity is 110 rpm.3 mL that take a sample at regular intervals, and add 3 mL fresh buffer.Discharge doxorubicin concentration in the liquid with the determined by ultraviolet spectrophotometry different time, draw its release in vitro curve.
Figure 14 is MPEG- b-(PLA- Co-PAE) the release in vitro curve of (embodiment 1 product), in home (pH 7.4), drug releasing rate is all very slow: 3 h discharge and are lower than 20%, 148 h and discharge and be lower than 36%, effectively protect the loss of medicine in the blood of human body working cycle.In weakly acidic condition (pH 6.5), MPEG- b-(PLA- Co-PAE) carrier micelle just discharges 25%, 30 h at 2.5 h and has discharged 50%, 148 h and discharge 66%; Acid reinforcement (pH 5.0), the MPEG-of continuing b-(PLA- Co-PAE) carrier micelle just discharges 38%, 24 h at 1.5 h and has discharged 62%, 148 h and discharge near 90%.
Embodiment 11
Cytotoxicity experiment: the HepG2 cell is tiled on 96 orifice plates by 1 * 104 density, adds 200 μ L nutrient solutions, cultivate 24 h.With certain density free Zorubicin, blank micella and carrier micelle add in the entering orifice plate, upgrade developing medium.3 skies of the parallel repetition of each concentration.Blank is put into the ovum device, and 5 % CO2 and 37 ° of C keep 24 h and 48 h respectively.With 180 μ L fresh mediums and 20 μ L MTT solution replacement orifice plate medium, continue ovumization 4 h, with 200 μ L DMSO replacement orifice plate medium.Orifice plate is placed on vibration 15 min in 37 ° of C shaking tables, utilizes ELIASA to measure the absorbance A that 490 nm go out each hole then, calculate cell survival rate, estimate its cytotoxicity.
Figure 15 is blank MPEG- b-(PLA- Co-PAE) cytotoxicity figure.Can know that by figure along with the increase of block polymerization substrate concentration, cell survival rate has slight decline; When polymer concentration was 400mg/L, case study on implementation 1,2 and the corresponding cell survival rate of 3 products were 96.08 %; 95.14 % and 92.95% explain that this block polymer is almost non-toxic.Figure 16 and Figure 17 are the cytotoxicity figure behind three case study on implementation product carrier micelles and free Zorubicin 24 h and 48 h.Can know that by figure behind 24 h and 48 h, cell survival rate is that the concentration of pairing free DOX of 50 % (IC50) and carrier micelle is respectively 1.5 mg/L, 3.0mg/L, 1.8mg/L, 5.35mg/L and 0.42 mg/L, 1.0mg/L, 0.8mg/L, 1.75mg/L.

Claims (9)

  1. One kind based on gather- βThe pH response random copolymers of amino ester is characterized in that structural formula does
    Figure 2012100878750100001DEST_PATH_IMAGE001
    x=2~10，y=1~6，n=113。
  2. 2. according to claim 1 based on gather- βThe pH response random copolymers of amino ester is characterized in that the number-average molecular weight of said multipolymer is 10752 ~ 14536 g/mol.
  3. Claim 1 described based on gather- βThe preparation method of the pH response random copolymers of amino ester is characterized in that, may further comprise the steps:
    (1) preparation macromonomer A: under protection of inert gas and anhydrous condition,, react 12 ~ 36 h down at 80 ~ 120 ℃ with monomer, catalyst system A, initiator and solvent, remove solvent after, deposition, filtration, drying obtain macromonomer A;
    (2) preparation macromonomer B: macromonomer A is dissolved in the solvent; Add reductive agent; Be cooled to 0 ℃; Add acylating agent then, keep 0 ℃ to rise to behind reaction 2 h down and react 12 ~ 36 h under the room temperature again, then the macromonomer B after deposition, filtration, dry terminal hydroxyl acrylated;
    (3) preparation macromonomer D: hydrophilic macromonomer C is dissolved in the solvent; Add reductive agent; Be cooled to 0 ℃; Add acylating agent then, keep 0 ℃ to rise to behind reaction 2 h down and react 12 ~ 36 h under the room temperature again, then the macromonomer D after deposition, filtration, dry terminal hydroxyl acrylated;
    (4) preparation based on gather- βThe pH response linear random copolymer of amino ester: the macromonomer B that under protection of inert gas and anhydrous condition, respectively macromonomer D, diacrylate, diamines and step (2) is obtained is dissolved in the solvent; The mixing back is reacted 60 ~ 80 h down at 50 ~ 80 ℃, and deposition, filtration, oven dry obtain pH response linear random copolymer;
    The parts by weight of reactant are following in the said step (1):
    84.7 ~ 98.04 parts of monomers
    0.33 ~ 2.6 part of catalyst system A
    1.62 ~ 12.7 parts of initiators;
    The parts by weight of reactant are following in the said step (2):
    61 ~ 91.32 parts of macromonomer A
    4.8 ~ 17 parts of reductive agents
    4.13 ~ 23.68 parts of acylating agents;
    The parts by weight of reactant are following in the said step (3):
    90.9 ~ 96.2 parts of hydrophilic macromonomer C
    1.9 ~ 5.45 parts of reductive agents
    1.9 ~ 3.35 parts of acylating agents;
    The parts by weight of reactant are following in the said step (4):
    20.36 ~ 41.81 parts of macromonomer D
    9.8 ~ 25.57 parts of diacrylates
    12.96 ~ 31.45 parts of diamines
    16.72 ~ 42.25 parts of macromonomer B.
  4. 4. preparation method according to claim 3 is characterized in that, said monomer is D, the L-rac-Lactide;
    Said diamines is 1,3-pair-4-piperidines propane;
    Said diacrylate is a 1,6 hexanediol diacrylate;
    Said hydrophilic macromonomer C is a mono methoxy polyethylene glycol, and its structural formula is following:
    Figure 331991DEST_PATH_IMAGE002
    N=113 wherein.
  5. 5. according to claim 3 or 4 described preparing methods, it is characterized in that catalyst system A is the toluene solution of stannous octoate described in the step (1);
    The said initiator of step (1) is 1, the 4-butyleneglycol;
    The said solvent of step (1) is toluene or methyl-phenoxide;
    Step (2) and (3) said reductive agent are triethylamine;
    Step (2) and (3) said acylating agent are acrylate chloride;
    Step (2) and (3) said solvent are methylene dichloride;
    The said solvent of step (4) is a chloroform.
  6. 6. preparation method according to claim 5 is characterized in that, the said solvent that removes of step (1) is that reaction product is reduced pressure rotary evaporation except that desolvating;
    Deposition described in the step (1) is that the product that removes behind the solvent is dissolved in chloroform or the methylene dichloride, solution is joined in the mixing solutions of 0 ℃ of cold methanol being equivalent to 10 times of its volumes and water again, and cold methanol and water volume ratio are 1:1;
    Step (2) and (3) said deposition are that reacted solution is joined in the 0 ℃ of cold normal hexane that is equivalent to 10 times of its volumes;
    The said deposition of step (4) is that reacted solution is joined in the 0 ℃ of cold diethyl ether that is equivalent to 10 times of its volumes.
  7. 7. claim 1 is described based on gathering-micelle medicine carrying system of the poorly water soluble drugs that the pH of β amino ester response random copolymers makes.
  8. 8. the preparation method of the micelle medicine carrying system of the described poorly water soluble drugs of claim 7; It is characterized in that; Be will based on gather-pH of β amino ester response random copolymers and poorly water soluble drugs be dissolved in the organic solvent, stirs behind 6 h with the pH damping fluid 24 h postlyophilizations of dialysing, obtain; Said organic solvent is N or DMSO 99.8MIN. or acetone, and said pH damping fluid is the buffered soln of pH value 7 ~ 9.
  9. 9. the micelle medicine carrying system of the described poorly water soluble drugs of claim 7 carries and APPLICATION IN TRANSMISSION at the bag of slightly water-soluble cancer therapy drug.
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CN103709343A (en) * 2013-12-16 2014-04-09 三棵树涂料股份有限公司 Block polymer with pH/temperature/light triple responsiveness and preparation method thereof
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CN103709343A (en) * 2013-12-16 2014-04-09 三棵树涂料股份有限公司 Block polymer with pH/temperature/light triple responsiveness and preparation method thereof
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CN108363901A (en) * 2018-02-08 2018-08-03 中国石油大学(华东) A kind of analogy method of pH response types block copolymer PAE-PEG loadings and release naphthalene molecule
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