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Polymer 55 (2014) 2369e2379

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Polymer

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Effect of atom transfer radical polymerization macroinitiator onproperties of poly(meth)acrylate-based pentablock type ofthermoplastic elastomers

Ravikumar Muppalla a,1, Swati Srivastava b,1, Partha Roy b, Suresh K. Jewrajka a,c,*

aReverse Osmosis Discipline, CSIReCentral Salt and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, IndiabMolecular Endocrinology Laboratory, Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee 247 667, Uttarakhand, IndiacAcSIReCentral Salt & Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat, India

a r t i c l e i n f o

Article history:Received 14 January 2014Received in revised form9 March 2014Accepted 13 March 2014Available online 21 March 2014

Keywords:Macroinitiator effectPIB-containing pentablock copolymerIn vitro oxidative stability and cellularinteraction

* Corresponding author. Reverse Osmosis Disciplinrine Chemicals Research Institute, Bhavnagar 3640022782566511.

E-mail address: skjewrajka@csmcri.org (S.K. Jewra1 Contributed equally.

http://dx.doi.org/10.1016/j.polymer.2014.03.0190032-3861/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

We report well controlled synthesis of novel tri-component [polyisobutylene (PIB), poly(n-butyl acrylate)(PnBA) and poly(methyl methacrylate) (PMMA)] pentablock copolymers (PMMA-b-PnBA-b-PIB-b-PnBA-b-PMMA) by Atom Transfer Radical Polymerization (ATRP) using PIB as a macroinitiator. The surfaceproperties (hydrophobicity, in vitro oxidative stability and cellular interaction) and the bulk properties(phase separation and mechanical properties) of the PIB-containing pentablock copolymers werecompared with PMMA-b-PnBA-b-PDMS-b-PnBA-b-PMMA (where PDMS ¼ polydimethylsiloxane) andconventional PMMA-b-PnBA-b-PMMA copolymers synthesized by PDMS and PnBA macroinitiatorsrespectively. It is revealed that type of ATRP macroinitiator (with low glass transition temperature) in-fluences the properties of resultant pentablock copolymers in terms of phase separation, mechanicalproperties in vitro oxidative stability, cytocompatibility and cell proliferation. Pentablock copolymerssynthesized by PIB macroinitiator exhibited superior overall properties compared to pentablock co-polymers synthesized by PDMS macroinitiator and neat triblock copolymer synthesized by PnBA mac-roinitiator. Among the copolymers tested, one with composition PIB:PnBA:PMMA ¼ 10:64:26 (w/w)exhibited best mechanical property, oxidative stability and cytocompatibility. The newly designed PIB-containing pentablock copolymer may be useful where softness, flexibility, processability and bio-stability/cytocompatibility are desired.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Thermoplastic elastomers (TPEs) are used for biomedical ap-plications. For example, biostable/biocompatible copolymers arepreferable for application in long-term implantable devices. Tri-block copolymer of polystyrene (PSt) and PIB is a classic example ofbiostable/biocompatible TPE which was synthesized by carboca-tionic polymerization [1e4]. The PSt-b-PIB-b-PSt TPE was identifiedas a successful soft material for stent coating application due to itsenhanced biostability/biocompatibility [1,2]. Segmented TPEs suchas PDMS-based polyurethanes [5,6] and PIB-based polyurethanes[7,8] were also reported to be biostable and biocompatible. The

e, CSIReCentral Salt and Ma-, Gujarat, India. Tel./fax: þ91

jka).

enhanced biostability of PDMS-based and PIB-based TPEs wasattributed to the hydrophobicity and inertness of both PDMS andPIB backbones.

Triblock poly(meth)acrylates such as PMMA-b-PnBA-b-PMMA isa conventional TPE synthesized by the living anionic polymeriza-tion (LAP). Later on, the copolymer was successfully synthesized bythe ATRP [9,10]. However, copolymer synthesized by ATRP had lowstorage modulus, high complex viscosity, high order-disordertransition temperature and poor ultimate tensile strength andelongation at break, compared to the LAP analogue. These differ-ences were attributed to the slow initiation of MMA by PnBAmacroinitiator used in ATRP which yielded broad PDI [9].

From synthesis point of view, fully poly(meth)acrylate-basedTPEs are beneficial due to (i) wide range of properties owing totunable glass transition temperature (Tg) of different poly(meth)acrylates [ranging from 250 �C for poly(isooctyl acrylate) up to190 �C for poly(isobornyl methacrylate)] [9,11], (ii) easy synthesisvia Living Radical Polymerization (LRP) [9], (iii) tunable mechanical

R. Muppalla et al. / Polymer 55 (2014) 2369e23792370

properties by changing the structure from simple linear to radial[12], (iv) easy surface modification via LRP process [13,14] and (v)superior oxidative and UV-resistant properties compared topoly(diene)-based copolymers [9,15].

Despite the better oxidative stability of poly(meth)acrylatescompared to poly(diene), poly(meth)acrylates also undergooxidative degradation [16]. Indeed, accelerated oxidative degrada-tion test revealed that poly(meth)acrylates-based TPEs are alsooxidatively/hydrolytically vulnerable [13]. Such type of degradationof poly(meth)acrylates-based TPE might limit their in vivo appli-cation where biostability is highly desirable. Previous publicationsreported the use of fully poly(meth)acrylate-based triblock co-polymers in stent coating application. Long-term biostability/biocompatibility of such copolymers was not evaluated [17,18].Synthesis of PIB-b-PMMA block copolymers was reported bycombining living cationic polymerization and coupling reactions[19]. Block copolymers of PIB and poly(methyl methacrylate) or PStwere also synthesized by combining living cationic polymerizationand ATRP [20]. Typically, 70e85% (w/w) soft block is necessary toachieve reasonable thermoplastic properties. Of course, TPE ex-hibits better mechanical property when it contains middle softblock and ends hard blocks. Hence, synthesis of biostable/biocompatible and predominantly poly(meth)acrylate-containingelastomer is desirable for particular biomedical application.

To enhance the cytocompatibility and to address the problem ofpoor in vitro oxidative stability of conventional PMMA-b-PnBA-b-PMMA copolymer, we propose to use a macroinitiator capable ofmodifying the surface of the copolymer, without negativelyaffecting the mechanical properties. Thus the focus of this workwas the synthesis of predominantly poly(meth)acrylate-containingTPEs with improved properties as far as biomedical application isconsidered.

Herein, we report novel effect of ATRP macroinitiators (softmiddle blocks) such as PnBA, PDMS and PIB respectively on prop-erties (phase separation behaviour, mechanical property, in vitrooxidative stability and cellular interaction) of respective co-polymers. It is demonstrated that covalent attachment of moderateamount of PIB (w10 wt%) into poly(meth)acrylates-based copol-ymer greatly improved the properties for further application.

2. Experimental section

2.1. Materials

Allyl-terminated PIB (Allyl-PIB-Allyl) with NMR-derived mo-lecular weight (Mn,NMR ¼ 5300 g/mol) and polydispersity index(PDI) ¼ 1.1 was kindly provided by Kaneka Corporation, Japan andused as received. nBA (Aldrich, 98%) and MMA (Aldrich, 96%) werewashed with 5% aqueous NaOH solution, dried over anhydrousCaCl2, vacuum distilled and stored under nitrogen at �5 �C. Trie-thylamine (Spectrochem, India) was stored over NaOH pellets and4-�A molecular sieves. Dinonyl-2,20-dipyridyl (dNbpy) (97%), 2-bromoisobutyryl bromide (EBiB) (98%), 9-borabicyclo[3.3.1]non-ane (9-BBN, 0.5 M in THF) were obtained from Aldrich and used asreceived. Catalysts CuCl (BDH, 98%) and CuBr (Aldrich, 98%) werepurified by washing with 10% (w/v) HCl and HBr in water respec-tively followed by washing with methanol and diethyl ether in aSchlenk tube under a nitrogen atmosphere and stored in desicca-tors. p-Xylene, toluene (98%, SRL, India), CoCl2 (Spectrochem, In-dia), H2O2 (30% solution in water, Rankem, India) were used asreceived. Tetrahydrofuran (THF) (Rankem, India) was distilled oversodium and used. The triblock copolymer (triblock)PnBA/PMMA(PMMA-b-PnBA-b-PMMA) and PDMS-containing PMMA-b-PnBA-b-PDMS-b-PnBA-b-PMMA pentablock copolymer [(penta-block)PDMS/PnBA/PMMA] were synthesized as reported earlier [13].

2.2. Synthesis

Pentablock copolymers containing PIB as one of the soft blockswere synthesized using PIB-based difunctional macroinitiator (Br-PIB-Br). The Br-PIB-Br macroinitiator was prepared from Allyl-PIB-Allyl via two step reaction (Supplementary content).

2.2.1. Synthesis of Br-PnBA-b-PIB-b-PnBA-Br [(triblock)PIB/PnBA-2]macroinitiator containing 14 wt% PIB

Liquid triblock macroinitiator was synthesized by ATRP of nBAusing Br-PIB-Br as macroinitiator. A typical example for the syn-thesis of (triblock)PIB/PnBA-2 macroinitiator is as follows: Br-PIB-Br(2 g, 3.77 � 10�4 mol), was taken in round bottom flask providedwith a B-24 standard joint and a stir bar. Next, p-xylene (1.2 mL),nBA (13.4 g, 0.104 mol) previously purged with nitrogen wereintroduced into the flask under nitrogen atmosphere. The flask wasthen closed with a rubber septum which was secured by a copperwire and stirred for 20 min. The septum was then reopened undernitrogen atmosphere, CuBr (0.12 g, 8.3 � 10�4 mol) and dNbpy(0.68 g, 0.0016 mol) were added. The flask was again closed byseptum. After formation of homogenous maroon colour solution,the flask was kept in an oil bath at 90 �C and stirred for 5 h. Themixture was diluted by toluene and the solution was passedthrough silica/alumina column to remove copper catalyst. Thetoluene solution was concentrated by rotary evaporator andprecipitated in methanol. Next, the isolated polymer was againdried in a vacuum oven for 48 h at 60 �C, and weighed. The con-version was 79%. The molecular weights (Mn,GPC and Mn,NMR)calculated from Gel Permeation Chromatography (GPC) and NMR,were 27,700, 38,000 g/mol respectively. PDI of the resultantcopolymer was 1.27. Kinetics analysis was performed by carryingout four sets of polymerization using exactly the same recipe andthe polymerizations were terminated at different time.

2.2.2. Synthesis of pentablock PMMA-b-PnBA-b-PIB-b-PnBA-b-PMMA [(pentablock)PIB/PnBA/PMMA-2] copolymer containing 10 wt%PIB

A typical procedure for the synthesis of (pentablock)PIB/PnBA/PMMA-2 copolymer is as follows. Previously synthesized (triblock)PIB/PnBA-2macroinitiator (3 g, 7.87 � 10�5 mol) was taken in a round bottomflask provided with stir bar. Next, p-xylene (1.5 mL) and MMA (2.5 g,0.025 mol) previously purged with nitrogen were injected into theflask using a gastight syringe. The fluxwas then closedwith a rubberseptum,whichwas securedwith Cuwire. Themixturewas stirred for5 h for complete dissolution of the macroinitiator. The septum wasreopened under nitrogen atmosphere, CuCl (0.017 g,1.73�10�4mol)and dNbpy (0.14 g, 3.46�10�4mol)were then quickly added into theflask under nitrogen atmosphere. The flask was filled with nitrogenand was again sealed by septum. After formation of homogenousmaroon colour solution, the flask was kept in an oil bath at 90 �C andstirred for 55min. The polymer was then isolated, purified and driedas described for triblock copolymer. The conversion was 45%, andMn,GPC, Mn,NMR and PDI of the copolymer were 38,000, 51,400 g/moland1.35 respectively. Kinetics analysiswasperformedbycarryingoutfour sets of polymerization using exactly the same recipe and thepolymerizations were terminated at different time.

2.3. Characterization

2.3.1. Mn and PDIThe Mns of the copolymers were determined by both GPC and

NMR analyses. The GPC was performed using a Waters model 2695separation module coupled with Waters 2414 refractive index de-tector and Waters Ultra Styragel columns of 10.000, 1000, 500 �Apore size which were preceded by a prefilter. HPLC grade THF

R. Muppalla et al. / Polymer 55 (2014) 2369e2379 2371

(Spectrochem, India) was used as an eluent at a flow rate of 1 mL/min. The copolymer solutions were purified by passing throughbasic alumina column prior to inject into the GPC system to freethem from Cu salts. The copolymer solutions were then filteredthrough a prefilterefilter combination system compatible withorganic solvents. For calibration, polystyrene standards were used.

Bruker 200 MHz spectrometer was used to record 1H NMRspectra. NMR was recorded at 25 �C. CDCl3 was used as a solventand TMS as the internal reference.

The Mn of Br-PIB-Br was also obtained by NMR analysis,considering the bifunctionality of PIB. The Mn of Br-PIB-Br wasestimated to be 5300 g/mol from NMR by the following equation:

Mn;Br�PIB�Br ¼ hI1:1

6

ihI3:84

i�MIB

!þ ð2� 251:99Þ (1)

where MIB is the molecular weight of IB. I1.1 and I3.8 are the in-tensities of eC(CH3)2e present in the PIB backbone and eOeCH2e

present in the terminal ends of PIB chain respectively. The value252 is the molecular weight of bromo alkyl group.

The NMR derived Mns of both PMMA and PnBA blocks wereestimated by the following equations:

Mn;PMMA ¼ 2� I3:6 � RPIBI1:1

�MMMA (2)

Mn;PnBA ¼ 3� I4:15 � RPIBI1:1

�MnBA (3)

where I3.6, and I4.15 are the intensities for NMR signals of eOCH3and eOCH2 present in PMMA and PnBA blocks respectively. RPIB is

Fig. 1. NMR spectra of (a) Allyl-PIB-Allyl, (b) HO-PIB-OH, (c) Br-PIB

the repeat unit of PIB calculated from NMR.MMMA andMnBA are themolecular weights of respective monomers. Fig. 1 shows the NMRspectra of Allyl-PIB-Allyl, OH-PIB-OH, Br-PIB-Br, (triblock)PIB/PnBA-2macroinitiator, and representative (pentablock)PIB/PnBA/PMMA-2copolymer. Thus the Mns of the copolymers were estimated by 1HNMR (Fig. 1 and Fig. S1, Supplementary content). The monomerconversion during ATRP was also determined by NMR (Fig. S2,Supplementary content).

Others material characterizations including tensile properties,Dynamic Mechanical Analysis (DMA), Differential Scanning Calo-rimetric (DSC), water q measurements and SEM analyses wereperformed as reported earlier for PDMS-containing copolymers(Supplementary content) [13].

2.3.2. Accelerated oxidative degradation testAccelerated hydrolytic/oxidative degradation of samples under

both normal and pre-stretched conditions was investigated byexposure to aqueous CoCl2 solution (200 mL, 0.1 M) containingH2O2 (25%, w/v) for different time periods at 60 �C and concen-tration of radicals present in the solution was maintained relativelyconstant by changing the solution twice a week (Supplementarycontent for detailed experiment).

2.3.3. Cell culture study and evaluation of cell morphologyThe attachment of cells on the surface of soft pentablock co-

polymers, a fully silicon rubber (control) and tissue culture platewas tested under similar in vitro conditions. For the analysis of theattachment of cells on the surface, flat rectangular copolymer filmsof thickness 0.3e0.4 mm were used. The films were initially ster-ilized with 70% v/v ethyl alcohol for 1 h followed by washing threetimes in sterile phosphate buffer (PBS, pH ¼ 7.4) to remove alcohol.After that the copolymer films were UV sterilized for 8e10 h. Then

-Br, (d) (triblock)PIB/PnBA-2 and (e) (pentablock)PIB/PnBA/PMMA-2.

R. Muppalla et al. / Polymer 55 (2014) 2369e23792372

the films were cut in equal sizes and were placed in 24-well tissueculture plates (Corning, Germany). The films were initially equili-brated in the presence of 2 mL of culture media (DMEM-Highglucose) for 2 h followed by seeding of 5�106 mouse bonemarrowstem cells (BMMSCs) per well. The plates were then shaken gentlyfor 10 min to ensure that the cells were distributed uniformly, andthen placed inside a humidified incubator at 37 �C in a 5% CO2 at-mosphere. After a 4 h of attachment period, the cell loaded filmswere washed with culture medium to remove unattached orloosely attached cells and 1 mL of culture medium was added to itand incubated further for 4 h. Total cell attachment was analysed bynuclear staining of cells with fluorescent dye 40,6-diamidino-2-phenylindole (DAPI) and by observing through the fluorescencemicroscope (Axiovert 25, Zeiss, Germany).

Further, the morphology of the tightly attached cells on thecopolymer surface was determined. For morphological study, thecell seeded copolymer films were cultured for 1 day. After incu-bation, cells were washed and fixed in 4% glutaraldehyde. Furthercells were dehydrated in increasing gradient of ethanol (10e100%). After dehydration, films were preceded for SEM analysis asper earlier protocol [21]. Briefly, the surface of films was exam-ined after coating the specimens with gold particles in a sputtercoater for 3 min under vacuum and observing them at 1000�magnification under a scanning electron microscope (SEM, LEO435 VF, UK).

2.3.4. Cell viabilityThe cell viability on the copolymer films, silicon rubber

(control) and tissue culture plate was measured using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)assay (HiMedia, India). For this the samples were seeded withBMMSCs and cultured for various time periods. After completionof incubation, cells were washed twice with PBS and incubatedwith fresh culture medium containing MTT (0.5 mg/mL) at 37 �Cfor 4 h in dark. Then the unreacted dye was removed, and DMSOwas added to dissolve the intracellular insoluble purple formazanproduct into a coloured solution. The absorbance of this solutionwas quantified by photo spectrometry at 570 nm with a platereader (FLUO star Optima 413-1210, BMG LABTECH, Germany).The assay was performed after 2, 5 and 10 days of seeding. Cellsseeded on tissue culture plate were used as a positive control forthis study.

Cell viability was also determined by Trypan blue stainingbased live and dead cell counting. Viability assays measure thepercentage of a cell suspension that is viable. After 24 h of culturecells were trypsinized from each surface and cell suspension wasprepared (106 cells/mL). A 1:1 (v/v) dilution of the suspensionusing a 0.4% (w/v) Trypan blue solution was then prepared. Un-stained cells were counted using haemocytometer. The percentageof unstained cells to the total cells represents the percentage ofviable cells.

Table 1Compositions, Mns, and PDIs of various copolymers synthesized by ATRP.

Copolymer abbreviation Copolymer

(Triblock)PIB/PnBA-1 PnBA7.6-b-PIB5.3-b-PnBA7.6

(Triblock)PIB/PnBA-2 PnBA16.4-b-PIB5.3-b-PnBA16.4

(Triblock)PIB/PnBA-3 PnBA24.3-b-PIB5.3-b-PnBA24.3

(Pentablock)PIB/PnBA/PMMA-1 PMMA2.9-b-PnBA7.6-b-PIB5.3-b-PnBA7.6-b-PMMA2.9

(Pentablock)PIB/PnBA/PMMA-2 PMMA6.7-b-PnBA16.4-b-PIB5.3-b-PnBA16.4-b-PMMA6.7

(Pentablock)PIB/PnBA/PMMA-3 PMMA8.5-b-PnBA24.3-b-PIB5.3-b-PnBA24.3-b-PMMA8.5

(Triblock)PnBA/PMMA PMMA10.1-b-PnBA37-b-PMMA10.1

a wt% determined by NMR; NMR derivedMns are in the bracket; (triblock)PIB/PnBA-1, 2,-3 copolymers respectively.

2.3.5. Cell proliferation, acridine orange/ethidium bromide stainingand reactive oxygen species (ROS) test

Cell proliferation of mouse BMMSCs on various samples wasassessed by crystal violet assay. After seeding for 2, 5 and 10 days,cellefilm constructs were washed in PBS, fixed in 4% formaldehydefor 15 min. Cellefilm constructs were washed again and stainedwith 0.5% crystal violet for 30 min. After that, cells were washedthrice with PBS. Stain taken by the cells was then extracted in 100%methanol for 15 min. OD of the methanol extract was taken at540 nm. Acridine orange/ethidium bromide staining and ROSdetermination were also carried out to evaluate the cytocompati-bility of the copolymers (Supplementary content).

3. Results and discussion

3.1. Synthesis of (pentablock)PIB/PnBA/PMMA copolymers

PIB was selected as a macroinitiator for synthesizing (penta-block)PIB/PnBA/PMMA copolymers due to (i) its low surface energyalong with its excellent biostability and biocompatibility [1,2,7,8],(ii) PIB backbone is chemically inert compared to poly(meth)acry-lates [2,8] and (iii) unlike PDMS, PIB exhibited higher degree ofphase separation with PnBA part (vide infra). Table 1 summarizesthe compositions, abbreviations, Mns and PDIs of synthesized co-polymers. Scheme 1 shows the synthetic strategy for (penta-block)PIB/PnBA/PMMA copolymer starting from Allyl-PIB-Allyl.

First, (triblock)PIB/PnBA macroinitiators (Table 1, entries 1e3)were synthesized by ATRP of nBA using Br-PIB-Br (Mn ¼ 5300,PDI ¼ 1.1) as a macroinitiator and CuBr/dNbpy as a catalyst. Next,ATRP of MMA using (triblock)PIB/PnBA as macroinitiators and CuCl/dNbpy catalyst yielded (pentablock)PIB/PnBA/PMMA copolymers(Table 1, entries 4e6) with well-defined Mns and narrow PDIs. Thereason for the use of mixed halogen system was discussed earlier[22]. Optimized macroinitiator to Cu(I) and dNbpy ratio to achievefast ATRP, yielding copolymers with narrow PDI was found to be1:2.2:4.4. Fig. 2A and B shows the kinetics of disappearance of nBAand MMA respectively when Br-PIB-Br and (triblock)PIB/PnBA-2were used as macroinitiators. The apparent propagation rateconstants ðk0psÞ for the ATRP of nBA and MMA were found to bew8 and w1.5 times higher than the k0ps of corresponding mono-mers when the macroinitiator to Cu(I) and dNbpy ratio wasmaintained to 1:2.2:4.4 instead of 1:1.6:3.2 during ATRP whichyielded linear Mn vs. conversion plots (Fig. 2C and D). The PDIvalues of the copolymers were also somewhat lower when themacroinitiator to Cu(I) and dNbpy ratio was 1:2.2:4.4. The wellcontrolled polymerization with enhanced propagation rate athigher catalyst concentration is attributed to the fast activationand deactivation reaction by Cu(I) and in situ generated Cu(II)complexes respectively. Much greater effect of Cu(I) concentrationon k0p for the ATRP of nBA compared to that of ATRP of MMA maybe attributed to the (i) faster activation of Br-terminated polymerchains (all the dormant species) by higher concentration of CuBr/

aPIB (wt%) aPnBA (wt%) aPMMA (wt%) Mn GPC (NMR) PDI

26 74 e 18,000 (20,500) 1.2714 86 e 27,700 (38,000) 1.2710 90 e 41,500 (53,900) 1.2520 58 22 24,000 (26,000) 1.3110 64 26 38,600 (51,400) 1.357 70 23 57,500 (71,000) 1.320 64 36 57,000 (�) 1.30

and 3 are the macroinitiators for the synthesis of (pentablock)PIB/PnBA/PMMA-1, -2, and

Scheme 1. Strategy for synthesis of (pentablock)PIB/PnBA/PMMA.

R. Muppalla et al. / Polymer 55 (2014) 2369e2379 2373

dNbpy for the former case compared to that of Cl-terminatedpolymer chains by higher concentration of CuCl/dNbpy catalystfor the later case and (ii) higher rate of deactivation of propagatingspecies with the in situ generated CuCl2/dNbpy during ATRP ofMMA compared to in situ generated CuBr2/dNbpy during ATRP ofnBA with the higher concentration of respective Cu(I) catalysts.Such combined effect should not much influencing the k0p for the

Fig. 2. Kinetic plots (A and B) and evaluation of Mn with conversion of monomers (C and Dmonomer conversions and Mn values were determined by 1H NMR. Plots: (1) [initiator]:[Cu

ATRP of MMA even when the concentration of CuCl/dNbpy washigher.

Fig. 3 shows the GPC traces of Br-PIB-Br, corresponding (tri-block)PIB/PnBA macroinitiator, and (pentablock)PIB/PnBA/PMMA withtwo different compositions (Table 1, entries 4 and 5). Gradual in-crease ofMns from Br-PIB-Br to (triblock)PIB/PnBA macroinitiator andto (pentablock)PIB/PnBA/PMMA indicates efficient initiation of

) for the synthesis of (triblock)PIB/PnBA and (pentablock)PIB/PnBA/PMMA copolymers. The(I)]:[dNbpy] ¼ 1:1.6:3.2 and (2) [initiator]:[Cu(I)]:[dNbpy] ¼ 1:2.2:4.4.

Fig. 3. (A) GPC traces of (1) Br-PIB-Br (Mn ¼ 5500 g/mol, PDI ¼ 1.1), (2) (triblock)PIB/PnBA-1(Mn ¼ 18,000 g/mol, PDI ¼ 1.27) and (3) (pentablock)PIB/PnBA/PMMA-1(Mn ¼ 24,000 g/mol, PDI ¼ 1.31). (B) GPC traces of (1) Br-PIB-Br (Mn ¼ 5500 g/mol,PDI ¼ 1.1), (20) (triblock)PIB/PnBA-2 (Mn ¼ 27,700 g/mol, PDI ¼ 1.27) and (30) (penta-block)PIB/PnBA/PMMA-2 (Mn ¼ 38,600 g/mol, PDI ¼ 1.35). The starting macroinitiator (Br-PIB-Br) was the same for synthesis of both (pentablock)PIB/PnBA/PMMA-1 (trace 3, Fig. A)and (pentablock)PIB/PnBA/PMMA-2 (trace 30 , Fig. B).

R. Muppalla et al. / Polymer 55 (2014) 2369e23792374

respective macroinitiators and formation of well-defined penta-block copolymers. On the other hand, NMR derived Mns were closeto the theoretical determined values but were somewhat higherthan the GPC derived values due to fact that the GPC calculationswere based on narrow polystyrene standard for calibration.

Fig. 4. DSC curves (extended scale from �100 �C to 20 �C) of (a) PnBA homopolymer,(b) PIB þ PnBA mechanical blend, (c) PIB macroinitiator, (d) (triblock)PIB/PnBA-2, (e)(triblock)PIB/PnBA-1, (f) (pentablock)PIB/PnBA/PMMA-1, and (g) (pentablock)PIB/PnBA/PMMA-2.

3.2. Phase separation and mechanical properties of copolymerssynthesized by PIB, PDMS and PnBA soft macroinitiators

Phase separation behaviour of (pentablock)PIB/PnBA/PMMA wasstudied by DSC (Fig. 4) and DMA (Fig. 5) analyses. As expected,PnBA (Mn ¼ 32,000 g/mol) and PIB (Mn ¼ 5300 g/mol) homopol-ymers exhibited Tgs at �57 �C and �76 �C respectively (Fig. 4,curves a and c). The mechanical blend of PnBA and PIB (70:30 wt/wt) exhibited two Tgs (curve b), similar to the Tgs of PnBA and PIBhomopolymers. The (triblock)PIB/PnBA-2 and (triblock)PIB/PnBA-1 alsoshow Tg at �57 �C (Fig. 4, curves d and e). Appearance of Tg at�57 �C for PnBA homopolymer, mechanical blend and (triblock)PIB/PnBA indicates phase separation between PnBA and PIB. However,the Tgs of PnBA part of pentablock copolymers were somewhatshifted towards higher temperature side compared to (triblock)PIB/PnBA. For example, PnBA part of (pentablock)PIB/PnBA/PMMA-1 and(pentablock)PIB/PnBA/PMMA-2 shows Tgs at �52 �C and �47 �Crespectively (Fig. 4, curves f and g). Hence, the PnBA phase wassomewhat influenced by the hard PMMA matrix in the pentablockcopolymer. This may be attributed to the partial solubilization ofPMMAwithin the PnBA part because of higher degree of miscibilitybetween PnBA and low molecular weight PMMA attached to bothends of PnBA. The Tg of PIB (Mn ¼ 5300 g/mol) centre block was notvisible in the DSC curves of the copolymers. This may be ascribed tothe much lower molecular weight of the PIB block and relativelylower amount of PIB present in the copolymers compared to otherblocks. Mechanical blend of PnBA (Mn ¼ 37,000 g/mol) and PIB(Mn ¼ 5300 g/mol) homopolymers (70:30 wt/wt) showed fainttransition (Tg) for PIB and relatively strong transition for PnBAwhereas the blend showed Tg for PnBA and the transition due to PIBwas not prominent when PnBA to PIB ratio was 75:25 in the blend.Duong et al. reported that the Tg of hydrophobic soft block (PDMSwith lower Mn) in a copolymer was not visible in DSC curve whenthe wt% of PDMS was lower than 39% [23].

Thermoplastic nature of (pentablock)PIB/PnBA/PMMA copolymersis evident from storage moduli vs. temperature plots of

representative (pentablock)PIB/PnBA/PMMA-2 and (pentablock)PIB/PnBA/PMMA-3 (Fig. 5A). The copolymers exhibited rubbery regionabove the Tg of PnBA. Fig. 5A also indicates that the storagemodulusvalues of (pentablock)PIB/PnBA/PMMA-2 is higher than (pentablock)PIB/PnBA/PMMA-3 at wide range of temperature due to greater softnessassociated with the later since the former copolymer containsrelatively higher amount of PMMA. Although, the storage modulivs. temperature plots for two different pentablock copolymersshow single Tg at lower temperature side, the loss modulus vs.temperature plots (Fig. 5B) indicate a strong transition at �43 �Cand a weak transition (hump) at �62 �C. This fact suggestsappearance of Tg at �43 �C for PnBA part which is the majorcomponent and probable appearance of another Tg at�62 �C for PIBpart which is the minor component of the copolymers. The tandelta vs. temperature plots also indicates appearance of such typeof transitions (Fig. S3, Supplementary content).

Thus the phase separation behaviour of (pentablock)PIB/PnBA/PMMA and (triblock)PIB/PnBA (macroinitiator) is substantiallydifferent compared to previously reported (pentablock)PDMS/PnBA/

PMMA and (triblock)PDMS/PnBA respectively. The later two copolymersshowed single Tg for PnBA/PDMSmatrix [13,24]. The (triblock)PDMS/

PnBA showed single Tg at temperature �69 �C whereas (triblock)PIB/PnBA copolymer showed Tg at �57 �C (Table S1, Supplementarycontent). Such type of phase separation of low surface energy andbiostable/biocompatible PIB from rest of the relatively higher sur-face energy components (PnBA and PMMA) is beneficial as far asoxidative stability and biocompatibility are considered (see later).However, some degree of phase mixing between PIB and PnBAcannot be overruled due to covalent connections.

Fig. 5C shows the stressestrain curves of (pentablock)PIB/PnBA/PMMA-1, (pentablock)PIB/PnBA/PMMA-2 and (pentablock)PIB/PnBA/PMMA-3. The (pentablock)PIB/PnBA/PMMA-1 (curve 3) exhibits a high value ofelastic modulus and the strain hardening is not prominent afterw170% strain which is ascribed to the initial deformation of hardPMMA outer block followed by slippage of short rubbery chains(Mn ¼ 26,000 g/mol) of the copolymer. In contrast, (pentablock)PIB/PnBA/PMMA-2 (curve 1) and (pentablock)PIB/PnBA/PMMA-3 (curve 2)show almost linear relationship between stress and strain and theformer copolymer exhibited higher stress and modulus comparedto later copolymer. This may be attributed to the presence of lower

Fig. 5. (A) Storage modulusetemperature plots, (B) Loss modulusetemperature plots and (C) tensile stressestrain plots. Curves: (1) (pentablock)PIB/PnBA/PMMA-2, (2) (pentablock)PIB/PnBA/PMMA-3 and (3) (pentablock)PIB/PnBA/PMMA-1.

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amount of total soft PIB/PnBA matrix and higher amount of PMMAin (pentablock)PIB/PnBA/PMMA-2. Tensile stress ofw4MPa, along withstrain >350% may be suitable for in vivo applications which in-cludes soft tissue replacement, and coating of medical grade de-vices [25].

Hence, (pentablock)PIB/PnBA/PMMA-2 synthesized for this workexhibited superior tensile property (3.5 MPa stress and 400%elongation at break) compared to (pentablock)PDMS/PnBA/PMMA(2.2 MPa stress and 260% elongation at break) and (triblock)PnBA/PMMA (2.8 MPa stress and 180% elongation at break) copolymerswith comparable PDI (1.35) and hard PMMA (29 wt%) content [13](Table S1, Supplementary content). This effect may be attributed tothe low average molecular weight entanglement of PIB (w8900 g/mol) compared to PDMS (w12,000 g/mol) and PnBA (w28,000 g/mol) [10]. Thus the entanglement molecular weight of (triblock)PIB/PnBA part should be lower than PnBA and (triblock)PDMS/PnBA.

3.3. Surface characterization and in vitro oxidative stability ofcopolymers synthesized by PIB, PDMS and PnBA soft macroinitiators

Surface hydrophobicity of different (pentablock)PIB/PnBA/PMMAand a representative (triblock)PnBA/PMMA was assessed by water q

measurements. The q value increases with increasing PIB content inthe copolymer (Fig. S4, Supplementary content). The (penta-block)PIB/PnBA/PMMA copolymers (containing 7.5e20 wt% PIB)exhibited average q of 93�e100� whereas the (triblock)PnBA/PMMAexhibited average q of 89�. The higher contact angle of copolymerswith increasing amount of PIB may be attributed to the hydro-phobization of copolymer surface by PIB due to lower surface

energy of PIB compared to other components. The reported surfaceenergies of PIB, PMMA, and PnBA are w30 mN/m, w43 mN/m, andw39 mN/m respectively [26,27]. Preferential surface coverage byPIB in PIB-based polyurethanes/polyureas was reported earlier byone of us [7,8].

Representative (triblock)PnBA/PMMA, (pentablock)PIB/PnBA/PMMA-1, and (pentablock)PIB/PnBA/PMMA-2 were submerged inaqueous CoCl2/H2O2 for 30 and 40 days at 60 �C and the con-sequences of this treatment were analysed by SEM and GPCanalyses. The strong oxidizing action of CoCl2/H2O2 solution andits use for evaluation of in vitro oxidative stability was reportedin the literature [28,29]. Fig. 6 (left) shows the SEM images ofsurfaces of copolymers after subjected to oxidative degradationtest under un-stretched state. As expected, the surface of (tri-block)PnBA/PMMA (row A, 30 and 45 days) shows severe cracking[13]. On the other hands, surfaces of (pentablock)PIB/PnBA/PMMA-2(row B) and (pentablock)PIB/PnBA/PMMA-1 (row C) after 45 days ofdegradation show no evidence of cracking. However, some sur-face unevenness is seen for both the copolymers after subjectedto degradation. Magnified (12.84 K�) SEM image of (penta-block)PIB/PnBA/PMMA-1 also showed no sign of cracking (Fig. S5,Supplementary content). In contrast, although, the PDMS con-taining pentablock copolymer exhibited much superior oxida-tive stability compared to (triblock)PnBA/PMMA but showed somesurface erosion [13].

Fig. 6D shows GPC traces of (pentablock)PIB/PnBA/PMMA-2 beforeand after subjected to degradation for up to 30 days. After exposureto oxidative solution, the copolymer yielded superimposed GPCtraces (traces bef) with the GPC trace (trace a) of pristine

R. Muppalla et al. / Polymer 55 (2014) 2369e23792376

(pentablock)PIB/PnBA/PMMA-2. However, some broadening in the GPCtraces (traces def) was observed towards higher elution volumeside.

In biological system, Environmental Stress Cracking (ESC) wasidentified as one of the phenomenon responsible for degradation ofan implant. The ESC phenomenon was earlier discussed elsewhere[28,29]. Therefore, pre-stretched (pentablock)PIB/PnBA/PMMA-2 and(pentablock)PDMS/PnBA/PMMA were submerged in aqueous CoCl2/H2O2 for 20 days at 60 �C. SEM images (Fig. 7) of two different sitesof each representative (pentablock)PDMS/PnBA/PMMA (images a, a0)and (pentablock)PIB/PnBA/PMMA-2 (images b, b0) indicate that thelater copolymer surfaces show some erosion. SEM images of(pentablock)PIB/PnBA/PMMA-2 after degradation test show that w30%area had little surface erosion and hole formation (image b) but nosurface cracking was observed. The rest of the surface (image b0)remained almost smooth after the degradation test under pre-stretched state. A magnified (22 K�) SEM image of (penta-block)PIB/PnBA/PMMA-2 film taken after the degradation test did notshow any crack formation (Fig. S6, Supplementary content). How-ever, (pentablock)PDMS/PnBA/PMMA surface shows erosion and crackformation (images a and a0) after subjected to degradation in pre-stretched state. The better oxidative/hydrolytic stability of PIBcontaining copolymer is due to coverage of surface by oxidativelystable PIB. Since, PIB contains alternating secondary and quaternarycarbons which make the PIB backbone oxidatively stable [2]. Asdiscussed above, poly(meth)acrylates are oxidatively/hydrolyticallyvulnerable. A probable mechanism of degradation is the abstractionof hydrogen from carbon atom of ester side group (a-methylene ormethyl) by hydroxyl radicals generated from CoCl2/H2O2. Combi-nation of the side chain radical with a hydroxyl radical followed byacid hydrolysis causes degradation of poly(meth)acrylates. Suchtype of degradation mechanism for poly(meth)acrylates and poly-carbonates was reported in the literature [2,30]. Particularly forPnBA, there is also possibility of abstraction of hydrogen from ter-tiary carbon of polymer backbone by the hydroxyl radical followedby addition of hydroxyl radical or oxygen which may cause

Fig. 6. SEM images of (triblock)PnBA/PMMA (0 wt% PIB, row A), (pentablock)PIB/PnBA/PMMA-2 (1(control) and after (30 and 45 days) subjected to accelerated in vitro oxidative treatment b(pentablock)PIB/PnBA/PMMA-2 copolymer before and after accelerated in vitro oxidative treatmsamples for 5, 10, 15, 20 and 30 days respectively.

backbone degradation as was reported for polyethylene underoxidative environment [31].

3.4. Cellular interaction with the surfaces of copolymerssynthesized by PIB, PDMS and PnBA macroinitiators

Several clinical studies have showed that mesenchymal stemcells (MSCs) play a significant role in tissue repair and homoeo-stasis. In addition to being a progenitor cell population with self-renewing and multipotent differentiation capabilities, MSCs haveunique immune modulatory properties, making them even moreattractive for regenerative medicine [32]. Hence in this study wehave used bone marrow derived MSCs. Silicon rubber which wasreported to be biocompatible was used to compare the cellularinteraction properties [33]. The (pentablock)PIB/PnBA/PMMA-2 wasselected for this study due to its superior mechanical property andsuperior/comparable oxidative stability compared to other PIB-containing pentablock copolymers.

Fig. 8A shows optical images of BMMSCs cultured on varioussample surfaces for 24 h. Adherence of cells was found to bemaximum (w30e35% surface coverage) on (pentablock)PIB/PnBA/PMMA-2 and (pentablock)PDMS/PnBA/PMMA whereas surfaces of tissueculture plate, silicon rubber (control) and (triblock)PnBA/PMMAshowed somewhat lower cells adhesion (w20e25% of surfacecoverage). The cell attachment study thus indicated superiorcytocompatibility of both PDMS and PIB containing pentablockcopolymers compared to (triblock)PnBA/PMMA.

The live/dead cell assay with acridine orange/ethidium bromidestaining of cell was also performed after culture for up to 5 days.The fluorescent images of the cells cultured on different copolymersamples indicated greater numbers of total viable cells on surface of(pentablock)PDMS/PnBA/PMMA and (pentablock)PIB/PnBA/PMMA-2compared to (triblock)PnBA/PMMA surface (Fig. S7, Supplementarycontent).

Cell morphology on the copolymers surfaces was investigatedby SEM after 24 h of culture (Fig. 8B). As depicted in Fig. 8B, the

0 wt% PIB, row B) and (pentablock)PIB/PnBA/PMMA-1 (20 wt% PIB, row C) recorded beforey CoCl2/H2O2 at 60 �C. Scale bar 2 mm and magnifications 2e3 K�. (D) GPC traces ofent by CoCl2/H2O2 at 60 �C. Trace a before treatment and traces bef are for the treated

Fig. 7. SEM images recorded at two different sites of each (pentablock)PIB/PnBA/PMMA-2 and (pentablock)PDMS/PnBA/PMMA surfaces after subjected to accelerated in vitro oxidativetreatment by submerging in CoCl2/H2O2 under pre-stretched (70% elongation) condition. Images a and a0 for (pentablock)PDMS/PnBA/PMMA and images b and b0 for (pentablock)PIB/PnBA/PMMA-2 copolymers.

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tissue culture plate, (pentablock)PDMS/PnBA/PMMA and (penta-block)PIB/PnBA/PMMA-2 surfaces show healthy morphology withhigher degree of cell spreading compared to silicon rubber and(triblock)PnBA/PMMA.

Cell proliferation of mouse BMMSCs on various copolymer filmsurfaces was also carried out and compared with silicon rubber(control) and tissue culture plate. Fig. 9, bar diagram A shows thequantitative of total cellular proliferation for all the samples. The(pentablock)PIB/PnBA/PMMA-2 showed the highest cell proliferationafter 2 days of culture, which further increased significantly after day5 suggestive of appreciable cytocompatibility of the copolymers.

Further, MTT assay was performed to study the cytocompati-bility of our newly designed soft copolymers. Fig. 9, bar diagram Bshows that after 2 days of incubation, cell viability over all kind ofsamples is almost equal. But after 5 days of incubation, cellularviability decreases for all copolymers as well as silicon rubber, andwith minimum reduction in (pentablock)PIB/PnBA/PMMA-2 (cellviability 80% � 4.2). After day 10, cellular viability was furtherdecreased for all the samples but (pentablock)PIB/PnBA/PMMA-2showed non-significant decrease in viability when compared withviability of cells after day 5 suggesting superior cytocompatibility ofthe selected copolymer compared to other copolymers. As weknow, MTT assay depicts the cellular physiology of cells, it isprobable that after reaching confluency, cells were stressed andhence showed less response to MTT for all the samples. Repeatedexperiment also showed that the cells seeded on tissue cultureplate also showed somewhat reduction (w10%) of viability in MTTassay after 10 days of culture. The above explanation was alsosupported by Trypan blue staining based live and dead cellcounting assay (Fig. 9, bar diagram C) carried out for 24 h. The(pentablock)PIB/PnBA/PMMA-2 still showed somewhat better cellviability compared to other copolymers.

The better cytocompatibility of (pentablock)PIB/PnBA/PMMA-2 wasalso evident from ROS generation as determined by DCFH-DA assay

(Fig. S8, Supplementary content). When cells are in oxidative stresscondition, they generate ROS. Prolonged generation of ROS causescell toxicity. Cells treated with H2O2 are positive control andshowed maximum amount of ROS generation after 24 h. As shownin Fig. S8 in supplementary content (images def), all the threecopolymers causes substantially less ROS generation as confirmedby significantly lower levels of fluorescent intensities in those cellscompared to peroxide treated cells (histogram, Fig. S8,Supplementary content).

Thus, MTT assay, cell-attachment, cell morphology and cellproliferation of mouse BMMSCs on various copolymers samplesand silicon rubber (control), indicated that the (pentablock)PIB/PnBA/PMMA-2 showed better results. Superior oxidative stability andcytocompatibility of (pentablock)PIB/PnBA/PMMA-2 compared to sili-con rubber and (triblock)PnBA/PMMA is once again attributed to thesuperior biocompatibility and inertness of soft PIB which effectivelycovers the surface of the copolymer. Strictly, comparison between(pentablock)PIB/PnBA/PMMA-2 and (pentablock)PDMS/PnBA/PMMA showsthat the former copolymer exhibited higher oxidative stability andcomparable or somewhat better cytocompatibility than the latercopolymer.

4. Conclusions

It may be concluded that PIB-based ATRP macroinitiator facili-tates controlled and facile synthesis of surface modified predomi-nantly poly(meth)acrylates-based TPEs with improved propertiesas far as biomedical application is considered. The surface and bulkproperties of copolymers were greatly influenced by the startingATRP macroinitiator used for the synthesis. For example, (penta-block)PIB/PnBA/PMMA-2 synthesized by PIB-macroinitiator exhibitedsuperior mechanical property, in vitro oxidative stability, cellularproliferation and cytocompatibility compared to (triblock)PnBA/PMMA and (pentablock)PDMS/PnBA/PMMA synthesized by PnBA and

Fig. 8. (A) Optical images of live cells and (B) cell morphology observed under SEM after being cultured for 24 h. Sample surfaces: (a) tissue culture plate, (b) silicon rubber, (c)(triblock)PnBA/PMMA, (d) (pentablock)PDMS/PnBA/PMMA and (e) (pentablock)PIB/PnBA/PMMA-2.

Fig. 9. (A) Quantitative of total cell proliferation on various sample surfaces, (B) viabilities of BMMSCs treated with different samples and (C) Trypan blue staining based live anddead cell counting showing the viable cells present among total adhered cells on the respective surfaces after 24 h of culture. Samples: (a) tissue culture plate, (b) silicon rubber, (c)(triblock)PnBA/PMMA, (d) (pentablock)PDMS/PnBA/PMMA and (e) (pentablock)PIB/PnBA/PMMA-2.

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PDMS macroinitiators (soft block) respectively. The PnBA-b-PDMS-b-PnBA soft blocks formed single phase due to enhancedmiscibilitybetween PnBA and PDMS whereas PIB and PnBA exhibited phaseseparation. The reason of better overall properties (mechanical,oxidative stability and cell viability) of (pentablock)PIB/PnBA/PMMA-2(containing 10 wt% of PIB) is attributed to the comparatively lowerentanglement molecular weight, phase separation, inertness andcytocompatibility of PIB. Hence, the biocompatibility/biostability ofpoly(meth)acrylates-based TPEs surfaces can be tuned by varyingthe chemical structure of the ATRP initiator (from low molecularweight conventional initiator to PDMS-based macroinitiator to PIB-based macroinitiator). Future work will aim to synthesize pre-dominantly poly(meth)acrylate-based copolymers with complexarchitecture for further improvement of mechanical property bytaking advantage of use of PIB/PDMS type of macroinitiator.

Acknowledgement

CSIR-CSMCRI Communication No. 028/2013. We thank Depart-ment of Science and Technology (project grant number: SR/FT/CS-009/2010), Government of India for financial support. We alsothank the Centralized Analytical Facility, CSMCRI for analyticalsupport and particularly Jayesh Chaudhury and Harshad Brahmb-hatt for carrying out SEM and GPC analyses respectively. We thankMr. Tohru Nakashima, Kaneka Corporation, Japan for kindlyproviding the Allyl-terminated PIB (EP200A).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.polymer.2014.03.019.

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