SHORT COMMUNICATION

Giant vesicles prepared by nitroxide-mediatedphoto-controlled/living radical polymerization-inducedself-assembly

Eri Yoshida

Received: 25 July 2013 /Accepted: 13 August 2013 /Published online: 30 August 2013# Springer-Verlag Berlin Heidelberg 2013

Abstract Giant vesicles with several-micrometer diameterswere prepared by self-assembly induced by the nitroxide-mediated photo-controlled/living radical polymerization. Therandom block copolymerization of methyl methacrylate(MMA) and methacrylic acid (MAA) were performed usingpoly(methacrylic acid) (PMAA) as the prepolymer in anaqueous methanol solution to produce a PMAA-block -poly(MMA-random-MAA) random block copolymer (PMAA-b -P(MMA-r-MAA)). PMAA195-b-P(MMA0.817-r-MAA0.183)224formed spherical vesicles with a 4.74 μm diameter and0.108 μm wall thickness. A differential scanning calorimetryanalysis demonstrated that the vesicles had a bilayer structureconsisting of a hydrophilic PMAA surface and hydrophobicP(MMA-r-MAA) interface. The wet vesicles before air-dryingwere flexible and easily transformed by stress, whereas the dryvesicles were fragile and cracked. The vesicles in the solutionwere dissociated into much smaller vesicles by increasing thetemperature. They were also transformed by a further tempera-ture increase into hollow fibers and finally into membranesretaining the bilayer structure.

Keywords Giant vesicles . Polymerization-inducedself-assembly . Nitroxide-mediated photo-controlled/livingradical polymerization . Random block copolymer .

PMAA-block -poly(MMA-random-MAA) .Morphologytransition . Thermal stability . 4-Methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl

Introduction

Giant vesicles are micrometer-sized supramolecules with aclosed bilayer structure formed by self-assembling amphi-philes. The giant vesicles are plausible artificial models forbiomembranes composed of cells and organelles, such aserythrocytes, platelets, and mitochondria due to the similarityof their size and construction. In recent years, the importanceof the giant vesicles has been increasing for industrial appli-cations as microcapsules in drug and gene delivery systems,microreactors, and selective membranes [1–6]. A number ofstudies about the giant vesicles have been made using small-molecule natural and synthetic surfactants, like phosphatidyl-choline and cholesterol, for the purpose of preparing thevesicles and elucidating their formation mechanisms. Exam-ples of the preparations include electroformation [7–10], dial-ysis of lipid dispersion [11], hydrolysis of preamphiphiles[12–15], lipid-coated ice droplet hydration [16], and lipidswelling [17]. The studies also involved the thermal transitionof the vesicle morphology [18–20], the balance of hydrogen-bonding, electrostatic, and hydrophobic interactions in theself-assembly [21], and the self-reproducing system throughdehydrocondensation [22].

While small molecular amphiphiles have been utilized inthe vesicle studies, macromolecular amphiphiles of amphi-philic block copolymers have received scientific attentionwith regard to producing giant elastic vesicles different fromthose of the small molecular amphiphilies based onmembranetoughness and permeability [23–25]. Some block copolymergiant vesicles were prepared by methods similar to those usedfor the small molecular amphiphile vesicles. Discher andcoworkers prepared giant vesicles by electroformation usingpolyethyleneoxide (PEO)-block -polyethylethylene and PEO-block -polybutadine [25]. Eizenberg obtained the vesicles by

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 (2013) 291:2733–2739DOI 10.1007/s00396-013-3056-0

dialysis for a solution of polystyrene-block -poly(acrylic acid)[26], while Feijen also prepared them by dialysis of a disper-sion containing PEO-block -poly(DL-lactide) or PEO-block -poly(ε-caprolactone) [27]. Howse provided a templated for-mation based on the microphase separation by hydrationfollowed by dewetting of PEO-block -poly(butyleneoxide)[28].

In this report, a novel formation of giant vesicles ispresented using the nitroxide-mediated photo-controlled/liv-ing radical polymerization (photo-NMP)-induced self-assembly. A random block copolymer consisting of hydro-philic poly(methacrylic acid) (PMAA) and hydrophobicpoly(methyl methacrylate-random -methacrylic acid),P(MMA-r -MAA), was employed as an amphiphile. It hasbeen reported that the photo-NMP proceeds at room tem-perature by UV irradiation to produce a comparatively nar-row molecular weight distribution (Mw/Mn≈1.4) for methac-rylate monomers [29–41]. There are many publicationsabout the polymerization-induced self-assembly to ob-tain aggregates including vesicles, mainly using thenitroxide-mediated thermal controlled/living radical po-lymerization (thermal-NMP) [42, 43] and reversibleaddition-fragmentation chain transfer polymerization[44–49]. However, the self-assembly induced by thesepolymerizations produced nanometer-sized vesicles rath-er than micrometer-sized giant vesicles. This short com-munication describes the preparation of giant vesicles bythe photo-NMP-induced self-assembly of the randomblock copolymer amphiphile.

Experimental

Instrumentation

The photo-NMP was carried out using an Ushio opticalmodulex BA-H502, an illuminator OPM2-502H with ahigh-illumination lens UI-OP2SL, and a 500 W superhigh-pressure UV lamp (USH-500SC2, Ushio Co. Ltd.).1H NMR measurements were conducted using a JeolECS400 FT NMR spectrometer. Gel permeation chroma-tography (GPC) was performed using a Tosoh GPC-8020instrument equipped with a DP-8020 dual pump, a CO-8020 column oven, and a RI-8020 refractometer. Two gelcolumns, Tosoh TSK-GEL α-M were used with N ,N -dimethylformamide containing 30 mM LiBr and 60 mMH3PO4 as the eluent at 40 °C. Field emission scanningelectron microscopy (FE-SEM) measurements wereperformed using a Hitachi SU8000 scanning electron mi-croscope. Differential scanning calorimetry (DSC) wascarried out using a Shimadzu DSC-60 instrument equippedwith a TA-60WS system controller and a FC-60 nitrogenflow controller.

Materials

4-Methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl (MTEMPO)was prepared as reported previously [50]. MAAwas purifiedby distillation under reduced pressure. Methyl methacrylate(MMA) was passed through a column packed with activatedalumina to remove an inhibitor and distilled over calciumhydride. Methanol (MeOH) was refluxed over magnesiumwith a small amount of iodine and distilled. Acetonitrile wasdistilled over calciumhydride. 2,2′-Azobis[2-(2-imidazolin-2-yl)propane] (V-61) and distilled water were purchased from WakoPure Chemical 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 Ar gaswith over 99.999 vol% purity were purchased from TaiyoNippon Sanso Corporation.

PMAA prepolymer

MTEMPO (18.0 mg, 0.0966 mmol), V-61 (22.8 mg,0.0911 mmol), tBuS (24.0 mg, 0.0512 mmol), MAA (2.030 g,23.6 mmol), andMeOH (4mL) were placed in a test tube joinedto a high vacuum valve. The contents were degassed severaltimes using a freeze–pump–thaw cycle and charged with N2.The polymerization was carried out at room temperature for 5 h20 min with irradiation by reflective light using a mirror with a500 W high-pressure mercury lamp at 9.4 ampere. MeOH(11 mL) and distilled water (5 mL) degassed by bubbling Arfor 15 min were added to the product under a flow of Ar. Afterthe product was completely dissolved in the aqueous MeOHsolution, part of the mixture (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 collected by filtra-tion and dried in vacuo for several hours to obtain a polymer(72.5 mg).

Random block copolymer

MMA (1.03 g, 10.3 mmol) degassed by bubbling Ar for15 min and the aqueous MeOH solution (10 mL) containingthe PMAA prepolymer and unreacted MAA (2.865 mmol)were placed in a test tube joined to a high vacuum valve. Thecontents were degassed several times using a freeze–pump–thaw cycle and charged with N2. The polymerization wascarried out at room temperature and 600 rpm for 14 h withirradiation. A mixed solvent (MeOH/H2O=3/1 (v /v ), 40 mL)was added to the resulting dispersion to precipitate aggregates.The aggregates were cleaned with the mixed solvent by arepeated sedimentation–redispersion process. The resultingaggregates were stored in the presence of a small amount ofthe mixed solvent.

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Random copolymer

MTEMPO(4.5mg, 0.0242mmol),V-61 (5.7mg, 0.0228mmol),tBuS (6.0 mg, 0.0128mmol), MMA (1.87 g, 18.7 mmol), MAA(0.457 g, 5.31 mmol), MeOH (0.5 mL), and acetonitrile (1 mL)were placed in a test tube joined to a high vacuum valve. Thecontents were degassed several times using a freeze–pump–thawcycle and charged with N2. The polymerization was carried outat room temperature for 24 h with irradiation. Dichloromethane(8 mL) and MeOH (1 mL) were added to dissolve the viscousproduct. The solutionwas poured into petroleum ether (500mL).The precipitate was collected by filtration and dried to obtain apolymer (2.313 g).

SEM observations

The aggregates of the random block copolymer were dried in airand subjected o the FE-SEM measurements. The morphologyof the giant vesicles was determined using FE-SEM at 1.0 kVwithout coating. The size distribution of the vesicles was esti-mated as reported previously [51].

Results and discussion

In order to prepare the PMAA prepolymer for obtaining ablock copolymer amphiphile, the photo-NMP of MAA wasperformed in MeOH using V-61 as the initiator, MTEMPO asthe mediator, and tBuS as the accelerator. The polymerizationwas carried out for 5 h 20 min at room temperature by UVirradiation to produce PMAA at a 76 % conversion. Theconversion was determined by 1H NMR based on the signalintensity of the methyl and methylene protons at 0.9–2.2 ppmfor the resulting polymer and themethyl protons at 1.90 ppm forthe remaining unreacted monomer [52]. The molecular weight(Mn) andmolecular weight distribution (Mw/Mn) of the PMAAwere estimated to be Mn=17,100 and Mw/Mn=1.79 by GPCbased on PMAA standards. Based on the GPC analysis, thedegree of polymerization (DP) for the PMAA was calculatedto be DP=195.

The PMAA prepolymer solution containing the remainingunreacted MAA monomer was used for the block copolymer-ization with MMA. The block copolymerization wasperformed at 600 rpm for 14 h in an aqueous MeOH solutionat MeOH/H2O=3/1 (v /v). The colorless solution containingMMA and the remaining MAA at the initial molar ratio ofMMA/MAA=0.782/0.218 turned cloudy within 2 h and then awhite dispersion formed after the 14-h polymerization. TheMMA conversion by the block copolymerization was 86 %based on the 1H NMR using the signal intensity of the methylprotons at 3.54–3.70 ppm for the resulting copolymer and at3.74 ppm for the remaining unreacted MMA. The MAA con-version was estimated to be 69 % based on the signal intensity

of the α-methyl protons at 1.90 ppm for MAA and 1.92 ppmfor MMA, coupled with the MMA conversion. This estimationof the conversions suggests the formation of a random blockcopolymer, PMAA-b -P(MMA-r -MAA). The MMA/MAAmolar ratio for the P(MMA-r-MAA) random copolymer blockwas 0.817/0.183 based on the respective conversions. TheMAA ratio of the random copolymer block was slightly lowerthan the initial ratio of the solution due to its low reactivityduring the copolymerization with MMA [53, 54]. It wasassumed that no deactivation of the PMAA prepolymeroccurred during the block copolymerization, and the DP ofthe random copolymer block was determined as DPtotal=224(DPPMMA=183 and DPPMAA=41) based on the consumedmonomer concentrations and the propagating chain concen-tration equal to the initial concentration of MTEMPO,resulting in the formation of the random block copolymer,PMAA195-b-P(MMA0.817-r-MAA0.183)224.

GPC analysis demonstrated that the giant vesicles includedno PMAA prepolymer. The GPC profiles of the random blockcopolymer and the prepolymer are shown in Fig. 1. Themolecular weight and its distribution of the random blockcopolymer were Mn=50,500 and Mw/Mn=1.70, respectively,by GPC based on PMMA standards. The theoretical molecularweight of the random copolymer block was Mn=21,800 basedon themonomer conversions. Hence, the total molecular weightof the block copolymer was calculated to be Mn=50,000 usingthe theoretical molecular weight of the random copolymerblock and the molecular weight of the PMAA prepolymer,Mn=28,200 estimated by GPC using PMMA standards. Thesevalues are in good agreement with molecular weight of theblock copolymer between the experimental and the theoreticalvalues.

15 20Retention time (min)

PrepolymerRandom block copolymer

Fig. 1 GPC profiles of the PMAA-b-P(MMA-r-MAA) random blockcopolymer and the PMAA prepolymer

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a

e

2 µm

10 µm

10 µm

10 µm

10 µm

d

c

b

Fig. 2 SEM images of the giantvesicles (a), the magnification(b), the cracked vesicle (c), thecollapsed vesicles (d), and thevesicle with a more complicatedinside structure (e)

100 200

Temperature (ºC)

3.0

2.0

1.0

End

othe

rm (m

W)

PMAA

First scanSecond scan

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

P(MMA-r-MAA)

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

Fig. 3 DSC spectra of the giantvesicles, the PMAA prepolymer,and a P(MMA-r-MAA)random copolymer(MMA/MAA=0.759/0.241,Mn=94,800, Mw/Mn=2.31)

2736 Colloid Polym Sci (2013) 291:2733–2739

The aggregates produced by the block copolymerizationwere isolated and purified as white precipitates by arepeated sedimentation–redispersion process. The FE-SEMobservations of the precipitates revealed that the blockcopolymerization-induced self-assembly produced giantspherical vesicles (Fig. 2a). The vesicle size and its distributionwere Dn=4.74 μm and Dw/Dn=1.31, respectively. Dents andvery small holes were observed on the surface of the vesiclesunder magnification (Fig. 2b). The hollows observed throughthese very small holes and the crack in the particles suggestedthe formation of vesicles. The average wall thickness of thevesicles was estimated to be 0.108 μm based on the crack(Fig. 2c). The fully extended molecular length of the chainslmax of PMAA195-b -P(MMA0.817-r -MAA0.183)224 was

0.106 μm based on the DPtotal of the block copolymer [55].The wall thickness based on the lmax was 0.212 μm. It issuggested that the random copolymer block chains had shrunkin the vesicle bilayer. The PMAA chains should have alsoshrunk due to the air-drying process and cover the bilayer ofthe random copolymer blocks. On the other hand, the wetvesicles before air-drying were flexible because the collapsedvesicles were obtained without cracks by pushing the wet pre-cipitates using a spatula (Fig. 2d). In addition, a few vesicles withmore complicated inside structures were also observed (Fig. 2d).

The DSC analysis demonstrated that the giant vesicleshad a surface consisting of the PMAA blocks and interfaceof the random copolymer blocks. The DSC spectra of thegiant vesicles, coupled with the PMAA prepolymer and a

30 µm

30 µm

30 µm

3 µm

20 µm

30 µm

15 µm30 µm

30 µm

45°C

25°C

30°C

35°C

40°C

45°C

40°C

50°C

Fig. 4 The morphologytransition of the giant vesiclesby increasing the temperature

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P(MMA-r-MAA) random copolymer are shown in Fig. 3.The giant vesicles had a glass transition temperature (Tg) at164.5 °C, close to that of the PMAA (Tg=162.7 °C), and noobservation was made for the Tg based on the random copoly-mer during the first scanning. The Tg shifted to 137.9 °C duringthe second scanning, and was based on the random copolymer(Tg=139.0 °C). This Tg shift indicates that the giant vesicleswith the PMAA block chains gathering on the surface weremelted and dissociated by annealing. During the second andsubsequent scanning, no observation was made of the Tg for thePMAA blocks due to the fact that the random copolymer blocksprevented the PMAA blocks from crystallizing.

The thermal stability of the giant vesicles in the aqueousMeOHwas also investigated. Figure 4 shows the morphologytransition of the giant vesicles by increasing the temperature.The giant vesicles were dissociated at 35 °C into much smallervesicles and were partly fused. The fused vesicles formedmembranes and hollow fibers at 40 °C based on the observa-tion of the very small holes on the surface.Most of the vesicleswere transformed into membranes at 45 °C, although a smallnumber of vesicles were still observed. Some vesicles had abowl-like or ring-like structure, suggesting that the formationof membranes resulted not only from the fusion of the vesicles,but also from the expansion of the very small holes on thesurface due to an increase in the internal pressure at the hollows.The transformation into membranes was completed and novesicles were observed at 50 °C. The confirmation of the vesiclefragments and very small holes on the membrane surface sup-ports the fact that the membranes have a bilayer structure.

Conclusion

The photo-NMP-induced self-assembly produced micrometer-sized giant vesicles through the random block copolymerizationof MMA and MAA using the PMAA prepolymer. The giantvesicles of PMAA-b-P(MMA-r-MAA) had a bilayer structureconsisting of a hydrophilic PMAA surface and hydrophobicP(MMA-r-MAA) interface. The dry vesicles were fragile andeasily cracked, whereas the wet vesicles were flexible andtransformed by stress. The vesicles in solution were dissociatedinto much smaller vesicles by increasing the temperature andwere fused into hollow fibers, followed by the formation ofmembranes by increasing the temperature. This is the first studydemonstrating that the polymerization-induced self-assemblyproduced giant vesicles from the block copolymer amphiphileand that the vesicles were transformed into hollow fibers andthen membranes, retaining the bilayer structure by increasingthe temperature.

Acknowledgments The author is thankful for a JSPS Grant-in-Aid forScientific Research (Grant Number 25390003) and for a Shiseido FemaleResearcher Science Grant.

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