Preparation and characterization of thermo-responsive amphiphilic triblock copolymer anddrug release

Colloids and Surfaces B:Biointerfaces 72(2009)94–100Contents lists available at ScienceDirectColloids and Surfaces B:Biointerfacesj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c o l s u r fbPreparation and characterization of thermo-responsive amphiphilic triblock copolymer and its self-assembled micelle for controlled drug releaseTianhong Qu,Airong Wang,Jinfang Yuan,Jiahua Shi,Qingyu Gao ∗Institute of Fine Chemical and Engineering,Henan University,Kaifeng,475001,Henan,People’s Republic of Chinaa r t i c l e i n f o Article history:Received 14December 2008Received in revised form 24March 2009Accepted 24March 2009Available online 2April 2009Keywords:Thermo-responsive micelles Amphiphilic triblock copolymer Preparation Self-assemblyControlled drug releasea b s t r a c tAn amphiphilic thermo-responsive ABA triblock copolymer,poly(methyl methacrylate)-b-poly(N-isopropylacrylamide-co-poly(ethylene-glycol)methyl ether methacrlate)-b-poly(methyl methacrylate)(PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA),was designed and synthesized via reversible addition frag-mentation chain transfer (RAFT)polymerization,and subsequently characterized by FT-IR,1H NMR and GPC.The copolymer can disperse in water and self-assemble into nanoscaled micelles in a “flower-like”arrangement at room temperature;the hydrophobic PMMA tucks in the core while the hydrophilic and improved biocompatible P(NIPAM-co-PEGMEMA)forms a thermosensitive outer shell.The resulting micelles were investigated using fluorescence spectroscopy,dynamic light scattering technique (DLS)and transmission electron microscopy (TEM).The copolymer exhibited a lower critical solution temperature (LCST)of around 39◦C via optical transmittance measurements.Notably,there was no copolymer precip-itation observed at the LCST,which was propitious to in vivo use of the micelle.The micelles loaded with folic acid as a model drug showed a desired thermo-responsive drug release behavior.It was found that the rate and amount (maximum percentage 85%)of the drug release was much higher above the LCST than that (maximum percentage 36%)below the LCST.These results indicate that the thermosensitive triblock copolymer possesses promising potential applications as a “smart”drug carrier in biomedical science.©2009Elsevier B.V.All rights reserved.1.IntroductionIn recent years,stimuli-responsive polymeric micelles have been regarded as one of the most promising carriers for drug deliv-ery.These micelles,which are sensitive to environmental stimuli such as pH [1,2],ionic strength [3],temperature [4–6],ultravi-olet light [7],and magnetic field [8],have been developed and actively investigated for applications in effective cancer chemother-apy [1,2,4–12].Thermo-responsive polymeric micelles are used commonly because temperature is easy to be controlled and has practical advantages in vitro and in vivo [9–12].Core–shell (corona)type thermo-responsive polymeric micelles are formed by self-assembly of amphiphilic block copolymers having both thermo-responsive hydrophilic segments and hydrophobic seg-ments in aqueous media [4–6].Thermo-responsive polymeric micelles combine both passive targeting to tissue sites with their small size and the potential active targeting with their switchable physicochemical characteristic [11,12].The drug release mechanism of cancer chemotherapy using the thermo-responsive polymeric micelles is as follows.Polymeric micelles loaded with drugs in a∗Corresponding author.Tel.:+863783881589;fax:+863783881589.E-mail addresses:qingyugao@ ,huobolinzi@ (Q.Gao).nanosized range (<200nm)exhibit prolonged circulation in the systemic circulation by reducing nonselective reticuloendothelial system (RES)scavenge [13,14]while showing enhanced permeabil-ity and retention effects (EPR effects)at tumour sites for passive targeting below a critical temperature [15,16].Subsequently,the thermo-responsive outer shells of the micelles undergo a phase-transition and shrink by local heating up to the critical temperature at the target sites.This transition of micelle properties may induce selective drug activity at the heated target site.Simultaneously,this strategy can achieve temporal drug delivery control by localized temperature increments.To date,a series of thermo-responsive polymers have been studied as potential pharmaceutical delivery agents [5,6,17–20].Most thermo-responsive (co)polymers are synthesized from poly(N-isopropylacrylamide)P(NIPAM),since it undergoes a sharp coil-to-globule phase-transition at a critical temperature,namely,lower critical solution temperature (LCST)of 32◦C,close to phys-iological temperature,37◦C [21].Moreover,the LCST can be modulated via copolymerization with hydrophobic or hydrophilic monomers [22–25].However,in vivo use of such micelles may be hampered by the fact that at the LCST,the shell formed by the temperature-responsive block loses hydrophilicity and the whole copolymer molecule becomes hydrophobic,which could result in the copolymer precipitation and reduce their biocom-0927-7765/$–see front matter ©2009Elsevier B.V.All rights reserved.doi:10.1016/j.colsurfb.2009.03.020T.Qu et al./Colloids and Surfaces B:Biointerfaces72(2009)94–10095Scheme1.The formation and behavior of the thermosensitive polymeric micelles self-assembled from triblock copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA to temperature stimuli.patibility in the circulation[18,19].To overcome this limitation, we designed and synthesized an amphiphilic thermo-responsive ABA triblock copolymer consisting of poly(methyl methacrylate)-b-poly(N-isopropylacrylamide-co-poly(ethylene glycol)methyl ether methacrylate)-b-poly(methyl methacrylate)(PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA)by reversible addition fragmentation chain transfer(RAFT)polymerization.Here,we introduced a hydrophilic biocompatible long side chain poly(ethylene gly-col)methyl ether methacrylate monomer into P(NIPAM)segments to raise the LCST and improve the biocompatibility of the thermo-responsive polymeric micelles.At room temperature, the ABA triblock copolymer can self-assemble into nanoscaled micelles adopting a“flower-like”arrangement with hydropho-bic PMMA A-blocks at the core and a improved biocompatible hydrophilic P(NIPAM-co-PEGMEMA)B-block on the shell in water (Scheme1).The controlled release of folic acid(FA)model drug using the thermo-responsive micelles was also studied in detail. The novel thermo-responsive PMMA-b-P(NIPAM-co-PEGEMA)-b-PMMA polymeric micelles seem to be of great promise as a“smart”drug delivery system.2.Materials and methods2.1.MaterialsS,S -Bis(␣,␣ -dimethyl-␣ -acetic acid)trithiocarbonte(BDATC) was synthesized according to a literature[26].N-Isopropylacrylamide(NIPAM),poly(ethylene glycol)methyl ether methacrylate(PEGMEMA,Mn=475g/mol)were purchased from Acros Organics and used as received.Methyl methacrylate (MMA)and1,4-dioxane were procured from Shanghai Chenfu Chemical Co.,Ltd.and Tianjin No.1Chemical Reagent Factory, respectively,and were distilled under reduced pressure before use. N,N -Azobisisobutyronitrile(AIBN)obtained from Beijing Chemical Industry was used after recrystallization with95%ethanol.Folic acid(FA)was obtained from Acros Organics(Geel,Belgium).All other analytical agents and solvents were used as received.2.2.Synthesis of the triblock copolymerThe triblock copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA was synthesized in two steps via RAFT polymerization. Firstly,PMMA-CTA-PMMA was prepared using BDATC as a chain transfer agent(CTA):MMA(7.52g),BDATC(0.1417g)and AIBN (0.0137g)were dissolved in1,4-dioxane(15ml)in a25ml of reaction tube.The mixed solution was degassed by bubbling with nitrogen for30min and sealed.The polymerization was carried out at75◦C for 4.5h,and quenched by cooling in ice water bath.The precursor polymer was precipitated out from excessive methanol and purified twice using methanol before drying under vacuum.Secondly,the triblock copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA was synthesized in the presence of PMMA-CTA-PMMA as a macromolecular chain transfer agent: NIPAM(2.5g),PEGMEMA(0.5508g),PMMA-CTA-PMMA(1.0g), AIBN(0.0041g)were dissolved in1,4-dioxane(18ml)in a25ml of reaction tube.The treatment and polymerization of the mixture are similar to thefirst step.The polymerization was stopped by freezing after12h.The product was precipitated out from exces-sive cold diethyl ether and purified twice in diethyl ether and warm water,respectively,before drying under vacuum.Finally,the resulting copolymer MMA-b-P(NIPAM-co-PEG-MEMA)-b-PMMA was collected.The synthesis of the triblock copolymer PMMA-b-P(NIPAM-co-PEGMEM A)-b-PMMA is illustrated in Scheme2.2.3.Characterization of the copolymerFT-IR spectrum of the copolymer was recorded on AVATAR-360 FT-IR at a resolution of4cm−1using KBr pellet.1H NMR spectrum was recorded on a EW360L400nuclear mag-netic resonance(NMR)instrument at400MHz,using CDCl3as the solvent.The molecular weights and their distribution of the polymers were determined by gel permeation chromatography(GPC,Waters, MA,polystyrene standards)in THF at an elution rate of1ml/min at 25◦C.2.4.Fluorescence measurementsThe CMC of the triblock copolymer in deionized water was determined with afluorescent probe technique and pyrene was used as a hydrophobicfluorescent probe.The pyrene solution in acetone(6×10−4M,5␮m,prepared prior to use)was added to 5ml of aqueous polymer solutions with different concentrations (from1×10−5to0.2mg/ml).The acetone was then removed under reduced pressure at30◦C for2h,resulting in the same pyrene con-centrations(6×10−7M)in all of the aqueous polymer solutions. All solutions were equilibrated for24h at room temperature,prior to measurements.Fluorescence spectra were recorded by an F-7000fluorescence spectrophotometer(Hitachi,Tokyo,Japan).For the measurements,the scanning speed of pyrene excitation spec-tra was set at240nm/min.Excitation and emission slit widths were set at2.5nm and5.0nm,respectively.The emission spectra were recorded from350nm to550nm with an excitation wavelength of340nm.From the pyrene emission spectra,the intensity(peak height)ratios(I3/I1)of the third band(392nm,I3)to thefirst band (373nm,I1)were analyzed as a function of polymer concentra-tion.A CMC value was taken from the intersection of the tangent to the curve at the inflection with the horizontal tangent through the points at low concentrations.96T.Qu et al./Colloids and Surfaces B:Biointerfaces 72(2009)94–100Scheme 2.The synthesis of the triblock copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA.2.5.Micelles formation and characterizationThe micelle of the triblock copolymer was prepared by a dial-ysis method.8mg of triblock polymers were dissolved in 2ml of N ,N -dimethylformamide (DMF)at room temperature.The solution was put into a dialysis bag (MWCO =8000–14,000)and dialyzed against 1000ml distilled water at room temperature for 2days.The water was replaced overnight.After dialysis,the solution in the dial-ysis bag was collected and freeze-dried for 48h.3mg of lyophilized micelles were dispersed in 10ml distilled water.The micellar solu-tions were firstly kept for a week at room temperature,and then were characterized by dynamic light scattering instrument (DLS)and transmission electron microscopy (TEM)after filtering through a 0.45␮m pore-sized syringe filter.The size distribution of micelles was determined by Nano-ZS90laser nanogranulomertric analysis instrument (Malvern Instrument,Watts,UK).3replicates were per-formed for each measurement and an average value was obtained.TEM sample was prepared by dipping a copper grid with carbon film into the prepared aqueous nanoparticles solution.After deposition,the aqueous solution was blotted away with a strip of filter paper and dried in air.TEM observation of the micelles was conducted on a JEM-100cX II instrument (J.P.)operating at an acceleration voltage of 80kV.2.6.Optical transmittance measurementsOptical transmittance of aqueous polymer solutions at various temperatures was measured at 500nm with a UV–vis spectrom-eter (UV-540,US).Sample cell was thermostated in a refrigerated circulator bath at various temperatures from 25◦C to 45◦C prior to measurements.The LCST values of polymer solutions were deter-mined at the temperatures showing an optical transmittance of 50%.2.7.Drug loading and in vitro releasePMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA copolymers (8mg)and FA (4mg)were dissolved in 2ml of DMF,respectively,and both solutions were mixed.The mixed solution was poured into a dialysis bag (MWCO =8000–14,000)and dialyzed against 1000ml distilled water for 48h.To determine the drug loading content (DLC)and entrapment efficiency (EE),the drug-loaded micellar solution waslyophilized and then dissolved in DMF and finally measured with a UV–vis spectrophotometer at 286nm,using a standard calibration curve experimentally obtained with FA/DMF solutions.The DLC and EE of the micelles was calculated based on the following formulas [1,6,27]:DLC (wt.%)=mass of drug encapsulated in micelles mass of drug-loaded micelles×100(1)EE (%)=mass of drug encapsulated in micelles the initial mass of drug before dialysis×100(2)To study in vitro drug release,the dialysis bags were directly immersed into phosphate buffer solutions (PBS)at pH 7.4,and tem-perature was controlled at 25◦C or 37◦C.Periodically,aliquots of 5ml of buffered solution outside the dialysis bag are removed for UV–vis analysis and replaced with the same volume fresh PBS in order to hold the volume of solutions constant.The amount of drug released from micelles at different temperatures was measured by UV absorbance at 286nm.3.Results and discussion3.1.The composition of the PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA copolymersThe FT-IR spectrum of the triblock copolymers is shown in Fig.1.Absorbance of N H,OCHN ,N C and CH(CH 3)2bending frequency in PNIPAM segments occurs at 3443cm −1,1652cm −1,1555cm −1and 1384cm −1,respectively.The bands at 2975cm −1and 1149cm −1are the characteristic bands of C O C in PEGMEMA units.1728cm −1is the absorbance of C O stretch vibration of ester groups.1H NMR was used to further characterize the structure of the PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA copolymer.The struc-tural formula of the copolymer and the 1H NMR spectrum of the triblock copolymer are shown in Fig.2.All chemical shifts are given in parts per million (ppm)relative to the solvent signal.Clearly,all characteristic 1H NMR signals of PMMA segments (ıa 3.07,ıb 1.57,ıc 1.38),PNIAM segments (ıd 1.76,ıe 2.01,ıf 7.97,ıg 3.95,ıh 1.08),PEGMEMA segments (ıi 1.85,ıj 1.37)could be clearly observed.T.Qu et al./Colloids and Surfaces B:Biointerfaces72(2009)94–10097Fig. 1.FT-IR spectrum of PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA triblock copolymer.Fig. 2.1H NMR spectrum of the block copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA.The molecular weight(Mn)and molecular weight distribu-tion(MW/Mn)of the triblock copolymer were determined by GPC system equipped with laser light scattering detector(LSD) and deflection refractive index detector(DRID).The Mn and its distribution of PMMA-CTA-PMMA and PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA is summarized in Table1,respectively.From Table1,the molecular weight distribution of PMMA-CTA-PMMA block is a little wider than that of the polymer obtained from a typ-ical RAFT polymerization.This is probably because the structure of MMA is similar to that of BDATC,which results in the decrease in chain transfer constant of BDATC.All results above show that the target copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA was obtained.Table1GPC data of PMMA-CTA-PMMA and triblock copolymer PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA(the feed molar ratio of NIPAM to PEGMEMA=20/1).Polymer Mn a Mw/Mn b PMMA-CTA-PMMA7,6501.3 PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA27,400 1.14a Weight-averaged molecular weight.b Number-averaged molecular weight.Fig.3.The plot of I3/I1ratio as a function of logarithm of polymer concentrations (log C)in aqueous solutions.3.2.Micelle formation of the triblock copolymersAmphiphilic polymers consisting of hydrophilic block and hydrophobic block can self-assemble to form core–shell(corona) structure micelles in aqueous media.The formation of micelles from PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA was examined by the detection of CMC relying on afluorescence technique.A polymeric concentration showing a discontinuous change influorescence intensity or in an intensity ratio(I3/I1)is defined as the CMC.Fig.3 shows the plot of I3/I1ratios as a function of logarithm of the triblock copolymer concentration(log C)in aqueous solutions.From the plot of the I3/I1ratio versus polymer concentrations,the I3/I1ratio has no obvious change below a certain concentration,while the I3/I1ratio increases remarkably above that concentration,which indicates the formation of micelles.Hence,this concentration of10.5mg/l is the CMC of PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA micelle,as cal-culated from Fig.3.The low CMC value means that the nanoscaled core–shell-corona micelles can be formed at a lower polymer con-centration,which is suitable for the drug delivery in dilute aqueous media such as bodyfluids.3.3.Temperature sensitivity of the polymeric solutionAn ideal LCST would be slightly higher than the normal human body temperature so localized temperature increments could induce the deformation of the micelles to trigger the release of enclosed drug molecules.It is well known that PNIPAM exhibits a LCST of around32◦C in aqueous solution.Especially,the LCST can be regulated via introducing hydrophobic or hydrophilic monomers hinging on a critical hydrophilic/hydrophobic balance between the chemical groups on the polymer.In this study,a hydrophilic comonomer poly(ethylene glycol)methyl ether methacrylate(PEG-MEMA)was employed to modulate the LCST of the copolymer.The LCST of the triblock copolymer increased with the feed molar ratio of NIPAM to PEGMEMA decrease.As shown in Fig.4,when the feed molar ratio is20/1,19/1and18/1,the LCST of the triblock copoly-mer is39◦C,40.9◦C and42.7◦C,respectively.The cause is that the formation of stronger hydrogen bonds between the thermosensi-tive segment and water resulting from the incorporation of a more hydrophilic PEGMEMA,which allowed the thermosensitive seg-ment dehydration more difficult,leading to an increase in the LCST. In addition,we also investigated the relationship between the LCST of the copolymer and its concentration.From Fig.5,the copoly-mer concentration has little effect on its LCST.Moreover,we also98T.Qu et al./Colloids and Surfaces B:Biointerfaces 72(2009)94–100Fig.4.LCST determined by UV–vis transmittance of 0.3mg/ml of aqueous triblock copolymer solution at 520nm.These triblock copolymers had different feed molar ratio of NIPAM to PEGMEMA:20/1(a);19/1(b);18/1(c),respectively.found that the transmittance of triblock copolymer solution did not decrease to zero but retained 15%at 50◦C (above the LCST)and no concomitant precipitation occurred.This can be explained by the core–shell-corona structure of the resulting micelles formed above the LCST (Scheme 1).Above the LCST,a thin hydrophilic corona was formed from the long side chain PEGMEMA units,which inhibited the further intermicellar aggregation and precipitation.3.4.Characterization and the effects of temperature on the polymer micellesFig.6shows the TEM image of the 0.3mg/ml of the polymeric micelle.It was observed that the micelles showed well-defined spherical shape with core–shell (corona)structure and nanosize ranging from 70nm to 100nm in the solid state.To investi-gate the hydrodynamic diameters and their distribution of the self-assembled micelles and the effect of temperature on the diameters,0.3mg/ml of the resulting polymeric micelles solutions were determined at different temperatures such as 25◦CandFig.5.LCST determined by UV–vis transmittance of aqueous triblock copolymer (feed molar ratio of NIPAM to PEGMEMA:20/1)solutions at 520nm.[Poly-mer]=0.5mg/ml ( ),0.25mg/ml (᭹)and 0.20mg/ml,respectively.Fig.6.TEM image of the polymeric micelles formed from self-assembly of PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA in aqueous solution at roomtemperature.Fig.7.Size distribution of the micelles self-assembled from PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA triblock copolymer in aqueous solution at different temperatures.45◦C by DLS,respectively.The results are diagramed in Fig.7and summarized in Table 2.It was shown that the micelles formed were well dispersed as nanoscaled micelles and had a narrow size distribution.From Fig.7and Table 2,average hydrated diam-eters of the polymeric micelles were about 150nm at 25◦C and 110nm at 45◦C,respectively.The sizes of micelles at 45◦C became much smaller than that at 25◦C.It is because that P(NIPAM-co-PEGMEMA)blocks as the hydrophilic corona extend freelyTable 2Diameter and polydistribution index (PDI)of the polymeric micelles in aqueous solution responding to the temperature.Temperature Diameter a ±sd (nm)PDI ±sd 25◦C 150.3±1.80.125±0.01445◦C110.6±0.90.214±0.061aDetermined by DLS.The averaged hydrodynamic diameter of micelles in water with copolymer concentration at 0.3mg/ml.T.Qu et al./Colloids and Surfaces B:Biointerfaces72(2009)94–10099Fig.8.Temperature-responsive drug release from PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA polymeric micelles in PBSs(pH7.4)at25◦C(a),37◦C (b)and41◦C(c),respectively.below the LCST(at25◦C),whereas the hydrophilic blocks shrink above the LCST(at45◦C).These results also prove that polymeric PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA micelles have an appropriate thermo-responsiveness and the potential application as an“intelligent”drug carrier for controlled drug release.3.5.Drug loading and in vitro drug releaseIt is well known that water-insoluble drugs could be phys-ically incorporated and stabilized in the micellar hydrophobic inner core by hydrophobic interaction[6,18].FA is an anti-cancer drug having poor solubility in distilled water or strong acidic (pH<4)aqueous solution,but it is soluble in weak acid(pH>6) or alkaline aqueous solution.In view of the thermo-responsive property of PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA micelles, we utilized self-assembly of the copolymer(Feed molar ratio (NIPAM/PEGMEMA)=20/1)and FA to prepare FA-loaded micelles in distilled water by a dialysis technique,and investigated their potential application in controlled drug release.In this case,FA was physically entrapped and stabilized in the hydrophobic cores of the micelles by hydrophobic interaction during dialysis against water. The experimental results showed that FA was successfully incorpo-rated into polymeric micelles,and the DLC and EE was about19% and38%,respectively.To evaluate the effect of the temperature stimulus on release of FA from the thermo-responsive micelles,PBSs with the same ionic strength(I=0.15)and pH7.4were used as dialysis aqueous solution in this work.The temperature was maintained at25◦C(below the LCST),37◦C(physiological temperature)and41◦C(above the LCST), respectively.The drug release profiles from micelles are shown in Fig.8.Curves a,b,and c were the release profiles of FA at25◦C(a), 37◦C(b)and41◦C(c),respectively.It was clearly observed that FA release rate and amount increased with increasing tempera-ture under a constant pH value(7.4)condition.For curves b(37◦C) and c(41◦C),there were both a similar burst release for initial5h, followed by a gradual release up to15h,followed by an almost constant release from the micelles for the studied period of45h. Moreover,the drug release amount(maximum percentage85%)at 41◦C(c,above the LCST)was larger than that(maximum percentage 58%)at37◦C(b,below the LCST).However,for curves a(25◦C),the release of FA was always very low and the maximum drug release percentage was no higher than36%.This can be explained by the core–shell(corona)micellar struc-ture.The thermo-responsive shell(corona)of the micelle is very important for drug release at a temperature above the LCST.The hydrophilic P(NIPAM-co-PEGMEMA)segments in the shells of the micelles that were fully extended below the LCST stabilized the drug in the micellar core.However,when the temperature was raised above the LCST,PNIPAM segments in the shells of the micelles tend to shrink and become hydrophobic and further underwent a microphase-separation.The thickness of the micellar corona layer decreased with the temperature increasing,whilst the motion of FA molecules was speeded up by the slack of hydrophobic inter-action between the core and drug due to the deformation of micelles at higher temperature,leading to the much faster drug release from the micelles above the LCST than that below the LCST.Moreover,the higher the temperature was,the greater the final release amount of the drug was.The initial burst release may be attributed to the release of unstable FA molecules dis-tributed in shells of the micelles.Based on the results above, the thermo-sensitive PMMA-b-P(NIPAM-co-PEGMEMA)-b-PMMA copolymer micelles would have a great potential application as an “intelligent”drug carrier in biomedicinefield.4.ConclusionsIn this study,we synthesized a novel thermo-sensitive amphiphilic ABA triblock copolymer composed of PMMA,P(NIPAM-co-PEGMEMA)and PMMA by RAFT polymerization.The structure and composition of the copolymer characterized by FT-IR,1H NMR and GPC.The nanosized thermo-responsive micelles with a core–shell(corona)structure and regularly spherical shape were obtained by self-assembly from the triblock copolymer in aque-ous solution.The property of the thermo-sensitive micelles such as CMC,LCST and size were investigated.The micelles loaded with FA showed a remarkable thermo-responsive drug release behavior, which indicates that the thermo-sensitive PMMA-b-P(NIPAM-co-PEG-MEMA)-b-PMMA copolymer micelles seems to be of great promise as a drug delivery system.AcknowledgementThanks for National Natural Science Foundation of China pro-viding the fund(Grant Number:50273010).References[1]P.Satturwar,M.N.Eddine,F.Ravenelle,J.C.Leroux,Eur.J.Pharm.Biopharm.65(2007)379–387.[2]K.Na,K.H.Lee,Y.H.Bae,J.Control.Release97(2004)513–525.[3]J.Christophe,A.Z.Lefaux,J.Polym.Sci.Part B:Polym.Phys.42(2004)3654.[4]F.Kohori,K.Sakai,T.Aoyagi,M.Yokoyama,Y.Sakurai,T.Okano,J.Control.Release55(1998)87–98.[5]J.E.Chung,M.Yokoyama,M.Yamato,T.Aoyagi,Y.Sakurai,T.Okano,J.Control.Release62(1999)115–127.[6]C.Y.Choi,S.Y.Chae,J.W.Nah,Polymer47(2006)4571–4580.[7]K.Sofia,K.Anatol,L.Jukka,K.Bengt,J.Appl.Polym.Sci.92(2004)2833.[8]B.S.Kim,J.M.Qiu,J.P.Wang,T.A.Taton,Nano Lett.5(2005)1987–1991.[9]N.Nishiyama,K.Kataoka,Pharmacol.Ther.112(2006)630–648.[10]D.Schmaljohann,Adv.Drug Deliv.Rev.58(2006)1655–1670.[11]N.Rapoport,Prog.Polym.Sci.32(2007)962–990.[12]S.Ganta,H.Devalapally,A.Shahiwala,M.Amiji,J.Control.Release126(2008)187–204.[13]H.M.Yang,R.Reisfeld,Proc.Natl.Acad.Sci.U.S.A.85(1988)1189–1193.[14]P.Thédrez,J.C.Saccavini,D.Nolibé,et al.,Cancer Res.49(1989)3081–3086.[15]T.N.Palmer,V.J.Caride,M.A.Caldecourt,J.Twickler,V.Abdullah,Biochim.Bio-phys.Acta797(1984)363–368.[16]H.Maeda,J.Wu,T.Sawa,Y.Matsumura,K.Hori,J.Control.Release65(2000)271–284.[17]X.W.Lia,W.G.Liua,G.X.Yea,B.Q.Zhanga,D.W.Zhua,K.D.Yaoa,Z.Q.Liub,X.Z.Sheng,Biomaterials26(2005)7002–7011.[18]H.Wei,X.Z.Zhang,H.Cheng,W.Q.Chen,S.X.Cheng,R.X.Zhuo,J.Control.Release116(2006)266–274.[19]Z.M.O.Rzaev,S.Dincer,E.Piskin,Prog.Polym.Sci.32(2007)534–595.100T.Qu et al./Colloids and Surfaces B:Biointerfaces72(2009)94–100[20]H.Wei,X.Z.Zhang,C.Cheng,S.X.Cheng,R.X.Zhuo,Biomaterials28(2007)99–107.[21]M.Heskins,J.E.Guillet,J.Macromol.Sci.Chem.A2(1968)1441–1455.[22]S.Q.Liu,Y.W.Tong,Y.Y.Yang,Biomaterials26(2005)5064–5074.[23]O.Soga,C.F.V.Nostrum,W.E.Hennink,Biomacromolecules5(2004)818–821.[24]J.S.Park,K.Kataoka,Macromolecules39(2006)6622–6630.[25]T.Mori,M.Nakashima,Y.Fukuda,K.Minagawa,M.Tanaka,Y.Maeda,Langmuir22(2006)4336–4342.[26]i,D.Filla,R.Shea,Macromolecules35(2002)6754–6756.[27]W.Y.Seow,J.M.Xue,Y.Y.Yang,Biomaterials28(2007)1730–1740.。