Controlled Synthesis of Polymeric Nanocapsules by RAFT-Based

Mar 25, 2010 - E-mail: [email protected] (J.P.A.H.); [email protected] (A.M.v.H.). .... Polymeric Hollow Particles for Encapsulation of Chemical Mole...
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Controlled Synthesis of Polymeric Nanocapsules by RAFT-Based Vesicle Templating Syed Imran Ali, Johan P. A. Heuts,* and Alex M. van Herk* Laboratory of Polymer Chemistry, Eindhoven University of Technology, The Netherlands Received December 15, 2009. Revised Manuscript Received February 22, 2010 Polymeric nanocapsules were synthesized by encapsulating extruded vesicles of dimethyldioctadecyl ammonium bromide (DODAB) using a reversible addition-fragmentation chain transfer (RAFT)-based encapsulation approach. Random copolymers containing acrylic acid and butyl acrylate units were first synthesized by RAFT in solution using dibenzyl trithiocarbonate (DBTTC) as the RAFT agent. These anionic copolymer chains were subsequently adsorbed onto the surface of cationic DODAB vesicles and then chain extended to form a polymeric shell by starved feed emulsion polymerization. Cryogenic transmission electron microscopy (cryo-TEM) characterizations demonstrate the successful formation of nanocapsules.

Introduction Polymeric nanocapsules are an increasingly important class of materials owing to their vast and rapidly growing fields of application ranging from materials science to nanoscale drug release and targeting systems in the biomedical field. Research toward their design and synthesis has resulted in a variety of methods to afford nanocapsules of various size ranges and compositions for different application areas.1,2 Conventional emulsion/miniemulsion approaches such as osmotic swelling,3 hydrocarbon encapsulation,4 and interfacial polymerization5 are promising and have been successful to varying degrees. Nanocapsules obtained by these approaches, however, have found limited applications and commercialization, mainly because of the associated disadvantages of these techniques. For instance, use of relatively harsh processing conditions and the presence of radicals renders the procedure unsuitable for the encapsulation of sensitive substances, and the incorporation of these substances after the formation of nanocapsules is often more difficult.1 Besides, control over the morphology, size, and wall thickness is often also relatively poor. After the emergence of reversible addition-fragmentation chain transfer (RAFT) polymerization in dispersed media,6 the *To whom correspondence should be addressed. E-mail: j.p.a.heuts@tue. nl (J.P.A.H.); [email protected] (A.M.v.H.). (1) McDonald, C. J.; Devon, M. J. Hollow latex particles: synthesis and applications. Adv. Colloid Interface Sci. 2002, 99, 181-213. (2) Xiong Wen, L.; Lynden, A. A.; Zichao, Y. Hollow micro-/nanostructures: Synthesis and applications. Adv. Mater. 2008, 20, 3987-4019. (3) Okubo, M.; Ito, A.; Hashiba, A. Production of submicron-sized multihollow polymer particles having high transition temperatures by the stepwise alkali/acid method. Colloid Polym. Sci. 1996, 274, 428-432. (4) McDonald, C. J.; Bouck, K. J.; Chaput, A. B.; Stevens, C. J. Emulsion polymerization of voided particles by encapsulation of a nonsolvent. Macromolecules 2000, 33, 1593-1605. (5) Crespy, D.; Stark, M.; Hoffmann-Richter, C.; Ziener, U.; Landfester, K. Polymeric nanoreactors for hydrophilic reagents synthesized by interfacial polycondensation on miniemulsion droplets. Macromolecules 2007, 40, 3122-3135. (6) Qiu, J.; Charleux, B.; Matyjaszewski, K. Controlled/living radical polymerization in aqueous media: homogeneous and heterogeneous systems. Prog. Polym. Sci. 2001, 26, 2083-2134. (7) van Zyl, A. J. P.; Bosch, R. F. P.; McLeary, J. B.; Sanderson, R. D.; Klumperman, B. Synthesis of styrene based liquid-filled polymeric nanocapsules by the use of RAFT-mediated polymerization in miniemulsion. Polymer 2005, 46, 3607-3615. (8) Luo, Y.; Gu, H. A General strategy for nano-encapsulation via interfacially confined living/controlled radical miniemulsion polymerization. Macromol. Rapid Commun. 2006, 27, 21-25.

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technique has also been tried for the synthesis of controlled morphologies such as hollow capsules.7,8 The technique offers an inherent advantage of control over molecular weight and the structure/composition of the polymer. Nanocapsules containing a polystyrene shell and an isooctane core were synthesized by interfacially confined RAFT-mediated miniemulsion polymerization.7 The approach uses a water-soluble initiator to provide surface activity to the (entering) radicals to anchor at the oil/water interface. The approach, although successful in synthesizing nanocapsules, was complicated and the synthesis of nanocapsules was only possible by controlling the polymerization kinetics, surface effects, and the use of a non-rate-retarding RAFT agent. Luo and Gu used a RAFT-based styrene maleic anhydride copolymer as a reactive surfactant to synthesize liquid filled nanocapsules.8 The copolymer block provides the anchoring to confine the RAFT-mediated polymerization to the interface, resulting in the synthesis of nanocapsules. An alternate route for the synthesis of hollow capsules is colloidal templating. In colloidal templating, the layer-by-layer approach which involves sequential deposition of polymer layers on a colloidal substrate, usually by electrostatic interactions, has been widely used to form polymer capsules with tailored properties for biomedical applications.9-13 Despite its simplicity and versatility, the approach suffers from some major shortcomings. For instance, to get hollow capsules of reasonable shell thickness, several layers need to be deposited, making the whole procedure tedious.12 As the polyelectrolyte layers are adsorbed by physical interactions, such hollow capsules are less stable and subsequent cross-linking is often needed to make physically more robust (9) Gleb, B. S.; Edwin, D.; Sean, D.; Heinz, L.; Frank, C.; Victor, I. P.; Helmuth, M. Stepwise polyelectrolyte assembly on particle surfaces: a novel approach to colloid design. Polym. Adv. Technol. 1998, 9, 759-767. (10) Edwin, D.; Gleb, B. S.; Frank, C.; Sean, A. D.; Helmuth, M. Novel hollow polymer shells by colloid-templated assembly of polyelectrolytes. Angew. Chem., Int. Ed. 1998, 37, 2201-2205. (11) Johnston, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F. Layer-by-layer engineered capsules and their applications. Curr. Opin. Colloid Interface Sci. 2006, 11, 203-209. (12) Germain, M.; Grube, S.; Carriere, V.; Richard-Foy, H.; Winterhalter, M.; Fournier, D. Composite nanocapsules: Lipid vesicles covered with several layers of crosslinked polyelectrolytes. Adv. Mater. 2006, 18, 2868-2871. (13) Fukui, Y.; Fujimoto, K. The preparation of sugar polymer-coated nanocapsules by the layer-by-layer deposition on the liposome. Langmuir 2009, 25, 10020-10025.

Published on Web 03/25/2010

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Scheme 1. Schematic Representation of the Synthesis of Vesicle-Templated Nanocapsules by Aqueous Starved Feed Emulsion Polymerization Using RAFT Copolymers as Stabilizers

nanocapsules for certain applications.12,14 Moreover, as the approach is based on material deposition on a decomposable colloidal template, a separate step is often required involving harsh chemical/ heat treatment to remove the template core which can sometimes induce rupture of the capsule.15 Use of “soft templates” such as emulsion droplets, biological cells, and surfactant vesicles is rapidly gaining interest, as it eliminates the need to remove the so-called sacrificial template16 and allows the pre-encapsulation of different substances before the formation of the nanocapsule. As soft templates, vesicles are very actively explored because of their unique morphology, easy incorporation of a variety of substances, and ease of preparation with controllable sizes.17-19 They form by the self-assembly of some natural lipids, double tailed synthetic surfactants and even from a mixture of different conventional single tailed surfactants. Vesicles are composed of spherically closed bilayer structures surrounding an aqueous core domain.20,21 They have been widely explored in drug delivery applications because of their intrinsic biocompatibility.22 Vesicles have been explored successfully as soft templates in the layer-by-layer approach.12,13 Here they offer many advantages over the conventional substrates, but, as described earlier, the process is tedious. In this paper, we report a RAFT-based approach23,24 to synthesize polymeric nanocapsules via vesicle templating. The approach is based on the adsorption of polyelectrolytes on vesicles. These small polyelectrolytes are synthesized by the RAFT process and have the advantage of carrying “living” (14) Pastoriza-Santos, I.; Sch€oler, B.; Caruso, F. Core-shell colloids and hollow polyelectrolyte capsules based on diazoresins. Adv. Funct. Mater. 2001, 11, 122-128. (15) Frank, C. Hollow capsule processing through colloidal templating and selfassembly. Chem.;Eur. J. 2000, 6, 413-419. (16) Fujimoto, K.; Toyoda, T.; Fukui, Y. Preparation of bionanocapsules by the layer-by-layer deposition of polypeptides onto a liposome. Macromolecules 2007, 40, 5122-5128. (17) Hubert, D. H. W.; Jung, M.; German, A. L. Vesicle templating. Adv. Mater. 2000, 12, 1291-1294. (18) Meier, W. Polymer nanocapsules. Chem. Soc. Rev. 2000, 29, 295-303. (19) Gomes, J.; Ruysschaert, T.; Germain, M.; Lindemann, M.; Fournier, D.; Winterhalter, M. Liposome based nanocapsules. Biophys. J. 2004, 86, 163A-163A. (20) New, R. R. C. Liposomes: a practical approach; IRL Press: Oxford, 1994; p 301. (21) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. Theory of self-assembly of hydrocarbon amphiphiles into micelles and bilayers. J. Chem. Soc., Faraday Trans. II 1976, 72, 1525-1568. (22) Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993; p 580. (23) Nguyen, D.; Zondanos, H. S.; Farrugia, J. M.; Serelis, A. K.; Such, C. H.; Hawkett, B. S. Pigment encapsulation by emulsion polymerization using macroRAFT copolymers. Langmuir 2008, 24, 2140-2150. (24) Ali, S. I.; Heuts, J. P. A.; Hawkett, B. S.; van Herk, A. M. Polymer encapsulated gibbsite nanoparticles: efficient preparation of anisotropic composite latex particles by RAFT-based starved feed emulsion polymerization. Langmuir 2009, 25, 10523-10533.

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RAFT moieties, allowing their further extension at the vesicle surface with desired monomers added at controlled rates leading to robust nanocapsules. This avoids the need to build several layers of differently charged copolymers to get reasonably thick polymer shells. Following the same approach as we recently used to encapsulate gibbsite platelets,24 we prepared short-chain anionic polyelectrolytes comprising randomly distributed butyl acrylate and acrylic acid units using the RAFT agent dibenzyl trithiocarbonate (DBTTC), choosing the most promising RAFT copolymer compositions we found in the previous study.24 These living polyelectrolyte chains were then adsorbed on the surface of cationic vesicles of dimethyldioctadecyl ammonium bromide (DODAB) and chain extended to get a polymer shell around the vesicles, leading to nanocapsules (Scheme 1). As will be shown in this paper, cryogenic transmission electron microscopy (cryoTEM) characterization of the resulting products reveals the successful synthesis of polymeric hollow capsules.

Experimental Section Materials. Dimethyldioctadecyl ammonium bromide (DODAB, Acros, >99%) was used as received. Monomers butyl acrylate (BA, Aldrich, 99%), methyl methacrylate (MMA, Aldrich, 99%), and acrylic acid (AA, Fluka, 99%) were distilled under reduced pressure prior to use. The water-soluble azo initiator 4,40 -azobis(4-cyanovaleric acid) (V-501, Fluka, 98%) and the nonionic surfactant Triton X-100 (TX-100, Aldrich, 99%) were used as received. N,N-Dimethylformamide (DMF, Biosolve), tetrahydrofuran (THF, Biosolve), ethanol (Biosolve), and methanol (Biosolve) were all used as received. Vesicle Preparation. Large unilamellar vesicles (LUVs) were prepared by a membrane extrusion method.25 Briefly, a preheated dispersion of 10 mM DODAB in Super-Q (Millipore) water was passed through three-stacked 200 nm polycarbonate filters (Millipore, hydrophilized PC filters) at 60 °C in four to five passes (nitrogen pressure 7 bar). In order to obtain smaller vesicles, part of the resulting dispersion was further passed through three-stacked 100 nm polycarbonate filters (Millipore, hydrophilized PC filters) at 60 °C in four to five passes. After extrusion, the vesicle dispersion was kept overnight at 60 °C before slowly cooling down to room temperature. Synthesis of RAFT Copolymers. Three RAFT copolymers, BA6-co-AA9, BA3-co-AA10, and BA6-co-AA4, containing different combinations of randomly distributed AA and BA units were synthesized and characterized as per a previously reported procedure.24 Table 1 lists their structural composition and properties, and a general synthesis scheme is depicted in Scheme 2. (25) Shingles, R.; McCarty, R. E. Production of membrane vesicles by extrusion: size distribution, enzyme activity, and orientation of plasma membrane and chloroplast inner-envelope membrane vesicles. Anal. Biochem. 1995, 229, 92-98.

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Table 2. Recipes for Nanocapsule Synthesisa

Table 1. Characterization of RAFT Copolymers NMR

GPCc

BAx-co-AAy

entry entry

DPna

FAAb

x

y

Mn,NMR (g/mol)

Mn,GPC (g/mol)

PDI

1 2 3

15 0.6 6 9 1700 1459 1.2 12 0.8 3 10 1400 1052 1.2 10 0.4 6 4 1350 1412 1.3 a Number-average degree of polymerization. b Mole fraction of AA in copolymer, measured by 1H NMR. c Values against PS standards.

Adsorption Studies. In eight different vials, calculated amounts of RAFT copolymer were transferred from a 10 mM aqueous stock solution and the volume was made up to 1 mL by adding water (Super-Q). Equal volumes of vesicle dispersion (10 mM) were then added dropwise into these vials under stirring. The pH of the dispersion was around 7. Zeta potential and particle size measurements were performed on these samples. Synthesis of Nanocapsules. Hollow latex particles were synthesized by starved feed emulsion polymerization performed in a 50 mL three-neck flask equipped with a magnetic stirrer bar and a heating bath. The recipes used for the experiments are summarized in Table 2. Briefly, the required amount of water (Super-Q) and RAFT copolymer (from a 10 mM stock solution) were transferred into the flask, and an equal volume of vesicle dispersion was then added dropwise under constant stirring at room temperature. The required amount of initiator V-501 was then added, and the dispersion was flushed with argon for 30 min. The reactor was subsequently heated to 70 °C using an oil bath followed by the addition of 0.2 g of deoxygenated monomer mixture (MMA/BA) at a rate of 0.01 g/min using a Dosimat autotitrator. After the completion of the monomer addition, the reactor was kept stirring at 70 °C for another 2 h. At regular intervals during the polymerization process, samples were collected for molecular weight and particle size determination. Surfactant Lysis Experiments. In order to probe the successful formation of nanocapsules, the stability of vesicles and vesicle-templated nanocapsules against surfactant lysis was investigated. An amount of 2 mL of vesicle or nanocapsule dispersion was (diluted appropriately to reduce optical density) added to a screw-capped quartz cuvette (1 cm optical path length) and heated and equilibrated at 50 °C. Repeated injections of 10 μL aliquots of a 200 mM Triton X-100 solution were added to the sample while stirring inside the cuvette. The optical density (as absorbance at 400 nm) was recorded at 2 min after every injection. Characterization. Size exclusion chromatography was performed using a Waters GPC equipped with a Waters model 510 pump and a model 410 differential refractometer. A set of two mixed bed columns (Mixed-C, Polymer Laboratories, 30 cm, 40 °C) was used. N,N-Dimethylformamide (with 5% LiBr) was used as the eluent, and the system was calibrated using narrow molecular weight polystyrene standards (range = 580-7 500 000 g/mol). The gel-to-liquid-crystalline phase transition temperature (Tm) of DODAB vesicles was determined spectrophotometrically from the dependence of the absorbance on temperature. Measurements were recorded using a Hewlett-Packard Photodiode Array UV spectrophotometer equipped with a Peltier heater/cooler. Samples were measured in a screw-capped quartz cuvette (1 cm optical path length) equipped with a magnetic stirring bar and an immersed thermocouple. Absorbance (at 400 nm) values were 7850 DOI: 10.1021/la904709c

RAFT copolymer

V-501 (mg)

DDI water (g)

Feed composition, MMA:BAb

1 2 3 4 5

BA6-co-AA9 7.0 8.0 10:1 6.7 8.2 10:1 BA3-co-AA10 15.2 2.2 10:1 BA6-co-AA4 7.0 8.0 10:0 BA6-co-AA9 7.0 8.0 7:3 BA6-co-AA9 a For all experiments: [RAFT copolymer]/[DODAB] = 3, 0.23 g of monomer (fed at a rate of 0.01 g/min). [RAFT]: [V-501] = 2 was used. Polymerization was carried out at a pH of around 7. DODAB vesicles (obtained by extrusion through 100 nm PC filters) of around 130 nm diameter (measured by DLS) were used. b Feed ratios in w/w.

recorded in the temperature range of 20-60 °C with 0.5 °C increments and allowing a thermal equilibration time of 2 min between measurements. X-ray diffraction (XRD) was measured using a Rigaku diffractometer operated at 40 kV and 35 mA. The XRD spectra were recorded in the range of 1° < 2θ < 10° using a step size of 0.02°/ point, a scan rate of 0.24° min-1 and employing Cu-KR1 radiation (1.54 A˚). Nanocapsules were separated from free polymer particles by centrifugation and were analyzed as dry powder. The particle size distribution and zeta potential (ζ) were determined at 25 °C by dynamic light scattering (DLS) using a Malvern Zetasizer Nano ZS instrument. The ζ potential was calculated from the electrophoretic mobility (μ) using the Smoluchowski relationship, ζ = ημ/ε, where κa . 1 (where η is the viscosity, ε is the dielectric constant of the medium, and κ and a are the DebyeH€ uckel parameter and the particle radius, respectively). Cryogenic transmission electron microscopy (cryo-TEM) measurements were performed on an FEI Tecnai 20, type Sphera TEM instrument equipped with a LaB6 filament operating at 200 kV. Images were recorded with a bottom-mounted Gatan CCD camera. The sample vitrification procedure was carried out using an automated vitrification robot (FEI Vitrobot Mark III). A 3 μL sample was applied on a Quantifoil grid (R 2/2, Quantifoil Micro Tools GmbH; freshly glow-discharged just prior to use), excess liquid was blotted away, and the formed thin film was shot into melting ethane. The grid containing vitrified film was immediately transferred to a cryoholder (Gatan 626) and observed at -170 °C.

Results and Discussion Synthesis and Characterization of Vesicles. DODAB, a double-chain cationic surfactant, was chosen as the vesicle forming surfactant because it is inexpensive, is commercially available, and is widely studied in membrane mimetic chemistry and DNA transfection. DODAB vesicle populations are usually stable and do not tend to form lamellar phases. In aqueous dispersion, DODAB molecules self-assemble above the gel-to-liquid crystalline phase transition temperature (Tm) into giant closed bilayer vesicle structures and bilayer fragments26 and large unilamilar vesicles can easily be obtained by dissipation of energy.27 (26) Feitosa, E.; Karlsson, G.; Edwards, K. Unilamellar vesicles obtained by simply mixing dioctadecyldimethylammonium chloride and bromide with water. Chem. Phys. Lipids 2006, 140, 66-74. (27) Lapinski, M. M.; Castro-Forero, A.; Greiner, A. J.; Ofoli, R. Y.; Blanchard, G. J. Comparison of liposomes formed by sonication and extrusion: Rotational and translational diffusion of an embedded chromophore. Langmuir 2007, 23, 1167711683.

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Figure 1. Cryo-TEM micrographs of vesicles obtained after extrusion of 10 mM DODAB dispersion through three-stacked (a) 100 nm (b) 200 nm polycarbonate filters.

Figure 2. Effect of temperature on absorbance (at 400 nm) of 10 mM DODAB vesicle solution. Vesicles used were obtained after extrusion through 200 nm (empty black square) and 100 nm (solid red square) three-stacked polycarbonate filters.

To obtain large unilamilar vesicles (LUVs) we use a membrane extrusion method, which represents a simple, reproducible and widely used method.27 In extrusion the amphiphile dispersion, containing bilayer fragments, is forced through small pores of a track-etched polycarbonate membrane filter, using either pressurized gas or syringe-based plunger set-ups at temperature well above the gel-to-liquid crystalline phase transition temperature (Tm).20 Vesicle size can be controlled by the pore size of the filters. Extrusion of DODAB dispersions was done using two different filter sizes: that is, 100 and 200 nm. The morphology of the resulting vesicles was characterized by cryo-TEM and appeared to be unilamilar (Figure 1). The vesicles obtained with the 100 nm filter are rather polydisperse (Figure 1a). The z-average diameter measured by DLS was found to be around 127 nm (PDI = 0.15), which is in reasonable agreement with the cryo-TEM micrograph. Interestingly, the vesicles are not perfectly spherical but have (28) Andersson, M.; Hammarstrom, L.; Edwards, K. Effect of bilayer phasetransitions on vesicle structure and its influence on the kinetics of viologen reduction. J. Phys. Chem. 1995, 99, 14531-14538.

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sharp corners and edges (unlike the “well-behaved” natural phospholipids), which is in agreement with what has been reported in the literature.28 The inability of DODAB to form smooth bilayers of high curvature at temperatures below Tm is likely caused by packing constraints and bilayer rigidity, resulting from the high headgroup charge density of the DODAB molecule compared to other phospholipids.28 Occurrence of inhomogeneities is also visible, indicating the presence of weak domains in the bilayer. Figure 1b shows the vesicles obtained by extrusion through 200 nm filters. The particle diameter of around 200 nm obtained by DLS closely corresponds to what is obtained from cryo-TEM visualization. The size distribution of these vesicles is also polydisperse (PDI = 0.18), but the vesicles have less sharp edges compared to the vesicles obtained with a 100 nm filter; this is likely due to a lower bending rigidity resulting from larger vesicle diameters. As a simple method to determine the main gel (Lβ)-to-liquid crystalline (LR) transition temperature (Tm) of both vesicle populations, the absorbances (at 400 nm) of the vesicle dispersions were recorded as a function of temperature (Figure 2). The DOI: 10.1021/la904709c

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and stability of vesicles. Since the interaction is mainly electrostatic, the adsorption of charged polymer on the vesicle surface may screen electrostatic repulsion forces, causing a decrease in colloidal stability leading to the destruction of the vesicles. The interaction depends on many parameters such as vesicle size and charge density, polyelectrolyte chain length, its composition, flexibility and charge density, and the ionic strength of the medium. Depending on the concentrations of polyelectrolyte and vesicleforming surfactant/lipid, the polyelectrolyte adsorption onto the vesicle of opposite charge may result in a polyelectrolyte-coated aggregate or in charge inversion if more polymer chains collapse on the vesicle surface than those necessary to neutralize it.32,33 Hence, a careful study of the RAFT copolymer adsorption onto the vesicles was conducted. In this work, we studied the adsorption of three different random RAFT copolymers: BA6-co-AA9, BA3-co-AA10, and BA6-co-AA4. For the adsorption studies, DODAB vesicles (obtained by extrusion through 100 nm PC filters) of around 130 nm diameter (measured by DLS) were used. The adsorption studies were performed at room temperature, well below the gelto-liquid crystalline transition temperature (Tm) of DODAB vesicles restricting the lateral and transbilayer mobility of the surfactant molecules. The vesicle dispersion was added to the copolymer solution to obtain mixtures of various compositions. Here the stoichiometric charge ratio parameter (ξ) is often used to express the composition of the mixture and is defined as32 ξ ¼

Figure 3. Normalized z-average diameter (d/d0) (a) and zeta potential (b) of DODAB vesicles as a function of stoichiometric charge ratio ξ for three different RAFT copolymers: (empty black square) BA6-co-AA9, (empty red up triangle) BA3-co-AA10, and (empty blue down triangle) BA6-co-AA4 (d0 is the z-average diameter of the blank vesicles).

decrease in the absorbance is explained by a fall in the refractive index upon temperature rise, which is caused by a decreasing density of the vesicle bilayer as a result of bilayer melting during the phase transition. A steep change was observed as the membrane undergoes a transition from ordered gel phase to liquid crystalline phase. The value of Tm, taken at the midpoint of this steep part of the curve,29 was found to be around 44 °C for both vesicle populations, which is in good agreement with values reported in the literature.30 Adsorption of RAFT Copolymers on DODAB Vesicles. Adsorption of charged polymers onto oppositely charged surfaces is a process that is mainly electrostatically driven and depends on many factors such as the charge density of the polymer, the ionic strength, and the resident ion on the surface of the substrate.31 In this work, we study the adsorption of anionic RAFT copolymers onto positively charged DODAB vesicles. The adsorption of polyelectrolytes onto the surface of oppositely charged vesicles potentially has large effects on the structure (29) Nascimento, D. B.; Rapuano, R.; Lessa, M. M.; Carmona-Ribeiro, A. M. Counterion effects on properties of cationic vesicles. Langmuir 1998, 14, 7387-7391. (30) Jung, M.; Hubert, D. H. W.; van Veldhoven, E.; Frederik, P.; van Herk, A. M.; German, A. L. Vesicle-polymer hybrid architectures: A full account of the parachute architecture. Langmuir 2000, 16, 3165-3174. (31) Schwarz, S.; Lunkwitz, K.; Kessler, B.; Spiegler, U.; Killmann, E.; Jaeger, W. Adsorption and stability of colloidal silica. Colloids Surf., A 2000, 163, 17-27.

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N acid 3 ½RAFT ½DODAB

ð1Þ

where Nacid is the total number of acrylic acid units in the RAFT copolymer chain (measured by NMR, Table 1), [RAFT] and [DODAB] are the molar concentrations of RAFT copolymer and DODAB, respectively. It should be noted that during the interaction of the vesicles with the RAFT copolymers only the surfactant molecules in the outer layer of the bilayer take part in the interaction.33 For the vesicles in this study (diameter >100 nm), the number of the surfactant molecules is considered to be approximately the same in both the layers of the bilayer because the effect of bilayer thickness (∼5 nm) can be neglected. This implies (from eq 1) that the point of zero charge corresponds to a charge ratio parameter (ξ) value of around 0.5. During the adsorption studies, the vesicle solution of known surfactant concentration was added dropwise to RAFT copolymer solutions of various copolymer concentrations to get the required values of the charge ratio parameter (ξ). The interaction between vesicles and the RAFT copolymers is accompanied by a neutralization of the vesicle surface charge which can be probed by the measurement of particle diameter and the zeta potentials using dynamic light scattering. Figure 4 shows the dependence of the z-average diameter and the ζ potential as a function of the charge ratio parameter (ξ), and it can be seen that, at low values of ξ (hence a low concentration of RAFT copolymer), the size of the vesicle-RAFT copolymer complexes is close to the size of the original vesicles. With an increasing charge ratio (hence an increasing concentration of RAFT copolymer), the z-average diameter of the vesicles increases and the ζ potential decreases until the vesicles start aggregation (32) Volodkin, D.; Mohwald, H.; Voegel, J.-C.; Ball, V. Coating of negatively charged liposomes by polylysine: Drug release study. J. Controlled Release 2007, 117, 111-120. (33) Volodkin, D.; Ball, V.; Schaaf, P.; Voegel, J.-C.; Mohwald, H. Complexation of phosphocholine liposomes with polylysine. Stabilization by surface coverage versus aggregation. Biochim. Biophys. Acta, Biomembr. 2007, 1768, 280-290.

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Figure 4. (a) Cryo-TEM micrograph of nanocapsules obtained by encapsulating DODAB vesicles (obtained by extrusion through 100 nm pore size filters) using RAFT copolymer BA6-co-AA9 and a feed composition ratio of MMA/BA = 10:1 for encapsulation. (b) High magnification image of a single nanocapsule.

around a charge ratio parameter 0.25. The region between charge ratios from about 0.25 to 0.9 is marked by the aggregation of the vesicles, leading to unstable particle diameters in the micrometer range. The measurement of particle diameters by DLS in this region is unreliable, and therefore, these values are not included in the graph. Aggregation of the vesicles is caused by the fact that, with increasing concentration, more RAFT copolymer is adsorbed onto the vesicles and the inherent positive charge of the vesicles decreases, resulting in a reduced electrostatic repulsion between the partially polymer-coated vesicles. The non-homogeneous overcompensation of the surface charge by the adsorbing copolymer chains causes the disruption of bilayers and vesicle aggregation.34 Aggregation occurs when the polyion covered domain of a vesicle interacts with the uncovered domain of another vesicle (‘‘charge patch’’ attraction).34,35 As a consequence, the stability of the vesicle dispersion is lost and the system flocculates. Further increasing the charge ratio parameter (>1) leads to a decrease in particle diameter until a plateau is reached and copolymer-covered anionically stabilized vesicles are obtained. At these values of charge ratio parameters, enough RAFT copolymer is adsorbed on to the surface of the vesicle to provide the required electrostatic repulsion. Figure 3b shows the dependence of the zeta potential values on the charge ratio during adsorption of the RAFT copolymer. The zeta potential first decreases to zero as the positive charge of the vesicles is totally neutralized by the adsorbed copolymer chains. With increasing charge ratio, the zeta potential then becomes negative within the precipitation region until a plateau is reached when excess of the copolymer is added. For the RAFT copolymers BA6-co-AA9 and BA3-co-AA10, the isoelectric point was obtained at the charge ratios of 0.6 and 0.7, respectively, and not at the expected value of 0.5. This difference can be attributed to the charge inversion phenomenon due to lateral correlation between adsorbing copolymer chains, causing the adsorption of more copolymer chains than necessary to just neutralize the vesicles.33 The lower value (0.29) of the charge ratio for the RAFT copolymer BA6-co-AA4 is likely caused by its short chain length and lower charge density (34) Walker, H. W.; Grant, S. B. Factors influencing the flocculation of colloidal particles by a model anionic polyelectrolyte. Colloids Surf., A 1996, 119, 229-239. (35) Miklavic, S. J.; Chan, D. Y. C.; White, L. R.; Healy, T. W. Double layer forces between heterogeneous charged surfaces. J. Phys. Chem. 1994, 98, 90229032.

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allowing the copolymer to take a more flat conformation on the vesicle surface.32,36-38 Preparation and Characterization of Nanocapsules. Vesicletemplated nanocapsules were synthesized by starved feed emulsion polymerization in order to avoid the buildup of monomer droplets in the system. Monomer droplets can potentially destabilize the RAFT copolymer coated vesicles39 and compete for the RAFT copolymer during the encapsulation reaction, thus reducing the colloidal stability.24 Besides, excess monomer can also give rise to the formation of polymer particles as a result of secondary nucleation. The used RAFT copolymers chosen for this study were all relatively short, as we have previously shown24 that the longer RAFT copolymers also lead to significant secondary nucleation. The anionic RAFT copolymer was first adsorbed on to DODAB vesicles to get “RAFT copolymer stabilized” vesicles (Scheme 1). The adsorbed RAFT copolymers were then chain extended to form a polymeric shell around the vesicle by the feeding of a monomer mixture comprised of MMA and BA in the presence of a nonoxidizing water-soluble initiator 4,40 -azobis(4-cyanovaleric acid) (V-501). The amount of RAFT copolymer was kept high enough to give almost double the isoelectric point charge ratio, because sufficient RAFT copolymer should be present in the aqueous phase to adsorb onto the growing surface during encapsulation to provide the required stabilization.23,24 Cryo-TEM was used to examine the morphology of the resulting structures in their wet state and the micrographs indeed reveal the formation of polymer shells around the vesicle, resulting in nanocapsules. Figure 4 shows the nanocapsules obtained by templating DODAB vesicles obtained by extrusion through a 100 nm pore size PC filter using RAFT copolymer BA6-co-AA9 and a monomer feed composition of MMA/BA = 10:1 (entry 1, (36) Eckenrode, H. M.; Dai, H.-L. Nonlinear optical probe of biopolymer adsorption on colloidal particle surface: Poly-L-lysine on polystyrene sulfate microspheres. Langmuir 2004, 20, 9202-9209. (37) Yaroslavov, A. A.; Rakhnyanskaya, A. A.; Yaroslavova, E. G.; Efimova, A. A.; Menger, F. M. Polyelectrolyte-coated liposomes: Stabilization of the interfacial complexes. Adv. Colloid Interface Sci. 2008, 142, 43-52. (38) Sennato, S.; Bordi, F.; Cametti, C.; Diociaiuti, M.; Malaspina, P. Charge patch attraction and reentrant condensation in DNA-liposome complexes. Biochim. Biophys. Acta, Biomembr. 2005, 1714, 11-24. (39) Kepczynsk, M.; Lewandowska, J.; Romek, M.; Zapotoczny, S.; Ganachaud, F.; Nowakowska, M. Silicone nanocapsules templated inside the membranes of catanionic vesicles. Langmuir 2007, 23, 7314-7320.

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Figure 5. Evolution of particle size as a function of conversion for the encapsulation of DODAB vesicles (obtained by extrusion through 100 nm pore size PC filters) using RAFT copolymer BA6-co-AA9 and a total of 0.24 g of monomer feed (MMA/ BA = 10:1). Particle sizes were determined by DLS (all PDIs were between 0.13 and 0.17).

Table 2). It can be seen that a thick layer of polymer is formed over the surface, resulting in a 1:1 replica of the original vesicle. Small amounts of free polymer particles are also observed which are probably because of secondary nucleation in the aqueous phase.24 From the cryo-TEM image, a thickness of around 40 nm is estimated for the polymer shell formed on the surface of the vesicles. As discussed earlier, the vesicle population obtained by the extrusion of a DODAB dispersion through a 100 nm pore size filter is rather polydisperse with a mean diameter around 130 nm (measured by DLS). The diameter of the nanocapsules measured by DLS is around 165 nm. One of the most important benefits of using the RAFT copolymer approach in synthesizing the nanocapsules is excellent control over size and the wall thickness/composition of the nanocapsule. The size of the nanocapsule can easily be controlled by employing the desired sized vesicles, and the wall thickness can easily be controlled by monomer feed profiles. Besides, the versatility of RAFT allows the use of a vast variety of monomer combinations to get nanocapsules of different compositions for various applications. Figure 5 shows the evolution of z-average particle diameter as a function of the amount of monomer added during the encapsulation reaction of 130 nm DODAB vesicles using RAFT copolymer BA6-co-AA9 and a monomer feed composition of MMA/BA = 10:1. A steady increase in particle diameter with the amount of added monomer is indicative of the polymer growth on the surface of the vesicles, leading to a final wall thickness of around 30-40 nm. As stated before, the size of the nanocapsule can be tuned by changing the size of the original template vesicles. Figure 6 shows nanocapsules obtained by templating DODAB vesicles obtained by extrusion through 200 nm pore size filters. The original average diameter of the vesicles, measured by DLS, was around 200 nm. To elucidate the structure of the vesicles and vesicle-templated nanocapsules, XRD scans were recorded. Figure 7 shows the XRD diffractograms of DODAB vesicles obtained by extrusion through 100 nm pore size PC filters and vesicle-templated nanocapsules obtained using RAFT copolymer BA6-co-AA9 and a feed composition ratio of MMA/BA = 10:1 for encapsulation. The XRD pattern of the DODAB vesicles reveals different 7854 DOI: 10.1021/la904709c

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Figure 6. Cryo-TEM micrograph of nanocapsules obtained by encapsulating DODAB vesicles (obtained by extrusion through 200 nm pore size PC filters) using RAFT copolymer BA6-co-AA9 and a feed composition ratio of MMA/BA =10:1 for encapsulation.

Figure 7. X-ray diffraction patterns of DODAB vesicles (obtained by extrusion through 100 nm pore size filters) and vesicle-templated nanocapsules obtained using RAFT copolymer BA6-coAA9 and a feed composition ratio of MMA/BA = 10:1 for encapsulation.

Bragg orders characteristic of a layered structure.40 Well-defined diffraction peaks can be observed, and an average lamellar distance (d) of 3.65 nm was calculated from the position of the first peak using the Bragg equation (nλ = 2d sinθ). This value can be identified as the DODAB 3 H2O monohydrate spacing41 and is in close agreement with values of 3.68 and 3.7 nm, previously reported by Jung et al.42 and Okuyama et al.,43 respectively. Taking into account that the length of a single DODAB molecule (40) Schulz, P. C.; Rodrı´ guez, J. L.; Soltero-Martı´ nez, F. A.; Puig, J. E.; Proverbio, Z. E. Phase behavior of the dioctadecyldimethylammonium bromidewater system. J. Therm. Anal. Calorim. 1998, 51, 49-62. (41) Okuyama, K.; Soboi, Y.; Hirabayashi, K.; Harada, A.; Kumano, A.; Kaziyama, T.; Takayanagi, M.; Kunitake, T. Single crystals of totally synthetic amphiphiles, dialkyldimethylammonium bromides. Chem. Lett. 1984, 2117-2120. (42) Jung, M.; German, A. L.; Fischer, H. R. Polymerisation in lyotropic liquidcrystalline phases of dioctadecyldimethylammonium bromide. Colloid Polym. Sci. 2001, 279, 105-113.

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Figure 8. (a) Normalized optical density for the surfactant titration of DODAB vesicles (solid black square) and vesicle-templated nanocapsules (solid red down triangle). (b) DLS traces showing the effect of Triton X-100 on the stability of DODAB vesicles and vesicletemplated nanocapsules: DODAB vesicles (solid black square), DODAB vesicles þ Triton X-100 (empty black square), vesicle-templated nanocapsules (solid red down triangle), and nanocapsules þ Triton X-100 (empty red down triangle).

is just 2.7 nm, this spacing implies that the surfactant molecules have to be considerably tilted to the bilayer surface.43 Similar peaks with greatly reduced intensities are observed in the XRD pattern of the nanocapsules, indicating the presence of an intact bilayer structure. The reduction in diffraction intensity is likely caused by the thick amorphous polymer layer around the vesicles and a possible bilayer deformation during encapsulation. The formation of stable nanocapsules can be confirmed by surfactant lysis experiments. Addition of monotailed surfactant to a vesicle dispersion is known to destabilize the vesicles, leading to their complete breakdown into mixed micelles.44 For surfactant lysis experiments, we used Triton X-100, a nonionic surfactant known for its excellent membrane solubilization properties.44 The formation of the stable nanocapsules can thus be probed by measuring the resistance toward disintegration using a suitable technique, such as optical density and DLS measurements. Figure 8a shows the optical density as a function of molar ratio of Triton X-100 and DODAB ([TX-100]/[DODAB]). The titration of blank DODAB vesicles resulted in a typical three-stage disintegration profile.44 The optical density first increases up to a molar ratio of [TX-100]/[DODAB] of 0.8. The increase in the optical density is likely caused by the incorporation of TritonX-100 surfactant molecules into the vesicles resulting in denser bilayer.44 The optical density then drops steadily, indicating the transition from vesicles to mixed micelles, until a molar ratio of 2:1 yields a complete transition to mixed micelles, resulting in an almost transparent dispersion. No appreciable change in the optical density was observed at higher molar ratios. When comparing this to the titration of the nanocapsule dispersions, no such vesicle to micelles transition behavior was observed in the case of nanocapsules. The slight decrease in optical density after every injection is only due to a dilution effect and a possible breakdown of some partially encapsulated vesicles.39 (43) Okuyama, K.; Soboi, Y.; Iijima, N.; Hirabayashi, K.; Kunitake, T.; Kajiyama, T. Molecular and crystal structure of the lipid-model amphiphile, dioctadecyldimethylammonium bromide monohydrate. Bull. Chem. Soc. Jpn. 1988, 61, 1485. (44) Alonso, A.; Urbaneja, M.-A.; Go~ni, F. M.; Carmona, F. G.; Canovas, F. G.; Gomez-Fernandez, J. C. Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100. Biochim. Biophys. Acta, Biomembr. 1987, 902, 237-246.

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The stability of the nanocapsules was further confirmed by DLS measurements. Figure 8b shows the size distribution of vesicles and nanocapsules before and after mixing with Triton X-100. The size distribution of vesicles is monomodal, averaging around a diameter of about 130 nm. After mixing with surfactant Triton X-100, the vesicle peak diminishes and a different peak averaging around a diameter of 6 nm appeared in the distribution, representing the DODAB/ Triton X-100 mixed micelles. Mixing Triton X-100 with the nanocapsules dispersion did not affect the particle size distribution appreciably, and the average particle diameter remains around 170 nm. Here the slight decrease in intensity is likely caused by the disintegration of some uncoated vesicles and/or bilayer fragments. In order to probe whether polymerization occurred under RAFT control, the molecular weight growth was followed during the polymerization using size exclusion chromatography (SEC). Because the reactions were conducted under starved feed conditions, the amount of monomer added into the system can be approximated to be the monomer converted. Samples were taken at different amounts of monomer added into the system, and the SEC measurements were carried out on the total polymer present. The theoretical number average molecular weights were calculated using the following equation: M n, th ¼

½M0 xm0 þ M RAFT ½RAFT0

ð2Þ

where [M]0 and [RAFT]0 are the starting concentrations of monomer and RAFT agent, respectively, x refers to the overall monomer conversion, and m0 and MRAFT are the molecular weights of the monomer and RAFT agent respectively. Figure 9 shows the evolutions of the molecular weight distribution and the number average molecular weight (Mn) as a function of the added amount of monomer for the nanocapsule synthesis reaction using DODAB vesicles obtained by extrusion through 100 nm filters, RAFT copolymer BA6-co-AA9, and a monomer feed composition MMA/BA = 10:1 (the amount of monomer added during encapsulation was increased to 0.92 g instead of 0.23 g in order to get enough polymer in the samples for the SEC measurements). The continuous increase of the number average molecular weight DOI: 10.1021/la904709c

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Figure 9. Molecular weight evolution during encapsulation of DODAB vesicles using RAFT copolymer BA6-co-AA9 and a monomer feed composition of MMA/BA = 10:1. (a) Molecular weight distributions scaled to conversion (= % monomer added). (b) Mn (9) and PDI (4). The straight line in (b) corresponds to the theoretical Mn versus conversion obtained by using eq 2. Experimental distributions and averages are all relative to polystyrene standards.

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(Mn) with conversion indicates that the polymerization happens under RAFT control. It can be seen that the obtained chain lengths are larger than the theoretically calculated values (using eq 2). A reason for this behavior may lie in the fact that only those RAFT copolymers that are adsorbed onto the vesicle will participate in the polymerization process,23 meaning that the number of growing chains is smaller than the available RAFT groups. The nonlinear increase in Mn with conversion is expected because, with increasing conversion, more RAFT copolymers will adsorb onto the growing particle from the aqueous phase, thus increasing the number of growing polymer chains and hence causing the negative deviation from the linear increase. Effect of RAFT Copolymer Composition. To probe the effect of the RAFT copolymer composition, encapsulation reactions of DODAB vesicles (obtained by extrusion through 100 nm PC filters) were performed using two more RAFT copolymers, BA3co-AA10 and BA6-co-AA4 and a monomer feed composition of MMA/BA = 10:1. In both cases, as shown by the cryo-TEM micrographs in Figure 10, encapsulation was successful, resulting in the formation of nanocapsules. Figure 10a shows the nanocapsules obtained by using RAFT copolymer BA3-co-AA10 (entry 2, Table 2). The surface of the polymer layer is not uniform, and there is some phase separation. Polymer segregation was also observed for the encapsulation reaction using RAFT copolymer BA6-coAA4 (Figure 10b and entry 3, Table 2). This observation can be explained in terms of the hydrophilic-hydrophobic balance of the RAFT copolymer and the fluidity of the DODAB bilayer. Interaction of the charged copolymers with the oppositely charged surfactant vesicles largely depends on both the electrostatic and the hydrophobic interactions.45 For better adhesion and vesicle stability, the copolymer needs to have both charged and hydrophobic units. Completely hydrophilic and flexible copolymers with a high linear charge density are known to destabilize the bilayer, causing the bilayer rupture and a change in the gel-to-liquid crystalline transition temperature.37,46 This occurs because the adsorption of such copolymers is accompanied by formation of loops and tails and the electrostatic repulsions of the charge units located within these areas can induce dynamic distortions in the bilayer structure.37 The presence of hydrophobic units with grafts such as alkyl groups increases the copolymer backbone association with the bilayer as these grafts can position inside the apolar bilayer, thereby

Figure 10. Cryo-TEM micrographs of the nanocapsules obtained using different RAFT copolymers for the encapsulation of DODAB vesicles (obtained by extrusion through 100 nm pore size filters): (a) BA3-co-AA10 and (b) BA6-co-AA4. Monomer feed composition ratio in the reactions was MMA/BA = 10:1. 7856 DOI: 10.1021/la904709c

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Figure 11. Cryo-TEM micrographs of the nanocapsules obtained by the encapsulation attempt of the DODAB vesicles (obtained by extrusion through 100 nm pore size filters) using two different monomer feed compositions: (a) MMA only and (b) mixture of MMA/BA = 7:3. RAFT copolymer BA6-co-AA9 was used for the encapsulation.

giving additional adhesion and enhancing the stability of the bilayer.37,45 For the encapsulation of DODAB vesicles using the RAFT copolymer BA3-co-AA10 (Figure 10a), the observed polymer segregation is likely because of the weakening and partial rupture of the bilayer during encapsulation, caused by the adsorption of highly charged RAFT copolymer BA3-co-AA10 chains. Polymer segregation in the case of RAFT copolymer BA6-co-AA4 (Figure 10b) is likely caused by an inadequate number of charged units to provide better adhesion of the RAFT copolymer chains to the vesicle surface during the encapsulation reaction. Polymer segregation can also be caused by the fluidity of the DODAB vesicles (Tm = 44 °C) at the reaction temperature (70 °C). Effect of Monomer Feed Composition. To study the effect of monomer feed composition, encapsulation of DODAB vesicles (obtained by extrusion through 100 nm PC filters) was performed using two more feed compositions (i.e., 100% MMA and MMA/ BA = 7:3) and RAFT copolymer BA6-co-AA9 (entries 4 and 5, Table 2). The morphology of the obtained products was observed by cryo-TEM. As can be seen from Figure 11, encapsulation of DODAB vesicles was successful in both cases, resulting in the formation of nanocapsules. The surface of the polymer layer on the vesicles is to some extent uneven in the case of nanocapsules obtained with an all MMA monomer feed (Figure 11a) which is presumably caused by aggregation of secondary polymer particles onto the vesicle containing particles and because of the relatively high glass transition temperature of the PMMA the polymer mobility not being high enough for leveling out the uneven surface. This observation is similar to what we previously observed for the encapsulation of gibbsite.24 Use of a higher butyl acrylate containing feed (MMA/BA = 7:3, Figure 11b) resulted in more spherical nanocapsules with a relatively smooth surface. This effect can be attributed to the composition of the encapsulating polymer. The copolymer shell formed with the feed composition containing more BA is likely to be more hydrophobic and with a low glass transition temperature (Tg). High interfacial tension and the surface energy of the hydrophobic copolymer cause a mini(45) Antunes, F. E.; Marques, E. F.; Miguel, M. G.; Lindman, B. Polymervesicle association. Adv. Colloid Interface Sci. 2009, 147-148, 18-35. (46) Diederich, A.; Bahr, G.; Winterhalter, M. Influence of polylysine on the rupture of negatively charged membranes. Langmuir 1998, 14, 4597-4605.

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mization of the surface area, leading to more spherical-shaped nanocapsules with smooth surfaces. The nanocapsules in the cryo-TEM micrograph of Figure 11b look darker because of small contrast difference between the BA rich copolymer and the vitreous ice.

Conclusions In this paper, we presented a simple RAFT-based vesicletemplating approach to synthesize polymeric nanocapsules. Anionic RAFT copolymers of BA and AA were first adsorbed on to the surface of DODAB vesicles (used as template) and then chain extended to form a polymeric shell. As revealed by the cryo-TEM characterizations, the approach was successful to encapsulate DODAB vesicles leading to the formation of nanometer-sized hollow capsules. The hydrophilic-hydrophobic balance and monomer feed composition were found to have significant effects on the encapsulation of DODAB vesicles. The best encapsulation results were obtained using RAFT copolymer BA6-co-AA9 with a monomer feed composition MMA/BA = 10:1. This is the same composition as was found previously to work best in the case of gibbsite encapsulation.24 The RAFT copolymer approach describes a simple emulsion polymerization based route to form hollow capsules. The approach is quite flexible and can easily be scaled up for industrial applications. The versatility of RAFT can allow the use of a variety of monomer/cross-linker combinations for various end uses. We consider the main advantage of the RAFT copolymer based encapsulation approach to be control over the wall thickness and the shell size, which can be simply achieved by tuning the amount of monomer fed and using a desirable size template, respectively. The nanocapsules are stabilized by a layer of negatively charged carboxylate groups of the RAFT copolymer chains, thus avoiding the need for any external surfactant. In the future, the approach will be extended to synthesize responsive nanocapsules and to encapsulate anionic vesicles of naturally occurring lipids (liposomes) using cationic RAFT copolymers. Acknowledgment. We gratefully acknowledge Dr. Brian S. Hawkett of the Key Centre for Polymers and Colloids, University DOI: 10.1021/la904709c

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of Sydney for encouraging us to use the RAFT copolymers for encapsulation. We also acknowledge Dr. Nico Sommerdijk and Paul Bomans of the Soft Matter cryo-TEM Research Unit of Eindhoven University of Technology for helpful discussions about cryo-TEM, Dr. Alexandra Mu~noz Bonilla of Polymer Chemistry Group of Eindhoven University of Technology for

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helpful discussions about synthesis and characterization of RAFT copolymers, and Mark Berix of the Materials and Interface Chemistry group of Eindhoven University of Technology for XRD analysis. S.I.A. is grateful for financial support by the Higher Education Commission, Government of Pakistan, under the HEC-NUFFIC program.

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