European Polymer Journal 46 (2010) 804–813

Contents lists available at ScienceDirect

European Polymer Journal

journal homepage: www.elsevier .com/locate /europol j

Synthesis of thermally responsive cylindrical molecular brushesvia a combination of nitroxide-mediated radical polymerization and‘‘grafting onto” strategy

Daniel Gromadzki a,c,*, Alexander Jigounov b, Petr Št�epánek a, Ricardas Makuška c

a Department of Supramolecular Polymer Systems, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic,Heyrovsky Sq. 2, 162 06 Prague 6, Czech Republicb Department of Structure Analysis, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovsky Sq. 2,162 06 Prague 6, Czech Republicc Department of Polymer Chemistry, Vilnius University, Naugarduko 24, LT-03225 Vilnius, Lithuania

a r t i c l e i n f o

Article history:Received 20 October 2009Received in revised form 30 November 2009Accepted 13 December 2009Available online 21 December 2009

Keywords:Nitroxide-mediated radical polymerization‘‘Grafting onto”Thermally responsive cylindrical brushesConformationWater-soluble polymer

0014-3057/$ - see front matter � 2009 Elsevier Ltddoi:10.1016/j.eurpolymj.2009.12.015

* Corresponding author. Address: Department ofmer Systems, Institute of Macromolecular CheSciences of the Czech Republic, Heyrovsky Sq. 2, 16Republic. Tel.: +420 296809262; fax: +420 2968094

E-mail address: d_grom@interia.pl (D. Gromadzk

a b s t r a c t

Loosely grafted amphiphilic molecular brushes consisting of a hydrophobic polystyrenebackbone and hydrophilic poly(ethylene glycol) monomethyl ether (MPEG) side chains,PS-MPEG, were synthesized by a novel two step method. In the first step, well-defined lin-ear poly(p-chloromethylstyrene) PCMS with the degree of polymerization DP � 200 andpolydispersity index, Mw/Mn = 1.4 was prepared by bulk nitroxide (TEMPO)-mediated rad-ical polymerization (NMP). In the second step, pendant p-chloromethyl units were coupledwith activated chains of poly(ethylene glycol) monomethyl ether (MPEG) during the courseof the Williamson etherification reaction. Using MPEG with Mn = 1100 g/mol over 50%repeating units on PCMS underwent nucleophilic substitution yielding a densely graftedbrush with theoretical molecular weight Mn,th � 123,000 g/mol. The molecular brush PS-MPEG assumed conformation of a stiff cylinder in both dilute toluene (non-selective goodsolvent for PS backbone and MPEG grafts) and water (selective for MPEG grafts) solutionsas determined by small-angle neutron scattering (SANS) and small-angle X-ray scattering(SAXS). The contour length L = 570 Å and L = 694 Å obtained by fitting the scattering datausing model of Sharp–Bloomfield, Pedersen–Schurtenberger and Kholodenko wormrevealed dimensions corresponding to theoretically estimated size of a single molecularbrush. It was found that PS-MPEG molecular brush in dilute aqueous solutions exhibitedlower critical solution temperature (LCST) in the physiological range.

� 2009 Elsevier Ltd. All rights reserved.

1. Introduction

Water-soluble amphiphilic copolymers exhibitingconformational changes in response to different stimulisuch as pH, salt, ionic strength, temperature or light have re-ceived a great deal of attention because of their promising

. All rights reserved.

Supramolecular Poly-mistry, Academy of2 06 Prague 6, Czech10.i).

applications in biomedical field (drug delivery carriers)and nanotechnology (sensors, actuators) [1]. Poly(N-isopro-pylacrylamide) (PNIPAM) exhibiting a lower critical solu-tion temperature (LCST) at about 34 �C became a landmarkpolymer in these areas and was extensively investigated inmany research groups throughout the world [2]. Tempera-ture induced coil-to-globule transition of PNIPAM in aque-ous solutions relies on an interbalance between varioustypes of interactions, mainly hydrogen bonding and hydro-phobic interactions [3]. In contrast to PNIPAM, poly(ethyl-ene glycol) – PEG also known as poly(ethylene oxide) –PEO is nontoxic, well-soluble in water and various organic

D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813 805

solvents, possesses many attractive properties includingbiocompatibility, non-immunogenicity and protein-repel-lent properties [4]. Indeed, some of PEG’s are already ap-proved for internal consumption by US FDA and are widelyused for PEG-ylation, i.e. covalent attachment of PEG chainsto another molecule (drug or therapeutic protein) to prolongcirculation time of therapeutics in a bloodstream. For exam-ple, PEG–interferon alpha is used as injectable treatment forhepatitis C while PEG-filgrastim (Neulasta) is used to treatneutropenia [5].

Since the discovery of facile methods of controlled/livingradical polymerization (CRP/LRP) the synthesis of polymersof well-defined composition, molecular weight and func-tionalities became possible [6,7]. Lutz et al. reported the syn-thesis of thermally-sensitive copolymers with a tunableLCST including physiological range by atom transfer radicalpolymerization (ATRP) of oligo(ethylene glycol) macromo-nomers of different chain length [8]. A comparative studyof these novel PEG-based copolymers and PNIPAM revealedthat upon heating and cooling cycle the former polymersexhibited reversible transition (coil-to-globule) with moreuniform thermal profile that was almost independent ofexternal conditions (concentration of the polymer or a salt).Similarly, Yamamoto et al. investigated the effect of struc-ture on thermo-responsive nature of well-defined oligo(eth-ylene glycol) methacrylate copolymers prepared by ATRP[9]. The LCST was tuned by copolymerization in the range26–52 �C. In the above cases thermo-responsive copolymerswere prepared by the so called ‘‘grafting through” approachwhich together with ‘‘grafting from” [10] and ‘‘grafting onto”[11] are commonly applied for the synthesis of polymerswith branched architecture [12]. For the pioneering contri-bution on the synthesis and conformation of PEG macromo-nomers [13,14] and the recent interest in thermo-responsive polymers with PEG side chains the reader is re-ferred to literature [15–19].

Both chain dimensions and the unique behavior of linearPEG of a wide range of molecular weights in aqueous [20]and methanolic [21] solutions were elucidated. Recent dataevidence that unexpected properties arise under construc-tion of new PEG architectures such as stars, combs and den-drimers [22], etc. For example, a new state of matter the,supersoft elastomer, was discovered when PEG chains werearranged into a dense brush architecture (molecular or bot-tle-brush) [23]. Such congestion of PEG side chains resultedin complete suppression of crystallization facilitating dop-ing with Li+ and ion conduction behavior [24]. Indeed, it isfrequently argued that PEG brush architectures may repre-sent an important advantage over linear counterparts forthe design of polymeric medium in lithium batteries [25].

The most common strategies for the synthesis of PS-MPEG comblike copolymers involve free-radical ‘‘graftingthrough” copolymerization [26,27] or deactivation of pen-dant reactive groups on the PS backbone during the courseof ‘‘grafting onto” process [28]. If polystyrenes containingmaleic anhydride moieties are used, the resulting combcopolymers besides PEG grafts additionally contain pH-sen-sitive carboxylic functionalities which result in a more com-plex solution behavior [29–31]. To achieve a precise controlover PS-MPEG comb architecture, the use of CRP techniqueslike ATRP [32], nitroxide-mediated radical polymerization

(NMP) [33] or reversible addition-fragmentation chaintransfer (RAFT) [34] became indispensable. Recently, Dwo-rak et al. reported the synthesis of amphiphilic core–shellPEO stars by deactivating arborescent PCMS with PEO chainscombining self-condensing ATRP and Williamson etherifi-cation reaction [35]. In aqueous solutions such amphiphilicstars presumably assumed globular shape; however, noinformation is given concerning their true conformation.In this work we have adopted the Williamson etherificationstrategy [36] to link MPEG chains to linear PCMS backbonethat was previously prepared by NMP. We report here on no-vel synthetic pathway for the preparation of well-definedamphiphilic graft copolymers, and conformation and solu-tion behavior of the synthesized loosely grafted amphiphilicmolecular brush PS-MPEG with respect to polymer concen-tration, temperature and salt.

2. Experimental

2.1. Materials

2,2,6,6-Tetramethylpiperidin-1-yloxy(TEMPO),2,20- azobis(isobutyronitrile) (AIBN), p-chloromethylstyrene (30:70 paraand meta isomers) and sodium hydride (NaH), acetic acid,petroleum ether were purchased from Aldrich. Poly(ethyleneglycol) monomethyl ether (MPEG) (Mn = 1100 g/mol) was ofFluka. Tetrahydrofuran (THF) was dried and distilled beforeuse.

2.2. Synthesis of linear poly(p-chloromethylstyrene)

Synthesis of linear poly(p-chloromethylstyrene) PCMSwas done by bulk nitroxide (TEMPO)-mediated radical poly-merization according to the procedure described previously[10]. Characteristics of the synthesized PCMS: Mn =30,000 g/mol, DP� 200, polydispersity index Mw/Mn = 1.4.

2.3. Synthesis of polystyrene-graft-poly(ethylene glycol)monomethyl ether (PS-MPEG)

Grafting of poly(ethylene glycol) monomethyl etherMPEG onto PCMS backbone was carried out using William-son etherification reaction [36]. In a typical modificationprocedure, 4.55 mmol (1.6 g) of MPEG were dissolved in5 ml THF and in another flask 4.50 mmol (0.11 g) of NaH in10 ml THF. MPEG solution was added dropwise to the stirredsolution of NaH, and the resulting solution was left understirring for 2 h. Then, this solution was added dropwise tothe stirred solution of PCMS prepared by dissolving of 0.4 gof PCMS (2.6 mmol of chloromethyl groups) in 6 ml of THF,where ([CMS]:[MPEG] = 1:1.75). The reaction mixture waspurged with nitrogen and kept stirring at 25 �C for 72 h. Thenthe reaction was neutralized with an equivalent amount(0.27 g) of acetic acid and precipitated into petroleum etherand filtered. Finally, the isolated product was purified byexhaustive dialysis against water for 1 week. The extent ofgrafted MPEG chains (degree of modification of PCMS, DM)was estimated by 1H NMR analysis: DM = 57%. Characteris-tics of the comb copolymer PS-MPEG used for conforma-tional, aggregation and solution characterization: number-

806 D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813

average molecular weight Mn,th � 123,000 g/mol, graftingdensity 57% of the backbone units, molecular weight of thegrafted MPEG 1100 g/mol, number of grafted MPEG perbackbone 112.

2.4. Characterization

1H NMR spectra of the copolymers were recorded on aUNITY INOVA VARIAN spectrometer (300 MHz, Varian) inCDCl3 and D2O (solution concentration 20–30 mg/ml). FT-IR spectra of the polymers in the form of potassium bromidepellets were recorded on Perkin–Elmer Paragon 1000PCinfrared spectrometer. The SEC measurements for the eval-uation of number- and weight-average molecular weights(Mn) and (Mw) were carried out on a PSS SDV 10,000 column(8 � 600 mm, Polymer Standard Service, Germany), filledwith 5 lm sorbent particles. THF, distilled and dried overmolecular sieves (4 Å), served as a mobile phase. Chromato-graphic data from refractometric and UV (254 nm) detectorswere treated by using the Data-Monitor system (Watrex,Czech Republic). For the determination of molecularweights, a universal calibration equation based on the dataof polystyrene standards (Merck, Germany) was used.

2.4.1. Light scattering measurementsThe measurements were performed at room tempera-

ture on an ALV CGE instrument equipped with a Uniphase22 mW He–Ne laser operating at a wavelengthk = 632.8 nm and an ALV 6010 multiple s digital correlator.The scattering measurements were performed in the angu-lar range 30–150� by steps of 10� and the counting timewas fixed at 200 s for each angle. The obtained intensityautocorrelation functions g2(t) from dynamic light scatter-ing (DLS) were analyzed using algorithm REPES (incorpo-rated in the GENDIST program) which provides the inverseLaplace transformation according to Eq. (1) [37].

g2ðtÞ ¼ 1þ bZ

AðsÞ expð�t=sÞds� �2

ð1Þ

where t is the delay time of the correlation function and bis an instrumental parameter. Apparent diffusion coeffi-cients were obtained by plotting the relaxation frequency,C (= s�1) versus q2 according to

D ¼ Cq2 ð2Þ

where q is the wave vector defined as

O N

CN

ClCl

200

25

O-ONa+

THF

Fig. 1. Synthesis of amphiphilic molecular brush by grafting of activated MPbackbone.

q ¼ 4pnk

sinh2

� �ð3Þ

where k is the wavelength of the incident laser beam(632.8 nm), h is the scattering angle and, and n is therefractive index of the medium.

2.4.2. Small-angle X-ray scattering (SAXS)SAXS experiments were performed using a pinhole

camera (Molecular Metrology SAXS System) attached to amicrofocused X-ray beam generator (Osmic MicroMax002) operating at 45 kV and 0.66 mA (30 W) and yieldinga beamline with wavelength k = 1.54 Å. The camera wasequipped with a multiwire, gas-filled area detector withan active area diameter of 20 cm (Gabriel design). Twosample-to-detector distances were used to cover the q-range of 0.007–1.1 Å�1. The scattering intensities wereput on absolute scale using a glassy carbon standard. Priorto fitting analysis a solvent scattering had been subtracted.

2.5.3. Small-angle neutron scattering (SANS)The experiment was performed at the Laboratoire Léon-

Brillouin at Commissariat á l’Énergie Atomique Saclay onthe spectrometer PAXY [38]. Measurements were per-formed with a 128 � 128 multidetector (pixel size0.5 � 0.5 cm2) using a nonpolarized, monochromatic inci-dent neutron beam collimated with circular apertures ata sample-to-detector distance of 7 m (with wavelengthk = 8 Å). The 2D scattering patterns were isotropic so thatthey were azimuthally averaged to yield the dependenceof the scattered intensity I(q) on the scattering vector q.

3. Results and discussion

3.1. Synthesis of loosely grafted amphiphilic molecular brushPS-MPEG

Loosely grafted amphiphilic molecular brushes PS-MPEG were synthesized by nitroxide-mediated radicalpolymerization followed by ‘‘grafting onto” Williamsonetherification reaction according to general syntheticscheme outlined in Fig. 1.

Preparation of linear poly(p-chloromethylstyrene)backbone was carried out by NMP in the presence of TEM-PO radical according to the procedure reported by Boutevin[39]. Unimodal SEC eluogram shown in Fig. 2 indicatedthat the length of the backbone was fairly well-controlled

OO O

O25

O N

CN

Cl

200

EG 1100 onto TEMPO-terminated p-chloromethylstyrene homopolymer

10 11 12 13 14 15 16 17 18 19 20

Mn = 30 000 g/mol

Mw/Mn = 1.4

Elution volume (mL)

Fig. 2. SEC trace of PCMS backbone prepared by nitroxide (TEMPO)-mediated radical polymerization.

8 7 6 5 4 3 2 1

3

2

ppm

1

Fig. 4. 1H NMR spectra of the loosely grafted amphiphilic molecularbrush PS-MPEG in CDCl3 (1), D2O (2) and D2O/CDCl3 mixture (70/30 v/v%)(3) acquired at 25 �C.

D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813 807

under NMP conditions. Number-average molecular weightMn of PCMS determined by SEC was close to 30,000 g/molcorresponding to the degree of polymerization (DP) around200. Both the measured molecular weight and estimateddegree of polymerization represent apparent value sinceSEC was calibrated with polystyrene standards. Neverthe-less, our light and X-ray scattering data on PCMS backbonein toluene indicate that the hydrodynamic size and radiusof gyration correlates well with the theoretical value of theFlory radius (RF = 60 Å) for polymer with DP = 200. Synthe-sis of PCMS with higher N in the presence of TEMPO wasnot anticipated because of considerable chain transfer tochloromethyl groups that leads to increased polydispersityof the backbone.

Fig. 3 shows FT-IR spectra of PCMS backbone (a) and ofthe comb copolymer PS-MPEG (b) which are helpful inqualitative assessment of the employed Williamson ether-ification strategy. The absorption band at 671 cm�1 isattributed to halogen bonds in the p-chloromethyl groups

4000 3000 2000 1000

b

wavenumber(cm-1)

a

1094

671

2945

Fig. 3. FT-IR spectra of linear backbone PCMS (a) and loosely graftedamphiphilic molecular brush PS-MPEG (b).

which upon coupling with activated chains of MPEG al-most completely disappeared (spectrum b). The polyethercombs show a characteristic ether absorption band ataround 1094 cm�1 and strengthening of the alkyl absorp-tion at around 2945 cm�1. The absence of peak corre-sponding to halogen units in the product may indicate onquantitative conversion of halogen units to MPEG units.However, IR spectroscopy at attenuated total reflectionmode delivers qualitative information from the surfacewhich in our case is densely covered with MPEG chainstherefore it is plausible that halogen units buried insidethe brush are not seen by this technique.

Structure and amphiphilic character of the synthesizedgraft copolymer was further judged by 1H NMR spectra(Fig. 4) taken in deuterated chloroform (good solvent forthe backbone and marginally good for MPEG grafts), deu-terated water (very good solvent for grafts and non-solventfor PS backbone) as well as in a mixture of deuterated chlo-roform and water (30/70 v/v). The spectrum in chloroformshows signals of protons in both aromatic polystyrenebackbone at 6.3 � 7.2 ppm and MPEG structural units at3.5 � 3.8 ppm (–OCH2CH2–) and at 3.4 ppm (–OCH3). Thegraft copolymer dissolved in water exhibit markedly dif-ferent behavior. Here, the signals of the grafted MPEGchains are well expressed while the aromatic signals are al-most invisible. This happens due to very good solvation ofthe hydrophilic MPEG chains by water molecules. In addi-tion, this result shows that MPEG chains act as sufficientprotecting shell for the glassy PS backbone. Interestingly,an addition of 30% v/v chloroform forming mixed solventwith water (a decrease of the solvent quality for MPEGgrafts) restores the aromatic region in the spectrum con-firming amphiphilic behavior of the synthesized graftcopolymer PS-MPEG.

Composition of the molecular brush PS-MPEG was cal-culated using the integrated peak area ratio of the signalat 4.5 ppm in 1H NMR spectrum in CDCl3 attributed to

-5.5 -5.0 -4.5 -4.0 -3.5 -3.0 -2.5-11.0

-10.5

-10.0

-9.5

-9.0

-8.5

-8.0

-7.5

ln I(

q)

ln q (Å-1)

q-1.39

Fig. 5. SANS curve of the amphiphilic molecular brush PS-MPEG intoluene at polymer concentration c = 1 � 10�3 g ml�1.

808 D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813

the protons of unreacted chloromethyl –CH2Cl groups onPCMS backbone to the signal at 3.4 ppm attributed to theprotons of methoxy –OCH3 groups in MPEG. The degreeof modification of PCMS by MPEG grafts (DM) was 57%which evidenced that ca. 112 –CH2Cl units in every back-bone were reacted with anionic MPEG 1100 yielding singlemacromolecules with Mn,th � 123,000 g/mol and weightfraction of PS segment, wPS = 0.24. DM (degree of modifica-tion, i.e. grafting density) is fairly good taking into accountthat conversion in grafting onto processes rarely exceed60% because of the hindered diffusion of side chains tothe backbone [12].

The actual grafting efficiency might be sufficient to in-duce steric constraints between anchored MPEG chainsthat by stretching along the direction normal to the surfacewould force the backbone to adopt worm-like conforma-tion. This can be justified using scaling arguments basedon the de Gennes theory [40] of grafted linear, flexiblechains in a good solvent. If the average distance d betweentwo graft sites is smaller than the coil size (the Flory ra-dius) RF (d < RF) of a polymer with N segments of length a

RF ¼ a � N3=5 ð4Þ

the chains will stretch out from the backbone and form adense PEG brush with thickness

r ¼ a � Nða=dÞ2=3 ð5Þ

For graft copolymer PS-MPEG assuming that N = 25(MPEG 1100) and a = 3.36 Å (monomer unit length assum-ing fully extended PEG in its lowest energy conformation)[41], the Flory radius is RF = 23.2 Å. The average distance dbetween two graft sites is simply estimated by knowingthe length of C–C bond in vinyl chain corresponding to2.5 Å and that every second chloromethyl unit of PCMSbackbone is linked to MPEG chain. This roughly yieldsd = 5 Å which is significantly smaller than the Flory radiusRF = 23.2 Å (d < RF) suggesting that the MPEG chains are in-deed in the ‘‘brush-like” regime. The thickness of thehydrophilic MPEG corona was estimated at r = 64.4 Åwhich is just above the minimum layer thickness of�50 Å needed to prevent the adsorption of bovine serumalbumin and immunoglobulin [21].

4. Conformation of the amphiphilic molecular brushPS-MPEG in toluene as non-selective good solvent

4.1. Small-angle neutron scattering (SANS)

Toluene is good solvent for both PS backbone and MPEGgrafts; therefore toluene is a solvent of choice for determina-tion of conformation and dimensions of the amphiphilicmolecular brushes [42]. The solution overlap concentration(c�) was estimated using relation [43] c�L ¼ Mw=NAL3 ¼1:66� 10�3 g mL�1 rather than by the space filling formula[44], where Mw is weight-average molecular weight, L isthe estimated contour length (ca. 50 nm) and NA is Avogadronumber. Fig. 5 shows the SANS curve of the synthesizedamphiphilic brush in 1 � 10�3 g mL�1 toluene solution.

The measured SANS intensity in the intermediate scat-tering region of the curve follows a power-law decay pro-

portional to q�1.39 which means that the overall shape ofthe brush can be described by a stiff cylinder with finite ra-dius while slope q�1 would correspond to the thin rod [45].The worm-like nature arises from the repulsion betweenMPEG side chains (excluded-volume effect) because thehigher density of the side chains is separated by a distancemuch smaller than their unperturbed dimension whichleads to steric overcrowding of the side chains and entropi-cally unfavourable chain extension of the backbone [12].This result supports our theoretical estimations based onthe theory of de Gennes discussed in Section 3. Unfortu-nately, due to a lack of scattering data at low q-range thedetermination of the radius of gyration by the Guinier plotwas not possible.

4.2. Dynamic light scattering (DLS)

Fig. 6 shows representative DLS measurement per-formed below the solution overlap concentration (c�). Sin-gle and quite narrow distribution of relaxation times wasobserved for investigated solution concentrations indicat-ing a population of fully dissolved polymer species.

The dependence of relaxation rate C on q2 is linear andpasses through the origin (Fig. 7), which implies that thepeaks are related to purely translational diffusion.

This result is consistent with observation of Bolisettyet al. [46] whose DLS experiments on bottle-brush havinga backbone six times longer compared to the present brushalso did not reveal any contributions of internal modes tothe dynamics such as rotation, bending or stretching.

5. Conformation of the amphiphilic molecular brushPS-MPEG in water

5.1. Small-angle X-ray scattering (SAXS) analysis

Fig. 8 shows SAXS scattering curve of the molecularbrush PS-MPEG in aqueous solution at the concentrationc = 1 � 10�3 g mL�1.

1E-4 1E-3 0.01 0.1 1 10 100 10000.00

0.15

0.30

0.45

τ Α

(τ)

log t,τ (ms)

g 2(t

)-1

0.00

0.05

0.10

0.15

Fig. 6. Typical autocorrelation function and distribution of relaxation times of the loosely grafted molecular brush PS-MPEG in dilute toluene solution atc = 2.5 � 10�4 g ml�1 and temperature of 25 �C (scattering angle h = 90�).

D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813 809

The slope in the intermediate scattering region of thedependence log I vs log q starts from �1 and slightly in-creases at larger q which again indicates cylindrical confor-mation of the molecular brush in water. The SAXS curvewas fitted to the model of worm-like chain using three dif-ferent theoretical functions, namely the Sharp–Bloomfieldequation [47], Kholodenko fit [48] and the model of Peder-sen–Schurtenberger [49]. Generally, Sharp–Bloomfieldexpression is valid only for flexible chains, thus we haveused version corrected by Yoshizaki and Yamakawa [50].

SSB ¼ SDebye þ4

15þ 7

15x� 11

15þ 7

15x

� �expð�xÞ

� �� 2lp

Lð6Þ

wherein x ¼ lpLq2=3, lp is the persistence length and L isthe contour length.

0 1 2 3 4 5 6 7

0.5

1.0

1.5

2.0

2.5

3.0

3.5

Γ x

103

(s-1

)

q2 x 1010 (cm-2)

Fig. 7. The dependence of the decay rate C on q2 of the molecular brushPS-MPEG in dilute toluene solutions.

Pedersen–Schurtenberger in their work have modifiedthis expression by exchanging the Debye function by thatof a flexible chain with excluded-volume effect; also secondpart of expression was reduced by coefficient depending onL/b (b = 2�lp is Kuhn length). Excluded-volume effect is veryimportant when it comes to analysis of polymer stiffness.In good solvents this effect leads to increase of chain dimen-sions and calculations using form factors that do not takeinto account this fact will be accompanied by errors in deter-mination of Kuhn length. By fixing contour length, L, at samevalue as for Sharp–Bloomfield we have observed the in-crease of persistence length value.

0.01 0.1 11E-3

0.01

0.1

q (Å-1)

log

I(q)

q-1

L = 570 Å

Fig. 8. Small-angle X-ray scattering curve of the molecular brush PS-MPEG in water. The solid line represents the best fit with the theoreticalSharp–Bloomfield model of worm-like chain to the scattering curve. Theinset shows a sketch of probable shape of the molecular brush withindicated contour length.

Table 1Fitting parameters obtained from the analysis of SAXS data of the molecularbrush PS-MPEG in water.

Model L (Å) Rcs (Å) lp (Å)

Sharp–Bloomfield 570 7.4 15.4Pedersen–Schurtenberger 570 7.4 16.9Kholodenko 694 5.6 18.0

810 D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813

Form factor resulting from Kholodenko approach is afunction of two components [43]:

Pðq; L; b;RcsÞ ¼ P0ðq; L; bÞ � Pcsðq;RscÞ:

where b is Kuhn length, L is Contour length and Rsc is theradius of cylindrical core. First component, P0, is designedto reproduce correctly the rigid-rod limit and the ran-dom-coil limit. Another component, Pcs, is the form factorfor flexible cylinders with a circular cross-section and auniform scattering length density of the cross-section.The resulting persistence length is slightly higher thanfor Pedersen–Schurtenberger, but this increase was ex-pected following by simulated curves obtained by Pötschkeet al. [51]. Fitting procedures were done using SASFit soft-ware [52]. Fitting parameters are listed in Table 1.

The contour length (L) of this worm-like macromoleculecan be roughly estimated taking the contour length permonomer unit 2.5 Å (trans-conformations) and the degreeof polymerization of the backbone DP � 200. This gives thevalue of L = 500 Å which is in reasonably good agreementwith the contour length L = 570 Å from SAXS analysis usingthe Sharp–Bloomfield model and not incompatible withthe value of L = 694 Å extracted from Kholodenko fit.

The characteristic bump in low q-range of a Kratky plot(I(q) q2 vs q) presented in Fig. 9 provides further evidenceof a branched, compact chain with high persistence length.

The latter value can be conveniently determined bygraphical analysis of the Kratky plot I(q) q2 vs q from theabscissa value where straight line intersect data points:lp = 1.91/q�. The persistence length lp = 13.6 determined

0.1 0.2 0.3 0.40

1

2

3

4

5

6

7

q (Å-1)

I(q)

q2 x

104

q*

Fig. 9. Kratky plot of the amphiphilic molecular brush PS-MPEG in waterat the concentration c = 1 � 10�3 g ml�1.

by the above method is in a good agreement withlp = 15.4 Å from Sharp–Bloomfield model [53].

A few important conclusions can be drawn from the re-sults presented above. First, hydrophobic polystyrenebackbone is well protected by hydrophilic MPEG shelland the rigid-rod conformation in water is preserved de-spite significant difference in the solvent quality with re-spect to the backbone. The estimated and measuredcontour length is roughly comparable to that of an individ-ual macromolecule suggesting the existence of cylindricalunimolecular micelles. This is certainly not surprising sincea lot of experimental data on micellization of graft copoly-mers indicate that graft copolymers in a solvent which isthermodynamically good for grafts and bad for the back-bone exhibit low aggregation number or do not associateat all remaining in unimolecular state [54].

6. Clusterization of molecular brush PS-MPEG in water

The clusterization behavior of MPEG chains in water iswell-known phenomenon and its origin was thoroughlydiscussed by Hammouda et al. [55]. They concluded thatinteraction of hydrophobic end-groups with methyl end-groups of MPEG is the major factor responsible for the clus-terization of MPEG chains in pure water. Therefore itwould be too straightforward to accept the existence ofsingle chains of the studied PS-MPEG in pure water. Forexample, Cheng et al. [32] based on their very recent SANSinvestigations on a series of similar amphiphilic PS-MPEGbrushes (DP of the polystyrene backbone was varied be-tween 8 and 85 units with 4 MPEG units attached to it)synthesized by ‘‘grafting through” ATRP have shown thatthe presence of hydrophobic bromine and phenyl end-group, originating from the initiators, induces brush end-to-end clusterization. Specifically, they found that brushwith DP = 8 formed clusters in toluene-d8, methanol-d4

and D2O independent of polymer concentration up to1 wt.% while lack of clusterization was observed for poly-mers with the degree of polymerization DP = 30, 40, 47,58 and 85 or it was gradually diminished in D2O withincreasing DP. Since the probability of end-to-end interac-tions decreases with increasing DP of the backbone one canassume that the contribution of end-groups in the studiedmolecular brush PS-MPEG with DP = 200 is rather low. Onthe other hand, the hydrophobicity of 2 methyl groupsfrom AIBN at one end of PCMS backbone and 4 methylgroups of TEMPO at the other one in the studied molecularbrush is certainly much higher than the hydrophobicity ofATRP polymers. To this end, one shall expect a significantcontribution of interactions between end-capped initiatorgroups and dangling methoxy groups of MPEG chains. Onthe other hand, numerous authors have studied aggrega-tion of PEG-grafted polymers in water and overall it seemsthat the aggregation of such polymers result mostly froman amphiphilic behavior [13,14,56,57]. In addition, recentresults on the behavior of MPEG-grafted polystyrene inwater suggest that solution concentration plays a criticalrole in the interaction between brush chains [32]. In orderto gain a better insight into this phenomenon solutions ofthe molecular brush PS-MPEG in deionised water at con-

0 20 40 60 80 100 120 1400

100

200

300

400

500

600

700

800

Ph

ase

sep

arat

ion

Rh

(nm

)

c x 104 (g mL-1)

semi- dilute regime

Fig. 10. Dependence of hydrodynamic radii on concentration of themolecular brush PS-MPEG in deionised water (open circles) monitored atscattering angle h = 90� at 25 �C. The solid circles represent measure-ments performed after 6 months.

0 1 2 3 4 5 6 70

1

2

3

4

5

Kc/

Rθ x

107 (

mol

/g)

q2 x 1014 (m-2)

Fig. 11. q2 Dependence of Kc=Rh for PS-MPEG in deionized water (opencircles) and in and in 0.1 N NaCl aqueous solution at concentrationc = 2.5 � 10�4 g ml�1.

46

48

50

h (n

m)

D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813 811

centrations below the chain overlap c� ranging from 1 to4 � 10�4 g ml�1, in the vicinity of c� (1 � 10�3 g ml�1) andabove c� (0.3–1 � 10�2 g ml�1) were prepared and sub-jected to DLS measurements. Fig. 10 represents behaviorof the molecular brush at various concentration regimes.In dilute solutions unimodal distribution of relaxationtimes (graph not shown) was observed with dimensionsclose to RH = 50 nm.

In the vicinity of the overlap concentration the size of themolecular brush was comparable with that in the dilute re-gime but increased abruptly above c� with visual indicationof clouding and macrophase separation. The hydrodynamicdimensions in the semi-dilute regime ranged from 400 to800 nm, i.e. sizes characteristic for large aggregates result-ing from strong polymer–polymer interactions which is inagreement with previous experimental observations [32].DLS measurements performed after 6 months revealed thatthe hydrodynamic sizes of the molecular brush remainedunchanged but were about 1.5 times larger in semi-diluteregime and slightly larger in the vicinity of c�. A curvatureof the angular dependence at the high q-region of the Zimmplot provides additional evidence of large aggregates inaqueous solutions (Fig. 11).

In contrast, no curvature is seen for PS-MPEG brush dis-solved in 0.1 N NaCl (very weak chaotrope) at the same con-centration suggesting that the addition of salt can be used tosuppress aggregation behavior or at least trigger conforma-tion changes of the studied brush in aqueous solutions.

25 30 35 40 45 50 55 60 65 70

40

42

44 LCSTR

T (oC)

Fig. 12. Dependence of the hydrodynamic radii of the amphiphilicmolecular brush PS-MPEG in aqueous solution at dilute concentrationregime (1 � 10�4 g ml�1) on temperature.

7. Effect of temperature on aqueous solutions ofmolecular brush PS-MPEG

As it was discussed above stable amphiphilic particles(micelle like aggregates) with hydrodynamic size ofRH � 50 nm were observed at 25 �C. On heating, the hydro-dynamic radius of these aggregates started to decrease at30 �C followed by a sharp drop around 38 �C (physiologicalrange). This is due to occurrence of the lower critical solu-tion temperature (LCST), i.e. disruption of hydrogen bond-

ing between MPEG chains associated with intramolecularcollapse of the brush (Fig. 12).

At around 44 �C the hydrodynamic radius of the molec-ular brush assemblies reached the minimal value with sizeRH = 40 nm and started to increase upon further heating.The driving force for the latter effect are increasing hydro-phobic interactions between brush macromolecules insolution. Noteworthy, macroscopic clouding or milk-whiteturbidity of the solution did not occur because of relativelylow brush concentration (c < 1 � 10�3 g ml�1). It is knownthat LCST of MPEG chains in aqueous solutions dependson molecular weight [58]. Generally, increase in molecularweight shifts LCST to higher temperatures. In addition, it isknown that substitution of hydroxyl end-groups on MPEGby a more hydrophobic groups leads to a large decrease ofLCST [59,60]. Keeping in mind these facts one can specu-late that grafting of MPEG and, at the same, consumptionof hydroxyl end-groups were the factors responsible fordecrease of LCST from ca. 99 �C (pure MPEG with 1100 g/

20 30 40 50 60

62

64

66

68

70

72R

h(n

m)

T (oC)

Fig. 13. Dependence of the hydrodynamic radii of the amphiphilicmolecular brush PS-MPEG in 0.1 M NaCl aqueous solution at diluteconcentration regime (1 � 10�4 g mL�1) on temperature.

812 D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813

mol) [49] down to the physiological range ca. 38 �C. In thecase of molecular brushes containing thermo-responsiveentities other factors such as the length of the side chainsand grafting density may affect both the LCST and kineticsof the chain collapse [61,62]. In order to elucidate effect ofall these parameters a systematic study with a set of newamphiphilic cylindrical brushes is required which is how-ever beyond the scope of the present work.

The effect of salt have profound influence on phase tran-sition in PNIPAM [63] and in PEG [64]. In addition, for allPEG-grafted polymers studied so far there is a strong evi-dence of the ‘‘salting out” effect in the presence of NaCl[8,65,66]. In all cases LCST was found to decrease in the pres-ence of NaCl. The dependence of hydrodynamic radii ontemperature for molecular brush PS-MPEG in 0.1 M NaClsolution in dilute concentration regime is presented inFig. 13. At 25 �C the hydrodynamic radius RH = 71 nm ofthe molecular brush PS-MPEG was larger compared toRH = 50 nm in salt-free solution. Upon heating the hydrody-namic size remained essentially unchanged up to LCSTwhich similarly to salt-free aqueous solution appeared inthe physiological range, namely at 37 �C. In solution withadded salt the transition was however less sharp; it occurredin temperature range from 37 �C to around 50 �C while forsalt-free solution it was within a few �C (see Fig. 13).

In addition, after the minimum value of hydrodynamicradius RH = 62 nm has been reached further heating didnot show any influence on the RH up to 55 �C. While thisis in contrast to situation observed in salt-free solutionswe do not have convincing explanation for this effect atthe present. In our future work it will be of relevance tostudy the effect of ion type (Hofmeister series) and ionconcentration on phase transition in aqueous solutionscontaining molecular brushes PS-MPEG.

8. Conclusion

It was demonstrated that the combination of nitroxide-mediated radical polymerization and ‘‘grafting onto” Wil-

liamson etherification reaction can be successfully usedfor the synthesis of loosely grafted amphiphilic molecularbrushes. The amphiphilic behavior of molecular brush PS-MPEG was validated by 1H NMR spectroscopy in solventsselective for the respective hydrophobic and hydrophilicsegments. Due to excluded-volume effect the looselygrafted brush PS-MPEG assumed a conformation of a stiffcylinder in toluene as evidenced by the power-law decayproportional to �q�1.39 on the measured SANS intensity.The conformation of a stiff cylinder was preserved in aque-ous solutions despite significant difference in solvent qual-ity with respect to hydrophobic polystyrene backbone. Thedimensions of investigated molecular brush, i.e. the cross-sectional radius Rsc, contour length L and persistencelength lp were obtained by fitting the SAXS scattering datato the models of worm-like chain using theoretical func-tions of Sharp–Bloomfield, Pedersen–Schurtenberger andKholodenko. Preliminary results indicate on strong cluster-ization of molecular brush PS-MPEG with increasing con-centration. It was found that molecular brush PS-MPEGin dilute aqueous solutions exhibits a fast temperature re-sponse in the physiological range that is only slightly af-fected by the presence of NaCl.

Acknowledgements

The authors would like to thank Mrs. M. Kreneviciene ofVilnius University for 1H NMR measurements, Dr. J. Pleštilfor SAXS and Dr. L. Noirez (LBB/CEA Saclay, France) forSANS and the Grant Agency of the Czech Republic for sup-port (Grant 202/09/2078). DG was a grateful recipient ofthe Marie Curie Fellowship within Marie-Curie Researchand Training Network Self-Organization under ConfinementMCRTN-SOCON (Contract No. MRTNCT-2004-512331) andthe Marie-Curie Industry-Academia Partnerships and Path-ways (PIAP-GA-2008-218242) funded by the EuropeanCommission.

References

[1] McCormick Charles L, editor. Stimuli-responsive water soluble andamphiphilic polymers (University of Southern Mississippi).American Chemical Society, Oxford University Press: Washington,DC; 2001.

[2] Dušek K, editor. Responsive gels. Volume transition. Adv Polym Sci1993;110:49.

[3] Schild HG. Prog Polym Sci 1992;17:163.[4] Chen J, Spear SK, Huddleston JG, Rogers RD. Green Chem 2005;7:64.[5] Zeuzem S. Nat Clin Pract Gastroenterol Hepatol 2008;5:610–22.[6] Braunecker WA, Matyjaszewski K. Prog Polym Sci 2007;32:93.[7] Matyjaszewski K, Gnanou Y, Leibler L, editors. Macromolecular

engineering: from precise macromolecular synthesis to macroscopicmaterials properties and applications. Weinheim: Wiley-VCH; 2007.

[8] Lutz JF, Akdemir Ö, Hoth A. J Am Chem Soc 2006;128:13046.[9] Yamamoto S, Pietrasik J, Matyjaszewski K. J Polym Sci A Polym Chem

2007;46:194.[10] Gromadzki D, Makuška R, Netopilík M, Holler P, Lokaj J, Janata M,

et al. Eur Polym J 2008;44:59.[11] Gromadzki D et al. Proceedings of the European polymer congress

2007, Portoroz, Slovenia.[12] Sheiko SS, Sumerlin BS, Matyjaszewski K. Prog Polym Sci

2008;33:759.[13] Ito K, Tomi Y, Kawaguchi S. Macromolecules 1992;25:1534.[14] Ito K, Tanaka K, Tanaka H, Imai G, Kawaguchi S, Itsuno S.

Macromolecules 1991;24:2348.[15] Han S, Hagiwara M, Ishizone T. Macromolecules 2003;36:8312.[16] Ali MM, Stöver HDH. Macromolecules 2004;37:5219.

D. Gromadzki et al. / European Polymer Journal 46 (2010) 804–813 813

[17] Lutz JF. J Polym Sci A Polym Chem 2008;46:3459.[18] Holder SJ, Durand GG, Yeoh CT, Illi E, Hardy NJ, Richardson TH. J

Polym Sci A Polym Chem 2008;46:7739.[19] Becer CR, Hahn S, Fijten MWM, Thijs HML, Hoogenboom R, Schubert

US. J Polym Sci A Polym Chem 2008;46:7138.[20] Devanand K, Selser JC. Macromolecules 1991;24:5943.[21] Sung JH, Lee DC, Park HJ. Polymer 2007;48:4205.[22] Neugebauer D. Polym Int 2007;56:1469.[23] Neugebauer D, Zhang Y, Pakula T, Sheiko SS, Matyjaszewski K.

Macromolecules 2003;36:6746.[24] Zhang Y, Costantini N, Mierzwa M, Pakula T, Neugebauer D,

Matyjaszewski K. Polymer 2004;45:6333.[25] Meyer WH. Adv Mater 1998;10:439.[26] Feng Li-B, Zhou S-X, You B, Wu L-M. J Appl Polym Sci

2007;103:1458.[27] Zhu G. Eur Polym J 2005;4:2671.[28] Meng F, Engbers GHM, Feijen J. J Biomed Mater Res A Appl Biomater

2004;40:49.[29] Dérand H, Wesslén B, Wittgren B, Wahlund K-G. Macromolecules

1996;29:8775.[30] Eckert AR, Webber SE. Macromolecules 1996;29:560.[31] Weber J, Boyko V, Arndt K-F. Macromol Chem Phys 2007;208:643.[32] Cheng G, Melnichenko YB, Wignall GD, Hua F, Hong K, Mays JW.

Polymer 2007;48:4108.[33] Zhao B, Li D, Hua F, Green DR. Macromolecules 2005;38:9509.[34] You YZ, Hong CY, Wang WP, Wang PH, Lu WQ, Pan CY.

Macromolecules 2004;37:7140.[35] Dworak A, Kowalczuk-Bleja A, Trzebicka B, Walach W. Polym Bull

2002;49:9.[36] Park JY, Acar MH, Akthakul A, Kuhlman W, Mayes AM. Biomaterials

2006;27:856.[37] Jakeš J. Collect Czech Chem Commun 1995;60:1781–97.[38] http://www-llb.cea.fr/spectros/spectro/paxy.html.[39] Lacroix-Desmazes P, Delair T, Pichot C, Boutevin B. J Polym Sci A

Polym Chem 2000;38:3845.[40] de Gennes PG. Adv Colloid Sci 1987;27:189.[41] Ratto TV, Langry KC, Rudd RE, Balhorn RL, Allen MJ, McElfresh MW.

Biophys J 2004;86:2430.

[42] Cheng G, Melnichenko YB, Wignall GD, Hua F, Hong K, Mays JW.Macromolecules 2008;41:9831.

[43] Teraoka Iwao. Polymer solutions: an introduction to physicalproperties. Polytechnic University Brooklyn, New York. NewYork: Wiley & Sons Inc.; 2002.

[44] de Gennes PG. Scaling concepts in polymer physics. Ithaca,NY: Cornell University Press; 1979.

[45] Roe RJ. Methods of X-ray and neutron scattering in polymerscience. New York: Oxford University Press; 2000.

[46] Bolisetty S, Airaud C, Xu Y, Müller AHE, Harnau L, Rosenfeldt S, et al.Phys Rev E 2007;75:040803(R).

[47] Sharp P, Bloomfield VA. Biopolymers 1968;6:1201.[48] Kholodenko AL. Macromolecules 1993;26:4179.[49] Pedersen JS, Schurtenberger P. Macromolecules 1996;29:7602.[50] Yoshizaki T, Yamakawa H. Macromolecules 1980;13:1518.[51] Pötschke D, Hickl P, Ballauff M, Astrand PO, Pedersen JS. Macromol

Theory Simul 2000;9:345.[52] http://kur.web.psi.ch/sans1/SANSSoft/sasfit.html.[53] Glatter O, Kratky O. Small angle X-ray scattering. Academic press;

1982.[54] Gromadzki D, Filippov S, Netopilík M, Makuška R, Jigounov A, Pleštil

J, et al. Eur Polym J 2009;45:1748.[55] Hammouda B, Ho DL, Kline S. Macromolecules 2004;37:6932.[56] Lutz JF, Weichenhan K, Akademir Ö, Hoth A. Macromolecules

2007;40:2503.[57] Hussain H, Mya KY, He C. Langmuir 2008;24:13279.[58] Saeki S, Kuwahara N, Nakata M, Kaneko M. Polymer 1976;17:685.[59] Crespy D, Rossi RM. Polym Int 2007;56:1461.[60] Jiang X, Zhao B. J Polym Sci A Polym Chem 2007;45:3707.[61] Plunkett KN, Zhu X, Moore JS, Leckband DE. Langmuir 2006;22:4259.[62] Yamamoto S, Pietrasik J, Matyjaszewski K. Macromolecules

2007;40:9348.[63] Zhang Y, Furyk S, Sagle LB, Cho Y, Bergbreiter DE, Cremer PS. J Phys

Chem C Nanomater Interfaces 2007;111:8916.[64] Shetty MA, Solomon MJ. Polymer 2009;50:261.[65] Skrabania K, Kristen J, Laschewsky A, Akdemir Ö, Hoth A, Lutz JF.

Langmuir 2007;23:84.[66] Magnusson JP, Khan A, Pasparakis G, Saeed AO, Wang W, Alexander

C. J Am Chem Soc 2008;130:10852.