SHORT COMMUNICATION

Morphology control of giant vesicles by manipulatinghydrophobic-hydrophilic balance of amphiphilic randomblock copolymers through polymerization-inducedself-assembly

Eri Yoshida

Received: 26 November 2013 /Accepted: 24 December 2013 /Published online: 14 January 2014# Springer-Verlag Berlin Heidelberg 2014

Abstract The morphology of giant vesicles composed ofamphiphilic poly(methacrylic acid)-block-poly(methylmethacrylate-random-methacrylic acid) random block copol-ymers, PMAA-b-P(MMA-r-MAA), was effectively con-trolled by manipulating the hydrophobic-hydrophilic balanceof the P(MMA-r-MAA) blocks through the self-assemblyinduced by the nitroxide-mediated photo-controlled/livingradical polymerization in an aqueous methanol solution. Themorphology was transformed from spherical vesicles intofibers and finally into membranes as the molar ratio of theMAA units in the hydrophobic P(MMA-r-MAA) block in-creased at a constant block length. The membrane morpholo-gy reverted to spherical vesicles by exchanging the MMAunits with more hydrophobic isopropyl methacrylate units at aconstant MAA ratio. These morphology transitions wereaccounted for by the change in the critical packing shape ofthe random block copolymers based on the variation in theextent of the hydrophobic block chains.

Keywords Morphology control . Giant vesicles .

Amphiphilic random block copolymers . Poly(methacrylicacid)-block-poly(methyl methacrylate-random-methacrylicacid) . Polymerization-induced self-assembly .

Nitroxide-mediated photo-controlled/living radicalpolymerization . Hydrophobic-hydrophilic balance .

Critical packing shape

Introduction

Morphology control of supramolecules formed by theself-assembly of amphiphiles is significant in understand-ing the geometric packing shapes and properties of theamphiphiles, equilibrium states during the self-assembly,kinetics of the morphology transformation, andinteraggregate interactions. These understandings for thecontrol of the morphology are useful in order to antici-pate the shapes of the aggregates consisting of the newlyprepared amphiphiles and to create supramolecules basedon the self-assembly of the designed amphiphilic mole-cules. The morphology of aggregates formed by the self-assembly of amphiphiles is, in general, determined bythe critical packing parameter, v/a0lc, for the geometricpacking properties of the amphiphiles, where v is thehydrocarbon chain volume, a0 is the optimal area of thehydrophilic head group of the amphiphile, and lc is thecritical length of the hydrocarbon chain [1]. The mor-phologies are transformed from spherical micelles (v/a0lc≤1/3) to nonspherical rod-like or cylindrical micelles(1/3<v/a0lc≤1/2) to vesicles or flexible bilayers (1/2<v/a0lc<1) to planar bilayers (v/a0lc≈1) and finally toinverted structures (v/a0lc>1).

Based on this critical packing parameter, studies onthe morphology control of the supramolecular aggre-gates have been made using small molecular amphi-philes based on the balance of hydrogen bonding, elec-trostatic, and hydrophobic interactions [2] and on theproportion of the amphiphiles composing the aggregates[3]. Amphiphilic block copolymers have been also uti-lized for the studies on the morphology control due topossessing broadly designable structures and providingvarious factors to control the morphology. The factors

E. Yoshida (*)Department of Environmental and Life Sciences, ToyohashiUniversity of Technology, 1-1 Hibarigaoka, Tempaku-cho,Toyohashi, Aichi 441-8580, Japane-mail: eyoshida@ens.tut.ac.jp

Colloid Polym Sci (2014) 292:763–769DOI 10.1007/s00396-013-3154-z

include the length of the blocks composing the amphi-philic copolymers [4–13], the molar ratio of the como-nomer units in the corona block [14, 15], water contentin the solution dissolving the copolymers [16, 17], saltconcentration [6, 18], pH [14], and rotating speed dur-ing the polymerization [18]. These factors wereemployed to control the morphologies of the aggregatesformed by the methods of film rehydration [7], self-assembly in a selective solvent for separately preparedblock copolymers [4–7, 16, 17], and polymerization-induced self-assembly using the reversible addition-fragmentation chain transfer polymerization [9–12, 14,15, 18] and thermal nitroxide-mediated polymerization[13]. This polymerization-induced self-assembly basedon these thermal polymerizations produced nanometer-sized aggregates.

In recent years, micrometer-sized giant vesicles have beenobtained by the polymerization-induced self-assembly usingthe nitroxide-mediated photo-controlled/living radical poly-merization (photo-NMP) in an aqueous methanol solu-tion for the amphiphilic random block copolymerconsisting of hydrophilic poly(methacrylic acid),PMAA, and hydrophobic poly(methyl methacrylate-

random-methacrylic acid), P(MMA-r-MAA) [19]. ThePMAA-b-P(MMA-r-MAA) random block copolymerproduced several-micrometer spherical vesicles of thebilayer structure with a hydrophilic surface of thePMAA blocks and an internal hydrophobic phase ofthe P(MMA-r-MAA) blocks. In this study, it was foundthat the morphology of the giant vesicles formed by therandom block copolymer was controlled by the MMA/MAA ratio of the P(MMA-r-MAA) block at a constantblock length. This short communication describes thenovel control of the morphology for the giant vesiclescomposed of the amphiphilic random block copolymersby manipulating the hydrophobic-hydrophilic balance ofthe random copolymer blocks.

Experimental

Instrumentation The photo-NMP-induced self-assembly wasperformed using an Ushio optical modulex BA-H502, anilluminator OPM2-502H with a high-illumination lens UI-OP2SL, and a 500-W super high-pressure UV lamp (USH-500SC2, Ushio Co. Ltd.). Proton nuclear magnetic resonance

Table 1 The random block copolymers prepared by the photo-NMP-induced self-assembly

OOH

211O

O

xO

OH

y

N

O

O

NH

N

PMAA-b-P(MMA-r-MAA)

Conversion (%)

MMAa MAAb

Molar ratioc

x y

DPd Mne Mw/Mne Morphologyf

85 64 0.883 0.117 349 50,100 1.85 V

87 77 0.782 0.218 362 69,700 1.66 V + F

87 78 0.701 0.299 358 98,100 1.71 MThe PMAA block: DP=211, Mn=18,490, and Mw/Mn=1.64 by GPC based on PMAA standards

V vesicle, F fiber, Mmembranea Estimated on the basis of the signal intensity of the methyl protons at 3.54–3.70 ppm for the resulting copolymer and at 3.74 ppm for the remainingunreacted MMA (Fig. 1a)b Estimated on the basis of the signal intensity of the α-methyl protons for the remaining unreacted monomers at 1.90 ppm for MAA and 1.92 ppm forMMA, coupled with the MMA conversioncMolar ratio of the P(MMAx-r-MAAy) blockdDP of the P(MMA-r-MAA) blocke Total molecular weight of the random block copolymers. The molecular weight and its distribution were estimated by GPC based on PMMA standardsf Determined by FE-SEM observations

764 Colloid Polym Sci (2014) 292:763–769

(1H NMR) measurements were conducted using Jeol ECS400and ECS500 FT NMR spectrometers. Gel permeation chro-matography (GPC) was performed using a Tosoh GPC-8020instrument equipped with a DP-8020 dual pump, a CO-8020column oven, and a RI-8020 refractometer. Two gelcolumns, Tosoh TSK-GEL α-M, were used with N,N-dimethylformamide (DMF) containing 30 mM LiBr and60 mMH3PO4 as the eluent at 40 °C. Field emission scanningelectron microscopy (FE-SEM) measurements were per-formed using a Hitachi SU8000 scanning electronmicroscope.

Materials 4-Methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl(MTEMPO) was prepared as reported previously [20].Methacrylic acid (MAA) was purified by distillation underreduced pressure. Methyl methacrylate (MMA) and isopro-pyl methacrylate (IPMA) were passed through a columnpacked with activated alumina to remove an inhibitor anddistilled over calcium hydride. The MMA and IPMA thuspurified were degassed with Ar for 15 min with stirringjust before use. Methanol (MeOH) was refluxed over

magnesium with a small amount of iodine and distilled.Distilled water and 2,2′-azobis[2-(2-imidazolin-2-yl)propane] (V-61) were purchased from Wako PureChemical Industries, Ltd. and used without further purifi-cation. (4-tert-Butylphenyl)diphenylsulfonium triflate (tBuS)was purchased from Sigma-Aldrich and used as received.Extrapure N2 gas with over 99.9995 vol% purity and Argas with over 99.999 vol% purity were purchased fromTaiyo Nippon Sanso Corporation.

Preparation of PMAA prepolymer: general procedure V-61(22.8 mg, 0.0911 mmol), MTEMPO (18.0 mg, 0.0966 mmol),tBuS (24.0 mg, 0.0512 mmol), MAA (2.030 g, 23.6 mmol),and MeOH (4 mL) were placed in a 30-mL test tube joined toa high-vacuum valve. The contents were degassed severaltimes using a freeze-pump-thaw cycle and then charged withN2. The polymerization was carried out at room temperaturefor 5.5 h with irradiation at 9.5 A by reflective light by using amirror with a 500-W high-pressure mercury lamp. MeOH(11 mL) and distilled water (5 mL) degassed by bubbling Arfor 15 min were added to the product under a flow of Ar. After

1.92.0 1.83.63.8 3.7

012345678910 ppm

a

CH3OHH2O

CHCl3

6.06.1

012345678910 ppm

b

CH3OHH2O

CHCl3

4.85.0 4.95.1

Fig. 1 aA 1H NMR spectrum ofPMAA211-b-P(MMA0.883-r-MAA0.117)349. Solvent: CD3OD-d4/CDCl3=3/1 (v/v). bA

1H NMRspectrum of PMAA212-b-P[(MMA0.696-r-IPMA0.304)0.7-r-MAA0.3]360. Solvent: CD3OD-d4/CDCl3=3/1 (v/v) in the presenceof CF3COOH

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the product was completely dissolved in the aqueous MeOHsolution, part of the mixture (ca. 1 mL) was withdrawn using asyringe to determine conversion and molecular weight of thePMAA prepolymer. The solution was poured into ether(50 mL) to precipitate a polymer. The precipitate was collect-ed by filtration and dried in vacuo for several hours to obtain apolymer (44.7 mg). The residual solution of the PMAAprepolymer was subjected to the preparation of the randomblock copolymer.

Synthes i s o f PMAA-b-P(MMA-r-MAA): genera lprocedure MMA (627.1 mg, 6.263 mmol), MAA (63.9 mg,0.7422 mmol), and the solution (4 mL) of the PMAAprepolymer (MAA conversion=74 %, Mn=18,490, andMw/Mn=1.64) containing unreacted MAA (1.240 mmol)were placed in a 30-mL test tube joined to a high-vacuumvalve under a flow of Ar. The initial molar ratio of themonomers wasMMA/MAA=0.760/0.240. The contents weredegassed several times using a freeze-pump-thaw cycle andcharged with N2. The polymerization was carried out at roomtemperature and 600 rpm for 10 h with irradiation at 9.1 A. Amixed solvent (MeOH/H2O=3/1 (v/v), 20 mL) was added tothe resulting dispersion to precipitate aggregates. Theaggregates were cleaned with the mixed solvent by arepeated sedimentation-redispersion process. Theresulting aggregates were stored in the presence of asmall amount of the mixed solvent.

Synthesis of PMAA-b-P(MMA-r-IPMA-r-MAA): generalprocedure MMA (168.5 mg, 1.683 mmol), IPMA(504.3 mg, 3.935 mmol), MAA (142.1 mg, 1.651 mmol),and the solution (4 mL) of the PMAA prepolymer (MAAconversion=78 %, Mn=18,530, and Mw/Mn=1.66) contain-ing unreactedMAA (1.019mmol) were placed in a 30-mL testtube joined to a high-vacuum valve under a flow of Ar. Theinitial molar ratio of the monomers was MMA/IPMA/MAA=0.203/0.475/0.322. The contents were degassedseveral times using a freeze-pump-thaw cycle andcharged with N2. The polymerization was carried outat room temperature and 600 rpm for 10 h with irradi-ation. A mixed solvent (MeOH/H2O=3/1 (v/v), 20 mL)was added to the resulting dispersion to precipitateaggregates. The aggregates were cleaned with the mixedsolvent by a repeated sedimentation-redispersion pro-cess. The resulting aggregates were stored in the pres-ence of a small amount of the mixed solvent.

SEM observations The aggregates obtained from the randomblock copolymers were dried in air and subjected to the FE-SEM measurements. The morphologies were determined bythe observation using FE-SEM at 1.0 kVwithout coating. Thesize distribution of the vesicles was estimated as reportedpreviously [21].

Results and discussion

The polymerization-induced self-assembly was performed bythe photo-NMP for the random block copolymerization ofMMAwith MAA using a PMAA prepolymer in an aqueousmethanol solution (MeOH/H2O=3/1v/v) at room temperatureby irradiation with a high-pressure mercury lamp. ThePMAA-b-P(MMA-r-MAA) random block copolymers ob-tained by this polymerization are listed in Table 1. The mono-mer conversions were determined by 1H NMR (Fig. 1). TheMMA/MAA ratios of the P(MMA-r-MAA) block were cal-culated on the basis of the conversions. The degree of poly-merization (DP) of the PMAA block was estimated using the

20 µm

a

b

c

Fig. 2 SEM images of the morphologies formed by the random blockcopolymers with the MMA/MAA ratio of a 0.883/0.117, b 0.782/0.218,and c 0.701/0.299 in the P(MMA-r-MAA) block

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molecular weights of the initiation radical generated fromV-61, the MTEMPO mediator, and the PMAA prepolymer.The molecular weight of the PMAA prepolymer was deter-mined by GPC based on the PMAA standards. The DP of theP(MMA-r-MAA) block was calculated using the monomerconversions and the concentration of the propagating chainequal to the initial concentration of MTEMPO based on theassumption that no deactivation of the propagating chain endoccurred during the polymerization. It was confirmed that thecopolymers contained no PMAA prepolymer based on theGPC analysis. The difference in the total molecular weight ofthe random block copolymers in spite of almost the same DPswas due to the GPC estimation based on the PMMA standardsfor the copolymers with the differentMMA/MAA ratios of therandom copolymer blocks.

The FE-SEM images of the morphologies formed by therandom block copolymers with the different MMA/MAAratios of the random copolymer block are shown inFig. 2. The copolymer with a 0.883/0.117 MMA/MAAratio produced spherical vesicles. The average diameterand the distribution of the vesicles were Dn=2.86 μmand Dw/Dn=2.88, respectively. As the MAA ratio in therandom copolymer block increased, the vesicular

morphology was changed into larger vesicles (Dn=5.77 μm, Dw/Dn=2.28) and fibers. The morphologywas finally transformed into bilayer membranes by afurther increase in the MAA ratio. The increase in theMAA ratio of the P(MMA-r-MAA) block reduced thehydrophobic interaction among the random copolymerblocks, causing the transition from spherical vesicles intomembranes.

The membrane morphology reverted to spherical vesiclesby partial exchange of theMMA units with more hydrophobicIPMA units at a constant MAA ratio of the random copolymerblock. The copolymers containing the IPMA units are listed inTable 2. The total molecular weight of the random blockcopolymers decreased with an increase in the IPMA ratiodespite almost the same DPs based on the GPC estimationusing PMMA standards. The morphologies formed by thecopolymers are shown in Fig. 3. The copolymer containingno IPMA units formed membranes with the average thicknessof 717 nm. The membranes became thinner with a 255-nmthickness by exchanging 30 mol% of the MMA units with theIPMA units.When the IPMA ratio reached ca. 50mol% of theMMA units, the thin membrane was transformed into spher-ical vesicles with Dn=3.65 μm and Dw/Dn=1.58. The

Table 2 The random block copolymers containing IPMA units

OOH

212O

O

pO

OH

0.3

N

O

O

NH

N

OO

q 0.7

PMAA-b-P(MMA-r-IPMA-r-MAA)

Conversion (%)

MMA IPMAa MAAb

Molar ratio

p q

DPc Mnd Mw/Mnd Morphologye

87 90 77 0.696 0.304 360 105,000 1.65 M

86 91 76 0.486 0.514 363 92,800 1.71 V

82 87 65 0.312 0.688 338 74,900 1.81 V

90 71 1.00 360 69,300 1.89 VThe PMAA block: DP=212, Mn=18,530, and Mw/Mn=1.66 by GPC based on PMAA standardsa Estimated by 1H NMR using the signal intensity of the methyne protons at 4.77–4.95 ppm for the resulting copolymer and at 5.00 ppm for theremaining unreacted IPMA (Fig 1b)b Estimated on the basis of the signal intensity of the vinyl protons at 6.04 ppm for IPMA and 6.07 ppm forMMA andMAA, coupled with the IPMA andMMA conversionsc DP of the P(MMA-r-IPMA-r-MAA) blockd Total molecular weight of the random block copolymers. The molecular weight and its distribution were estimated by GPC based on PMMA standardse Determined by FE-SEM observations

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observation of many collapsed vesicles with dents on thesurface implies that the vesicles were composed of thin andflexible membranes. The vesicles were further changed intosmaller spherical vesicles with Dn=1.17 μm and Dw/Dn=1.11 at a ca. 70 mol% IPMA ratio. Over a 70 mol% IPMAratio, almost no changes in the morphology and vesicular sizewere made with the exception of the observation of a smallamount of worm-like vesicles. The size of the vesicles wasDn=1.44 μm, and the distribution was Dw/Dn=1.12 for thecompleted exchange of the MMA units with the IPMA units.An increase in the more hydrophobic IPMA units increasedthe hydrophobic interaction among the random copolymerblocks, resulting in the transition of the morphology frommembranes into spherical vesicles.

The morphology transition from the spherical vesicles intomembranes by an increase in the hydrophilic units in therandom copolymer block and vice versa involves the variation

10 µm

a

b

c

d

e

Fig. 3 SEM images of themorphologies formed by therandom block copolymers withthe MMA/IPMA ratio of a1.00/0,b 0.696/0.304, c 0.486/0.514, d0.312/0.688, and e 0/1.00. TheMAA ratio in the randomcopolymer block was 0.3

Low HighMAA ratio

in P(MMA-r-MAA) block

MAA unitMMA unit

Fig. 4 The changes in the critical packing shape of the random blockcopolymers based on the variation in the extent of the hydrophobic blockchains

768 Colloid Polym Sci (2014) 292:763–769

in the critical packing shape of the random block copolymers[1]. The transition from the vesicles into membranes implies achange in the critical packing shape from a truncated cone intoa cylinder by an increase in the MAA units (Fig. 4). Byincreasing the hydrophilic units in the P(MMA-r-MAA)blocks, the hydrophobic random copolymer blocks becamemore stretched, causing expansion of the hydrophobic chainvolume, v. On the other hand, the reverse transition by theexchange of the MMA units with the more hydrophobicIPMA units indicated a reduction in v. The random copolymerblocks shrank by increasing the more hydrophobic units,resulting in a decrease in v.

Conclusion

The morphologies of the giant vesicles composed of theamphiphilic random block copolymers were well controlledby manipulating the hydrophobic-hydrophilic balance of thehydrophobic random copolymer block through the photo-NMP-induced self-assembly in an aqueous alcoholic solution.The morphologies of the copolymers were changed fromspherical vesicles into much larger vesicles, then into fibers,and finally into membranes with an increase in the ratio of thehydrophilic units of the random copolymer block at a constantblock length. The membrane morphology reverted to spheri-cal vesicles by introducing more hydrophobic units into therandom copolymer block. This morphology transition wasattributed to the changes in the critical packing shape of therandom block copolymers based on the variation in the extentof the hydrophobic block chains. An increase in the hydro-philic units increased the hydrophobic chain volume bystretching the random copolymer block chains, whereas anincrease in the highly hydrophobic units decreased the volumeby shrinking them. This study demonstrated that the morphol-ogy of the giant vesicles was effectively controlled bymanipulating the hydrophobic-hydrophilic balance of the

hydrophobic block chains and added this to a series of indicescontrolling the morphology of the supramolecules formed bythe molecular self-assembly.

Acknowledgments The author is thankful for a JSPS Grant-in-Aid forScientific Research (grant number 25390003).

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