Colloidal templating : a route towards controlled synthesis of
functional polymeric nanoparticles
Citation for published version (APA):
Ali, S. I. (2010). Colloidal templating : a route towards controlled synthesis of functional polymeric nanoparticles. Technische Universiteit Eindhoven. https://doi.org/10.6100/IR692089
DOI:
10.6100/IR692089
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Controlled Synthesis of Functional Polymeric
Nanoparticles
PROEFSCHRIFT
ter verkrijging van de graad van doctor aan de
Technische Universiteit Eindhoven, op gezag van de
rector magnificus, prof.dr.ir. C.J. van Duijn, voor een
commissie aangewezen door het College voor
Promoties in het openbaar te verdedigen
op woensdag 17 november 2010 om 14.00 uur
door
Syed Imran Ali
Dit proefschrift is goedgekeurd door de promotor:
prof.dr. A.M. van Herk
Copromotor:
dr.ir. J.P.A. Heuts
S.I. Ali.
Colloidal Templating: A Route Towards Controlled Synthesis of
Functional Polymeric Nanoparticles
A catalogue record is available from the Eindhoven University of
Technology Library
ISBN: 978-90-386-2376-4
Copyright © 2010 by Syed Imran Ali
All rights reserved. No part of this publication may be reproduced or
transmitted in any form or by any means without the prior written
permission of the copyright owner.
(In the name of God, the Beneficent, the Merciful)
Table of Contents
1 Introduction
11.1 General Introduction 2
1.2 Templating for the synthesis of latex particles 5
1.2.1 Layer-by-Layer Approach 7
1.2.2 Vesicle Polymerization 9
1.3 Reversible addition–fragmentation chain transfer (RAFT)
polymerization 9
1.3.1 RAFT in emulsion 11
1.3.2 RAFT-based “living” polyelectrolytes: possible means towards
template-directed synthesis 12
1.3.2 RAFT copolymer approach for colloidal templating 13
1.4 Aim and outline of the thesis 14
References 16
2
Synthesis and characterization of DODAB vesicles
252.1 Introduction 26
2.1.1 Hydration of vesicle forming surfactants 27
2.1.2 Vesicle formation methods 28
2.2 Experimental section 31
2.3 Results and discussion 33
2.3.1 Hydration behavior of DODAB 33
2.3.2 Membrane extrusion 35
2.3.3 Sonication 40
2.3.4 Thermal behavior 42
2.3.5 Morphology 47
2.3.6 Stability of vesicles against surfactants 51
2.4 Conclusions 52
References 53
3. Polymerization of hydrophobic monomers in the bilayers
of
DODAB
vesicles
57
3.1 Introduction 58
3.2 Experimental section 62
3.3 Results and discussion 66
3.3.1 Polymerization in DODAB vesicle bilayers 66
3.3.2 Swelling of the vesicle bilayers with monomer 67
3.3.3 Polymerization in the vesicle bilayers 73
3.3.5 Thermal behavior 78
3.4 Conclusions 81
References 83
4. Anisotropic composite latex particles by RAFT-based
starved feed emulsion polymerization
89
4.1 Introduction 90
4.2 Experimental section 94
4.3 Results and discussion 101
4.3.1 Synthesis and characterization of anionic random RAFT copolymers
101
4.3.2 Adsorption of RAFT copolymers on gibbsite 105
4.3.3 Preparation and characterization of anisotropic composite nanoparticles using gibbsite platelets as template
109
4.3.4 Cryo-electron tomography of the composite latex particles 124
4.3.5 Preparation of anisotropic composite nanoparticles using Na- montmorillonite platelets as template
126
4.3.6 Encapsulation reactions on Na-montmorillonite clay 131
4.3.7 Mechanism of encapsulation 134
4.4 Conclusions 134
References 136
5. Controlled Synthesis of Polymeric Nanocapsules by
RAFT-based Vesicle Templating
141
5.1 Introduction 142
5.2 Experimental section 144
5.3 Results and discussion 149
5.3.1 Synthesis and characterization of vesicles 149
5.3.2 Adsorption of RAFT copolymers on DODAB vesicles 151
5.3.3 Preparation and characterization of nanocapsules 155
5.3.4 Preparation and characterization of responsive nanocapsules 166
5.3.5 Responsive behavior of nanocapsules 173
5.4 Conclusions 175
References 177
Summary and Future Research
183List of Publications
Acknowledgements
189 191
Chapter 1
Introduction
Abstract: This chapter contains a brief general background of the research described in this thesis, together with the scope and outline of the thesis.
1.1 General
introduction
Bulk properties of materials can effectively be tailored by controlling the properties and structure at microscopic level, imparting new features to the well-known standard materials and hence to new applications.1 Small building blocks have been found to exhibit enhanced properties such as mechanical, optical, magnetic, and electronic properties, compared to the coarser materials of the same chemical composition. This resulted in a quest to develop structurally organized “nanostructured” materials with defined size, shape and geometry in nano to micrometer length scales possessing enhanced properties compared to bulk materials. Today materials science deals increasingly with nanostructures, that is, with structures of characteristic dimensions between 1 and 1000 nm. This has resulted in the emergence of “nanotechnology”, technology of materials possessing order in the nanometer range, as a new field of research.1, 2 Nanotechnology as a scientific field has expanded enormously over the last two decades, combining almost all scientific fields. Nature presents many examples of how nanotechnology works. From atom by atom construction of proteins in the liposomes inside cells, to the chloroplasts of plants that convert sunlight, carbon dioxide, and water into sugar and oxygen. Naturally occurring well organized materials, such as mineral crystals, self
assembled structures, cells etc have attracted great deal of interest and give rise to an
appealing and attractive concept; that is to mimic the structural organization naturally present in these system for the controlled construction of organized matter and one obvious way of achieving this goal is to use them as submicrometer templates for the construction of nanomaterials.3, 4
A route for enhancing nanostructure order5 is “template synthesis” which entails the preparation of a variety of micro- and nano-materials of a desired morphology. To mankind
the idea of templating is very old and utilized for centuries for the production of metal
objects, pottery, household goods and artworks with reliably and reproducibly.6 In the
broadest sense, a template can simply be considered as a shape and structure-directing agent
Figure 1. Schematic representation of the vesicle templating strategies (a) Morphosynthesis and (b) transcriptive synthesis.8
The first step in templating is to either fill or coat a preformed template with a soft precursor material to bring the material into the desired shape and form.6 In the former case, the material thickness is confined by the space available within the template and this approach is often termed as endo-templating or morphosynthesis. In the later case the material grows outside the template which only act as shape director and this approach is often referred to as exo-templating or transcriptive synthesis. In the second step the precursor material is fixed or hardens as per the shape and limitations of the template through a chemical or a physical process. Once the materials take the shape of the template, the latter can be removed leaving behind the material in a desired shape and size. The concept of exo and endo templating can easily be explained by the example of vesicle templating process for the construction of hollow particles8 as illustrated in Figure 1. The template approach has a clear advantage of controlling the size and shape in a fairly reproducible and time efficient way, in contrast to the other approaches, where such control is often more difficult to attain.
Figure 2. Potential template materials which can be used for the preparation of nano- and microstructures.
Analogous to the macroscopic casting of materials, the concept of templating and template-derived strategies attracted an enormous interest as a potentially powerful approach to assure well-defined morphological properties and structure on a nanometer length scale.6, 7 The availability of numerous colloidal substrates, in various shapes, sizes and properties, which can be used for both morphosynthesis and transcriptive synthesis for the preparation of well-defined nano and microstructures make the concept even more appealing. Many inorganic, organic, or biological materials covering length scales from several angstroms to several
micrometers offer great potential for the templated synthesis of materials.7, 9, 10 Notable
examples include, soft templates such as biological cell, virus particles, micro/miniemulsion droplets, molecularly self-assembled structures such as micelles, vesicles and other surfactant
mesophases, liquid crystals and hard templates such as mesoporous silica and silica particles,
clay minerals, nanotubes, metal nanoparticles and nanorods etc.6, 7, 10, 11 Templating allows
the design and synthesis of a wide range of nanostructures with specified geometry and surface characteristics, and with a wide range of potential applications. However, the key for a successful templated synthesis is to select a template which ensures the formation of a desired nanostructure and, if needed, can be easily removed without damaging the formed nanostructure.
1.2 Templating for the synthesis of latex particles
Controlled synthesis of polymeric nanoparticles with defined shape, structure, composition and with tailored properties is of immense scientific and technological interest and has been an intensive area of research in emulsion polymer science for the last few decades. Many synthetic methods have been developed such that a variety of particle morphologies are now possible.12, 13 Synthetic routes leading to particles with core-shell, hollow, micro-domain, and interpenetrating networks have been published.12 These advances have been based on a greater understanding of the thermodynamic and kinetic aspects of emulsion polymerization as well as more sophisticated approaches to emulsion processing.14,
15
However, most of these approaches are quite complicated, often requiring more than one production step. Besides, control over the particle nanostructure and shape is often relatively poor. The shape of latex particles has recently been recognized as an important parameter
that can significantly influence the chemical, physical, and rheological properties of the derived materials.16-19
Shape control of the polymer particles synthesized using approaches based on heterogeneous systems, such as emulsion, dispersion and suspension polymerization, is often difficult because of the inherent tendency to produce spherical shape colloids due to the minimization of the interfacial free energy between the growing particles and the aqueous medium. However, few approaches has been developed for the formation of non-spherical anisotropic polymer particles, mostly based on composite polymer particles by various seeded polymerizations systems.20-22
Synthetic routes to develop latex particles by combining emulsion polymerization with colloidal templating offer potential for the easy and reproducible synthesis of complex morphologies which can potentially broaden the application scope of the emulsion polymers. Interestingly, the concept of emulsion polymerization in itself can be broadly considered as a surfactant templated process where monomer is first solubilized in the surfactant micelles in an aqueous medium and later converted into a solid latex particle by initiating the polymerization by adding water-soluble initiator that provides the radicals which diffuses into the micelle. The depleting monomer is replenished inside the micelle by diffusion from the larger monomer droplets, and eventually a stable, spherical polymer particle is produced.12
With the availability of a vast variety of template materials, colloidal templating offers great potential for the easy and reproducible synthesis of complex latex morphologies, such as hollow capsules, core shell, multilayer/phase, rattles, polymer-inorganic composites and anisotropic particles. Especially important to mention are the flat latex particles and hollow capsules which are difficult to obtain by normal means, can possibly be obtained by templating on flat and hollow substrates, respectively. Thus, colloidal templating offers opportunities to introduce new levels of control in the production of latex particles which could help in broadening the application scope of the emulsion polymers.
Templated synthesis of latex particles is generally based on three steps:
1. Identification or synthesis of a potential core/substrate material with the desired properties, such as shape, size, reactivity and surface functionalities.
2. Coating or filling of the templating core material with organic, inorganic, or hybrid precursor materials such as monomer or preformed polymeric building blocks and subsequent fixation.
3. Removal of the templating core by chemical, physicochemical, or thermal treatment. Currently, two approaches based on the concept of templating have been used for the production of latex particles. A brief overview of these approaches is given in the next two sections.
1.2.1 Layer by Layer Approach
The layer-by-layer approach (L-b-L), first develop by Caruso et al.23, is an elegant way of combining the concept of colloidal templating and self-assembly. The general concept is based on alternate (layer-by-layer) deposition of material in the form of oppositely charged polyelectrolyte chains, mostly through electrostatic interaction.23-26 With new advances, a wide variety of materials have been explored as active building blocks for the L-b-L assembly, beyond simple polyelectrolytes. The most prominent examples include inorganic nanoparticles27, nanorods28, polymeric micelles29, dendrimers30 and carbon nanotubes.31 Depending on the end use of the particles, the core can be removed by chemical or thermal treatments to generate the hollow structure.
The layer-by-layer approach allows the control over determinant parameters such as size, composition, geometry, wall thickness, and uniformity. A huge variety of hollow spheres and other complex morphologies has been successfully produced with a wide range of micrometer and submicron inner diameters.
Figure 3. Concept of L-b-L approach for the synthesis of hollow capsules.32 Sequential deposition of positively (gray) and negatively (black) charged polyelectrolytes onto the surface of negatively charged colloid particles (a–e). After removal of the colloidal core (e) a suspension of polyelectrolyte capsules is obtained.
Despite simplicity and versatility, the L-b-L approach suffers from some shortcomings. For instance, to get a reasonable material thickness, several layers needs to be deposited rendering the whole procedure tedious.33 As the polyelectrolyte layers are adsorbed mostly by physical interactions, such particles are less stable and subsequent crosslinking is often needed to make physically more robust morphologies for certain applications.33, 34 Besides, as the approach is often 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 formed morphology.32 For the construction of nanocapsules, use of “soft templates” such as emulsion droplets, biological cells and surfactant vesicles is rapidly gaining interest as it eliminates the need to remove to so-called sacrificial template35 and allows the pre-encapsulation of different substances before the formation of nanocapsules.
1.2.2 Vesicle Polymerization
Vesicles are an important class of aggregates formed by surfactant molecules in aqueous dispersions. Due to the non-covalent interactions responsible for their formation, vesicles have only limited stability against many environment parameters and inherently return to their native lamellar phase state. A variety of techniques has been employed in order to increase vesicle stability. Their unique hollow morphology makes them ideal candidates as starting materials or templates for the synthesis of interesting morphologies such as polymeric hollow capsules. Vesicle polymerization takes advantage of the bilayer morphology of the surfactant aggregates to solubilize organic substances such as monomers. Subsequent polymerization of the monomers should give hollow spheres whose shape is a replica of the original bilayer structure (morphosynthesis). The shell is often cross-linked so as to afford rigid and stable capsules after extraction of the templating surfactant matrix (Figure 1). The only limitation in this approach is that the thickness of the nanocapsule wall is limited to the bilayer thickness. Pioneering work in this field has been done by Murtagh and Thomas.36 Since then, polymerizations in vesicle bilayers have attracted much interest and have been reported by several groups.37-41 Although it has been suggested by many authors that the polymerized vesicles are indeed hollow, another morphology where the polymer phase separation inside the bilayers gives rise to the parachute-like architectures, has also been reported.42 In addition to morphosynthesis, vesicles can also be used as templating materials for transcriptive synthesis to get inorganic capsules as described by Hubert et al.43 and in the layer-by-layer approach.33
1.3 Reversible addition–fragmentation chain transfer (RAFT)
polymerization
During the last decade, “living” or “controlled” radical polymerization techniques (CRP) have been developed and emerged as a powerful tool to engineer macromolecules having desired architectures.44-47 Since its invention by the Commonwealth Scientific and Industrial Research Organisation (CSIRO)48, 49, Reversible Addition–Fragmentation chain Transfer (RAFT) radical polymerization has developed into an elegant synthetic tool for the controlled
synthesis of various polymer architectures.44, 50 The versatility of RAFT allows the polymerization of a vast variety of monomers under different polymerization conditions. Contrary to the other controlled radical polymerization techniques such as ATRP, RAFT is based on degenerative chain transfer.47, 51, 52 RAFT agents are thiocarbonylthio species like dithioesters50, xanthates53, dithiocarbamates53, 54, and trithiocarbonates.55-57
Since RAFT is a free radical polymerization process conducted in the presence of a suitable thiocarbonylthio species, the mechanism can be described using the same steps used to describe a conventional free radical polymerization. As described in Figure 4, polymerization needs to be initiated using traditional initiators.44, 50, 58 The propagating macro radical, Pn·, adds to the RAFT agent (1) to form an intermediate species (2). With the correct
choice of RAFT agent, R. should be a better leaving group than Pn·. The intermediate species
(2) by fragmentation yield a new initiating radical R. and a dormant chain (3). Further propagation of the radical R. leads to a new polymeric (oligomeric) radical Pm·. This step is
often referred to as pre-equilibrium where the net result is the conversion of all RAFT agent molecules to macro RAFT agents (3). After the establishment of the pre-equilibrium, the polymerization enters the main chain equilibrium involving degenerative chain transfer of the thiocarbonylthio species between polymer chains Pn· and Pm·. By using a large ratio of RAFT
agent concentration to initiator concentration, termination events can be minimized and optimal controlled radical conditions are established, which results in control polymer growth with relatively low polydispersity.
1.3.1 RAFT in emulsion
Recently the research interest of CRP has been widened to its applications in emulsions.45,
46, 58
The requirement of less demanding conditions compared to the other CRP techniques makes RAFT well suited for the application in emulsion based systems and hence has been widely studied.58-62 An important feature of RAFT is its applicability to the synthesis of water-soluble (co)polymers50, 58 under various polymerization conditions with low polydispersity. This has paved the way for the controlled synthesis of water soluble polymers possessing various architectures such as homopolymers, block, triblock, gradient, statistical, alternating, graft, and star shaped copolymers. The use of amphiphilic block copolymers produced by RAFT as living polymeric surfactants for the emulsion polymerization allowed overcoming initial difficulties encountered during the application of RAFT in emulsion.58 The origin of these difficulties was the transport of highly hydrophobic RAFT agent to the locus of polymerization63, 64 (micelles) which was often overcome by using an additional solvent to facilitate the transport of the RAFT agents from the monomer droplets to the
particles.58, 65 Amphiphilic RAFT copolymers can self assemble to form micelles in the aqueous phase by hydrophilic-liphophilic interactions. These micelles can act as the precursors for particle formation upon entry of surface active z-meric radicals.58, 60, 66 The hydrophilic unit of the amphiphilic block copolymer provides the colloidal stability and the hydrophobic units grow further through the living RAFT moiety to form monodisperse polymer latex particles.62
1.3.2 RAFT-based “living” polyelectrolytes: possible means towards template-directed synthesis
The use of polyelectrolytes in colloidal templating, as described earlier, is an elegant approach for the synthesis of various nanostructured morphologies. A survey of the existing literature reveals that most of the studies were performed using biological or synthetic polyelectrolyte (synthesized by conventional routes) pairs, where control over some key parameters such as polymer composition/microstructure, molecular weight, molecular weight distribution, charge distribution and conformation was very limited. When synthesized by controlled radical polymerization techniques such as RAFT, these parameters can be relatively more controlled and this could pave the way to introduce various functionalities, allowing the preparation of specific combinations of multilayer assemblies with designed architecture and responsiveness to the external stimuli.67-70 Well-defined polymers synthesized by the RAFT approach have been exploited to synthesize a wide range of responsive and functional surfaces and nanoparticles.68-74 Apart from the controlled architecture, RAFT copolymers also have an added advantage of possessing a RAFT moiety which can be transformed easily to other useful functionalities such as to thiol75 or pyridyl disulfide.76
1.3.3 RAFT copolymer approach for colloidal templating
The living character of the RAFT-based charged polymers can be effectively utilized for the templated synthesis of a variety of morphologies. As already described, colloidal templating using conventional polyelectrolytes is a tedious procedure involving multiple steps for the sequential deposition of differently charged polyelectrolyte layers. Short-chain random copolymers containing hydrophilic (ionisable) and hydrophobic parts can be synthesized via RAFT.77 These living polyelectrolytes, named here as RAFT copolymers, can be adsorbed on the surface of a substrate by electrostatic and/or hydrophilic-hydrophobic interactions, where they can act as stabilizers, like normal polyelectrolytes. In this way, they potentially form a perfect means to encapsulate particles because the RAFT moiety they contain allows further growth on the surface of the substrate, which could possibly lead to the formation of a polymeric shell.
RAFT copolymers of various compositions having hydrophilic (e.g. acrylic acid or dimethylaminoethyl methacrylate) and hydrophobic (e.g. butyl acrylate, or styrene) parts can be synthesized using appropriate RAFT agents. These RAFT copolymers being random, unlike block copolymers, are less likely to form micelles and are expected to adsorb onto the surface of a template material, minimizing the formation of secondary polymer particles in the aqueous phase. Once on the substrate, the adsorbed RAFT copolymers can be chain extended to form a polymeric shell around the template particles. The RAFT-based approach has previously been used for the successful encapsulation of titanium dioxide and phthalocyanine blue pigment particles.77
There are many parameters that will likely influence the outcome of the RAFT copolymer based colloidal templating approach and hence need to be optimized. The key parameters include the chain length of the RAFT copolymers, their composition and structure, their concentration and the conditions used for the templating reactions. Besides, the interaction of the charged RAFT copolymer with the oppositely charged template material also needs to be optimized for the colloidal stability and hence the successful templating.
1.4
Aim and outline of the thesis
The aim of the work described in this thesis is to explore template-based synthesis of polymeric nanoparticles in emulsion-based systems. Two types of nanoparticle morphologies are selected for the templated synthesis:
(a) Flat latex particles (b) Polymeric nanocapsules
For the synthesis of flat latex particles, a RAFT-copolymer approach is explored on gibbsite and Na-montmorillonite platelets. Use of clay platelets for the templated synthesis of latex particles is, apart from leading to flat latex particles, also of great importance because of the wide and rapidly growing spectrum of applications of polymer-clay composite latex
particles.19 These particles play an important role in coatings, adhesives, impact modifiers,
and many other specialty materials.78, 79 Present approaches to synthesize polymer-clay nanocomposite latex particles in emulsion-based systems are rather complex, often requiring modification of clay.78-80 Besides, morphology of these particles is often uncontrolled, mostly yielding so-called armored latex particles (platelets being located at the particle surface) and
eventually isotropic nanofilled systems in the final applications. Exploring clay platelets for
the templated synthesis of flat latex particles also provide an alternate route for the true
encapsulation of the single clay platelets with morphology control. Disk-shape polymer clay nanocomposite latex particle will potentially induce anisotropy and provide additional control over the platelet orientation in the final polymeric film thus enhancing the coating properties.
For the synthesis of nanocapsules, the RAFT-copolymer approach is explored for the encapsulation of surfactant vesicles. Vesicle morphology, as depicted in Figure 1, can be used as template either by using their hydrophobic bilayer for polymerization reactions, or their outer surface can also be utilized for the growth of polymeric material. Exploring vesicles in templating approaches can possibly open new routes for the synthesis of nanocapsules which represent a special class of materials having diverse applications,
including encapsulation of various substances (for the controlled release of drugs, cosmetics, inks, and dyes), the protection of light-sensitive components, catalysis, coatings, composites, and fillers.13 Presently employed methods for the synthesis of nanocapsules such as osmotic swelling81, hydrocarbon encapsulation82, interfacial polymerization83 and layer-by-layer approach32 have been successful to varying degrees. However, nanocapsules obtained by these approaches have found limited applications and commercialization, mainly because of the associated disadvantages of these techniques which mainly include lengthy procedures, harsh processing conditions, rendering the approach unsuitable for the encapsulation of sensitive substances and poor morphology control.
The following steps were taken to achieve the aim of this work:
Use of surfactant vesicles in templating approaches requires an efficient synthesis protocol capable to provide stable vesicle dispersions of relatively narrow size distribution in a reproducible way. In Chapter 2, three simple and readily available techniques for the synthesis of vesicles of a commercially available surfactant dimethydioctadecyl ammonium bromide (DODAB) were explored. Vesicles obtained by membrane extrusion, bath sonication and by probe sonication were evaluated on the basis of properties most relevant for templating. These include their size, polydispersity, morphology, stability and thermal behavior. The chapter finally concludes membrane extrusion to be a suitable technique to provide stable vesicle populations for templating reactions.
In the next step, the stability of extruded vesicles was explored for endo-templating. As described in chapter 3, this was achieved by using the hydrophobic bilayer of the vesicles as a confined reaction space for the polymerization of hydrophobic monomers. The resulting vesicle-polymer hybrid morphologies were characterized by cryoTEM.
Chapter 4 describes a RAFT copolymer based templating/encapsulation approach for the synthesis of flat nanocomposite latex particles. Using the RAFT agent dibenzyl trithiocarbonate, a series of amphipatic living random RAFT copolymers with different combinations of acrylic acid and butyl acrylate units and with different chain lengths were synthesized. These anionic RAFT copolymers were used as living stabilizers for the flat, hexagonal gibbsite platelets and chain extended to form a polymeric shell by starved feed emulsion polymerization. CryoTEM characterization of the resulting composite latexes
demonstrates the formation of anisotropic (flat) latex particles with mostly one platelet per particle. Extending the approach further, via the use of cationic RAFT copolymers of dimethyl aminoethyl acrylate and butyl acrylate, the encapsulation of anionic Na-montmorilonite platelets is demonstrated.
Chapter 5 describes the templated synthesis of polymeric nanocapsules using the RAFT copolymer approach. Anionic RAFT copolymers of acrylic acid and butyl acrylate were adsorbed on cationic DODAB vesicles and later, using their “living” RAFT moieties, were chain extended to form nanocapsules. Finally, the concept was extended further for the synthesis of responsive nanocapsules. For this propose, RAFT copolymers adsorbed on DODAB vesicles were grown to form a crosslinked shell using a monomer feed comprising methyl methacrylate (MMA), tertiary butyl acrylate (t-BA) and the cross linker ethylene glycol dimethacrylate (EGDMA). Subsequent hydrolysis of the tertiary butyl ester units resulted in the formation of pH-responsive nanocapsules.
References
1. Wolde, A. T., Nanotechnology: towards a molecular construction kit. STT
Netherlands Study Centre for Technology Trends: Den Haag, 1998; p 357.
2. Ozine, G. A., and Arsenault, A. C., Nanochemistry: A Chemical Approach to
Nanomaterials. Royal Society of Chemistry: Cambridge, 2005.
3. Huczko, A., Template-based Synthesis of Nanomaterials. Applied Physics A:
Materials Science & Processing 2000, 70, (4), 365-376.
4. Holmberg, K., Surfactant-templated nanomaterials synthesis. Journal of Colloid and
Interface Science 2004, 274, (2), 355-364.
5. Wade, T. L.; Wegrowe, J. E., Template synthesis of nanomaterials. The European
Physical Journal Applied Physics 2005, 29, (1), 3-22.
6. Förster, S., Amphiphilic Block Copolymers for Templating Applications. In Colloid
7. Hentze, H. P.; Co, C.; McKelvey, C.; Kaler, E., Templating Vesicles, Microemulsions, and Lyotropic Mesophases by Organic Polymerization Processes. In
Colloid Chemistry I, 2003; pp 197-223.
8. Bourgeat-Lami, E., Colloidal Polymers Synthesis and Characterization, chapter 8.
CRC Press: 2003.
9. Zhou, Y.; Shimizu, T., Lipid Nanotubes: A Unique Template To Create Diverse One-Dimensional Nanostructures†Chemistry of Materials 2007, 20, (3), 625-633.
10. Wang, Y.; Angelatos, A. S.; Caruso, F., Template Synthesis of Nanostructured Materials via Layer-by-Layer Assembly†Chemistry of Materials 2007, 20, (3), 848-858.
11. Caruso, F., Hollow Inorganic Capsules via Colloid-Templated Layer-by-Layer Electrostatic Assembly. In Colloid Chemistry II, 2003; pp 145-168.
12. van Herk, A. M., Chemistry and technology of emulsion polymerisation. Blackwell Publishing: 2005.
13. McDonald, C. J.; Devon, M. J., Hollow latex particles: synthesis and applications.
Advances in Colloid and Interface Science 2002, 99, (3), 181-213.
14. Mock, E. B.; De Bruyn, H.; Hawkett, B. S.; Gilbert, R. G.; Zukoski, C. F., Synthesis of Anisotropic Nanoparticles by Seeded Emulsion Polymerization. Langmuir 2006, 22, (9), 4037-4043.
15. Herrera, V.; Palmillas, Z.; Pirri, R.; Reyes, Y.; Leiza, J. R.; Asua, J. M., Morphology of Three-Phase PS/PBA Composite Latex Particles Containing in Situ Produced Block Copolymers. Macromolecules 2010, 43, (3), 1356-1363.
16. Jiang, W.; Kim Betty, Y. S.; Rutka, J. T.; Chan Warren, C. W., Nanoparticle-mediated cellular response is size-dependent. Nature Nanotechnology 2008, 3, (3), 145-150.
17. Yongxing, H.; Jianping, G.; Tierui, Z.; Yadong, Y., A Blown Film Process to Disk-Shaped Polymer Ellipsoids. Advanced Materials 2008, 20, (23), 4599-4602.
18. Yin, Y.; Xia, Y., Self-Assembly of Monodispersed Spherical Colloids into Complex Aggregates with Well-Defined Sizes, Shapes, and Structures. Advanced Materials 2001, 13, (4), 267-271.
19. Lee, D. I., Nanostructured latexes made by a sequential multistage emulsion polymerization. Journal of Polymer Science Part A: Polymer Chemistry 2006, 44, (9), 2826-2836.
20. Okubo, Studies on suspension and emulsion. LI. Peculiar morphology of composite polymer particles produced by seeded emulsion polymerization. Journal of Polymer
Science Polymer Letters Edition 1982, 20, (1), 45.
21. Okubo, Studies on suspension and emulsion. XLVII. Anomalous composite polymer emulsion particles with voids produced by seeded emulsion polymerization. Journal
of Polymer Science Polymer Letters Edition 1981, 19, (3), 143.
22. Skjeltorp, Preparation of nonspherical, monodisperse polymer particles and their self-organization. Journal of Colloid and Interface Science 1986, 113, (2), 577.
23. Caruso, F.; Caruso, R. A.; Helmuth, M., Nanoengineering of inorganic and hybrid hollow spheres by colloidal templating. Science 1998, 282, (5391), 1111.
24. Edwin, D.; Gleb, B. S.; Frank, C.; Sean, A. D.; Helmuth, M., Novel Hollow Polymer Shells by Colloid-Templated Assembly of Polyelectrolytes. Angewandte Chemie
International Edition 1998, 37, (16), 2201-2205.
25. 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. Polymers for Advanced Technologies 1998, 9, (10-11), 759-767.
26. Johnston, A. P. R.; Cortez, C.; Angelatos, A. S.; Caruso, F., Layer-by-layer engineered capsules and their applications. Current Opinion in Colloid & Interface
Science 2006, 11, (4), 203-209.
27. Lee, D.; Rubner, M. F.; Cohen, R. E., All-Nanoparticle Thin-Film Coatings. Nano
Letters 2006, 6, (10), 2305-2312.
28. Srivastava, S.; Kotov, N. A., Composite Layer-by-Layer (LBL) Assembly with Inorganic Nanoparticles and Nanowires. Accounts of Chemical Research 2008, 41, (12), 1831-1841.
29. Cho, J.; Hong, J.; Char, K.; Caruso, F., Nanoporous Block Copolymer Micelle/Micelle Multilayer Films with Dual Optical Properties. Journal of the
American Chemical Society 2006, 128, (30), 9935-9942.
30. Vladimir, V. T., Dendritic Macromolecules at Interfaces. Advanced Materials 1998, 10, (3), 253-257.
31. Mamedov, A., Molecular design of strong single-wall carbon nanotube/polyelectrolyte multilayer composites. Nature Materials 2002, 1, (3), 190. 32. Caruso, F., Hollow Capsule Processing through Colloidal Templating and
33. Germain, M.; Grube, S.; Carriere, V.; Richard-Foy, H.; Winterhalter, M.; Fournier, D., Composite Nanocapsules: Lipid Vesicles Covered with Several Layers of Crosslinked Polyelectrolytes. Advanced Materials 2006, 18, (21), 2868-2871.
34. Pastoriza-Santos, I.; Schöler, B.; Caruso, F., Core-Shell Colloids and Hollow Polyelectrolyte Capsules Based on Diazoresins. Advanced Functional Materials 2001, 11, (2), 122-128.
35. Fujimoto, K.; Toyoda, T.; Fukui, Y., Preparation of Bionanocapsules by the Layer-by-Layer Deposition of Polypeptides onto a Liposome. Macromolecules 2007, 40, (14), 5122-5128.
36. Murtagh, J.; Thomas, J. K., Mobility and reactivity in colloidal aggregates with motion restricted by polymerization. Faraday Discussions of the Chemical Society 1986, 81, (1), 127-136.
37. Kurja, J.; Nolte, R. J. M.; Maxwell, I. A.; German, A. L., Free radical polymerization of styrene in dioctadecyldimethylammonium bromide vesicles. Polymer 1993, 34, (10), 2045-2049.
38. Nathalie, P.; Evelyne, N.; Annabelle, P.; Guy, L., Nanoparticles from vesicle polymerization: Characterization and kinetic study. Journal of Polymer Science Part
A: Polymer Chemistry 1996, 34, (5), 729-737.
39. Morgan, J. D.; Johnson, C. A.; Kaler, E. W., Polymerization of Equilibrium Vesicles.
Langmuir 1997, 13, (24), 6447-6451.
40. Gomes, J. F. P. d. S.; Sonnen, A. F. P.; Kronenberger, A.; Fritz, J.; Coelho, M. A. N.; Fournier, D.; Fournier-Noel, C.; Mauzac, M.; Winterhalter, M., Stable Polymethacrylate Nanocapsules from Ultraviolet Light-Induced Template Radical Polymerization of Unilamellar Liposomes. Langmuir 2006, 22, (18), 7755-7759. 41. Hotz, J.; Meier, W., Vesicle-Templated Polymer Hollow Spheres. Langmuir 1998,
14, (5), 1031-1036.
42. 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, (7), 3165-3174.
43. Hubert, D. H. W.; Jung, M.; German, A. L., Vesicle Templating. Advanced Materials 2000, 12, (17), 1291-1294.
44. Barner-Kowollik, C.; Buback, M.; Charleux, B.; Coote, M., L.; Drache, M.; Fukuda, T.; Goto, A.; Klumperman, B.; Lowe, A., B.; Mcleary, J., B.; Moad, G.; Monteiro, M., J.; Sanderson, R., D.; Tonge, M., P.; Vana, P., Mechanism and kinetics of dithiobenzoate-mediated RAFT polymerization. I. The current situation. Journal of
45. Cunningham, M. F., Living/controlled radical polymerizations in dispersed phase systems. Progress in Polymer Science 2002, 27, (6), 1039-1067.
46. Qiu, J.; Charleux, B.; Matyjaszewski, K., Controlled/living radical polymerization in aqueous media: homogeneous and heterogeneous systems. Progress in Polymer
Science 2001, 26, (10), 2083-2134.
47. Perrier, S., Macromolecular design via reversible addition-fragmentation chain transfer (RAFT)/xanthates (MADIX) polymerization. Journal of Polymer Science
Part A Polymer Chemistry 2005, 43, (22), 5347.
48. Chiefari, J.; Chong, Y. K.; Ercole, F.; Krstina, J.; Jeffery, J.; Le, T. P. T.; Mayadunne, R. T. A.; Meijs, G. F.; Moad, C. L.; Moad, G.; Rizzardo, E.; Thang, S. H., Living Free-Radical Polymerization by Reversible Addition−Fragmentation Chain Transfer: The RAFT Process. Macromolecules 1998, 31, (16), 5559-5562.
49. Davis, T., The quest for control in free-radical polymerization. Chemistry in Australia 1998, 65, (10), 12.
50. Lowe, A. B.; McCormick, C. L., Reversible addition-fragmentation chain transfer (RAFT) radical polymerization and the synthesis of water-soluble (co)polymers under homogeneous conditions in organic and aqueous media. Progress in Polymer Science 2007, 32, (3), 283-351.
51. Moad, G.; Rizzardo, E.; Thang, S. H., Living Radical Polymerization by the RAFT Process. Australian Journal of Chemistry 2005, 58, (6), 379-410.
52. Arnaud, F.; Marie-Thérèse, C., Experimental Requirements for an Efficient Control of Free-Radical Polymerizations via the Reversible Addition-Fragmentation Chain Transfer (RAFT) Process. Macromolecular Rapid Communications 2006, 27, (9), 653-692.
53. Lai, J. T.; Shea, R., Controlled radical polymerization by carboxyl- and hydroxyl-terminated dithiocarbamates and xanthates. Journal of Polymer Science Part A
Polymer Chemistry 2006, 44, (14), 4298.
54. Schilli, C.; Lanzendorfer, M. G.; Muller, A. H. E., Benzyl and Cumyl Dithiocarbamates as Chain Transfer Agents in the RAFT Polymerization of N-Isopropylacrylamide. In Situ FT-NIR and MALDI−TOF MS Investigation.
Macromolecules 2002, 35, (18), 6819-6827.
55. Liu, J.; Hong, C.-Y.; Pan, C.-Y., Dihydroxyl-terminated telechelic polymers prepared by RAFT polymerization using functional trithiocarbonate as chain transfer agent.
56. Lai, J. T.; Filla, D.; Shea, R., Functional Polymers from Novel Carboxyl-Terminated Trithiocarbonates as Highly Efficient RAFT Agents. Macromolecules 2002, 35, (18), 6754-6756.
57. Mayadunne, R. T. A.; Rizzardo, E.; Chiefari, J.; Krstina, J.; Moad, G.; Postma, A.; Thang, S. H., Living Polymers by the Use of Trithiocarbonates as Reversible Addition−Fragmentation Chain Transfer (RAFT) Agents: ABA Triblock Copolymers by Radical Polymerization in Two Steps. Macromolecules 2000, 33, (2), 243-245.
58. McLeary, J. B.; Klumperman, B., RAFT mediated polymerisation in heterogeneous media. Soft Matter 2006, 2, (1), 45-53.
59. Manguian, M.; Save, M.; Charleux, B., Batch emulsion polymerization of styrene stabilized by a hydrophilic macro-RAFT agent. Macromolecular Rapid
Communications 2006, 27, (6), 399.
60. Ferguson, C. J.; Hughes, R. J.; Pham, B. T. T.; Hawkett, B. S.; Gilbert, R. G.; Serelis, A. K.; Such, C. H., Effective ab Initio Emulsion Polymerization under RAFT Control.
Macromolecules 2002, 35, (25), 9243-9245.
61. Pham, B. T. T.; Nguyen, D.; Ferguson, C. J.; Hawkett, B. S.; Serelis, A. K.; Such, C. H., Miniemulsion Polymerization Stabilized by Amphipathic Macro RAFT Agents.
Macromolecules 2003, 36, (24), 8907-8909.
62. Ferguson, C. J.; Hughes, R. J.; Nguyen, D.; Pham, B. T. T.; Gilbert, R. G.; Serelis, A. K.; Such, C. H.; Hawkett, B. S., Ab Initio Emulsion Polymerization by RAFT-Controlled Self-Assembly. Macromolecules 2005, 38, (6), 2191-2204.
63. Charmot, D.; Corpart, P.; Adam, H.; Zard, S. Z.; Biadatti, T.; Bouhadir, G., Controlled radical polymerization in dispersed media. Macromolecular Symposia 2000, 150, (1), 23-32.
64. Uzulina, I.; Kanagasabapathy, S.; Claverie, J., Reversible addition fragmentation transfer (RAFT) polymerization in emulsion. Macromolecular Symposia 2000, 150, (1), 33-38.
65. Prescott, S. W.; Ballard, M. J.; Rizzardo, E.; Gilbert, R. G., Successful Use of RAFT Techniques in Seeded Emulsion Polymerization of Styrene: Living Character, RAFT Agent Transport, and Rate of Polymerization. Macromolecules 2002, 35, (14), 5417-5425.
66. Delaittre, G.; Nicolas, J.; Lefay, C.; Save, M.; Charleux, B., Surfactant-free synthesis of amphiphilic diblock copolymer nanoparticles via nitroxide-mediated emulsion polymerization. Chemical Communications 2005, (5), 614-616.
67. Morgan, S. E.; Jones, P.; Lamont, A. S.; Heidenreich, A.; McCormick, C. L., Layer-by-Layer Assembly of pH-Responsive, Compositionally Controlled (Co)polyelectrolytes Synthesized via RAFT. Langmuir 2006, 23, (1), 230-240.
68. Boyer, C.; Whittaker, M. R.; Nouvel, C.; Davis, T. P., Synthesis of Hollow Polymer Nanocapsules Exploiting Gold Nanoparticles as Sacrificial Templates.
Macromolecules 2010, 43, (4), 1792-1799.
69. Liu, J.; Tao, L.; Yang, W.; Li, D.; Boyer, C.; Wuhrer, R.; Braet, F.; Davis, T. P., Synthesis, Characterization, and Multilayer Assembly of pH Sensitive Graphene−Polymer Nanocomposites. Langmuir 2010, 26, (12), 10068-10075.
70. Kim, B.-S.; Gao, H.; Argun, A. A.; Matyjaszewski, K.; Hammond, P. T., All-Star Polymer Multilayers as pH-Responsive Nanofilms. Macromolecules 2008, 42, (1), 368-375.
71. Jia, Z.; Liu, J.; Boyer, C.; Davis, T. P.; Bulmus, V., Functional Disulfide-Stabilized Polymer−Protein Particles. Biomacromolecules 2009, 10, (12), 3253-3258.
72. Kakwere, H.; Perrier, S. b., Orthogonal "Relay" Reactions for Designing Functionalized Soft Nanoparticles. Journal of the American Chemical Society 2009, 131, (5), 1889-1895.
73. Cortez, C.; Tomaskovic-Crook, E.; Johnston, A. P. R.; Scott, A. M.; Nice, E. C.; Heath, J. K.; Caruso, F., Influence of Size, Surface, Cell Line, and Kinetic Properties on the Specific Binding of A33 Antigen-Targeted Multilayered Particles and Capsules to Colorectal Cancer Cells. ACS Nano 2007, 1, (2), 93-102.
74. Suchao-in, N.; Chirachanchai, S.; Perrier, S., pH- and thermo-multi-responsive fluorescent micelles from block copolymers via reversible addition fragmentation chain transfer (RAFT) polymerization. Polymer 2009, 50, (17), 4151-4158.
75. Zelikin, A. N.; Such, G. K.; Postma, A.; Caruso, F., Poly(vinylpyrrolidone) for Bioconjugation and Surface Ligand Immobilization. Biomacromolecules 2007, 8, (9), 2950-2953.
76. Zareie, H. M.; Boyer, C.; Bulmus, V.; Nateghi, E.; Davis, T. P., Temperature-Responsive Self-Assembled Monolayers of Oligo(ethylene glycol): Control of Biomolecular Recognition. ACS Nano 2008, 2, (4), 757-765.
77. Nguyen, D.; Zondanos, H. S.; Farrugia, J. M.; Serelis, A. K.; Such, C. H.; Hawkett, B. S., Pigment Encapsulation by Emulsion Polymerization Using Macro-RAFT Copolymers. Langmuir 2008, 24, (5), 2140-2150.
78. Majumdar, D.; Blanton, T. N.; Schwark, D. W., Clay-polymer nanocomposite
79. Sugama, T., Polyphenylenesulfied/montomorillonite clay nanocomposite coatings: Their efficacy in protecting steel against corrosion. Materials Letters 2006, 60, (21-22), 2700-2706.
80. Negrete-Herrera, N.; Putaux, J. L.; David, L.; De Haas, F.; Bourgeat-Lami, E., Polymer/Laponite composite latexes: Particle morphology, film microstructure, and properties. Macromolecular Rapid Communications 2007, 28, (15), 1567-1573. 81. Okubo, M.; Ito, A.; Hashiba, A., Production of submicron-sized multihollow polymer
particles having high transition temperatures by the stepwise alkali/acid method.
Colloid & Polymer Science 1996, 274, (5), 428-432.
82. 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, (5), 1593-1605.
83. 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, (9), 3122-3135.
Chapter 2
Synthesis and Characterization of DODAB
Vesicles
Abstract: In this chapter, three simple and readily available techniques, namely membrane extrusion, bath sonication and probe sonication, are explored for the synthesis of unilamellar vesicles of dimethydioctadecyl ammonium bromide (DODAB). The vesicles were characterized for their size, polydispersity, morphology, stability and thermal behavior. To determine the main gel-to-liquid crystalline phase transition temperature (Tm), absorbance
measurements were found to be a simple and fast method. Morphological characterizations were performed by cryoTEM. It was revealed that the behavior of DODAB is different from conventional lipids in terms of higher Tm (44 °C) and peculiar bilayer angularities. Upon
hydration at 60 °C, DODAB molecules spontaneously arranged in large vesicles and bilayer fragments due to unfavorable interactions with water. Sonication (bath and probe) of the hydrated dispersion yields small unilamellar vesicles with a broad size distribution. CryoTEM revealed that sonication gives vesicles with sharp edges, open bilayer fragments and lens-shaped morphology. As another tool to prepare unilamellar vesicles, membrane extrusion of hydrated DODAB dispersions was performed using two different polycarbonate filter sizes (100 nm and 200 nm). Extrusion resulted in the formation of stable unilamellar vesicle populations with the final size depending on the membrane pore diameter. Morphological characterization using cryoTEM revealed that the vesicles are unilamellar and have less sharp corners and edges, compared to sonicated vesicles. Extrusion was found to be a reproducible and simple method for the production of large unilamellar vesicles of the desirable size range and relatively low polydispersity.
2.1 Introduction
In this chapter we, investigate the preparation and characterization of vesicles formed by a synthetic surfactant, dimethydioctadecyl ammonium bromide (abbreviated as DODAB, structure shown in Figure 1), using commonly used vesicle forming protocols. DODAB is commercially available and a very widely studied double-chained cationic amphiphile. It belongs to a class of synthetic surfactants known as dioctadecyldimethylammonium salts
which are the first studied synthetic surfactants capable of forming vesicles in aqueous
solutions.1 Among this class of surfactants, dioctadecyldimethylammonium bromide and
chloride (DODAB and DODAC) are the two most widely used amphiphiles.2, 3 The
properties and structure of their vesicles largely depend on their concentration, solvent ionic strength, temperature and the method used for vesicle preparation.3 Vesicles can be formed
by simply dispersing the surfactant in water and warming the resulting mixture at a
temperature well above the gel-to-liquid crystalline transition temperature.4 For this work,
DODAB is chosen as vesicle forming surfactant because it is commercially available and a very widely studied cationic amphiphile for many different applications. Besides, a relatively high gel-to-liquid crystalline phase transition temperature (Tm) of its bilayers makes DODAB
a suitable candidate for many templating studies where bilayer strength is an obvious requirement.
N+ Br
In spite of being a commonly used vesicle forming amphiphile, the structure and properties of DODAB vesicles are still not fully understood. There are conflicting results reported in the literature about the properties of these vesicles such as size, main gel-to-liquid crystalline phase transition temperature (Tm), stability, morphology and structural organization (whether
they are closed or fragmented bilayers, unilamellar or multilamellar structures). It is however known that the properties of these vesicles such as size, shape, polydispersity, phase behavior, surface potential, degree of ionization, permeability and physical stability largely depend on the vesicle preparation method and the physical conditions of the solvent, such as temperature and ionic strength.2, 5-7 At temperatures above the main gel-to-liquid crystalline phase transition temperature (Tm), DODAB molecules self-assemble in aqueous solution into
stable giant unilamellar vesicles and large bilayer fragments.4, 8 Most of the reported procedures in the literature to prepare vesicles involve sonication1, 9, solvent (mostly chloroform) injection6 and membrane extrusion.6, 10 Membrane extrusion has an advantage of controlling the size of the vesicles by the pore size of the membrane filter.6 However, despite of being a very common vesicle preparation protocol for phospholipids, its use for DODAB is limited (due to the relatively high Tm).
The goal of this study is to evaluate the properties of unilamellar vesicles of DODAB prepared by membrane extrusion, bath and probe sonication, in order to determine if the method of preparation has any influence on selected bilayer properties. The most relevant properties of the vesicles to be used as template are their shape, size, polydispersity, morphology, stability and thermal behavior.
2.1.1 Hydration of vesicle forming surfactants
The first step in vesicle formation involves the hydration of the lamellar crystals and subsequent aggregation of the amphiphile molecules.11 When dried crystals of vesicle forming surfactant are allowed to come in contact with water, they swell to form a variety of liquid crystalline structures. The phenomenon is analogous to the lyotropic mesomorphism observed with soaps of long-chain acids and other amphiphatic molecules.11, 12 The driving force for this self assembly is the so-called hydrophobic effect.13 The three-dimensional configuration of the resulting hydrated crystals depends on the amount of water, degree of
hydration and the temperature.5, 14 In general, most vesicle forming amphiphiles form liquid crystalline phases (Lc) at low temperatures and at low hydration.12, 15 With increasing
hydration, amphiphiles adopt a phase in which the hydrocarbon chains are still ordered in the all-trans conformation, where they exhibit long-axis rotation. In the gel-phase, the head groups are normally disordered and the lateral correlation between the two halves of the bilayers is weak or non-existent.15 The thickness of the water layer between the two successive bilayers depends on factors such as water content, temperature, charge, size and polarity of the amphiphile headgroup. With increasing temperature, the mobility of the amphiphiles within the bilayers increases accompanied by the conformational changes of the tails (increased fraction of gauche conformation) causing an increase in the cross-sectional area of the chains. At a certain temperature (Tm), these chains melt to a liquid-like
conformation. This melting corresponds to a transformation of the gel-like phase into the liquid-crystalline phase (Lα). The gel-to-liquid- crystalline phase transition is an important
intrinsic property of the bilayer. In the liquid crystalline phase the lateral diffusion of the amphiphiles increases rapidly and the phase transition coincides with the thinning of the bilayer membrane.
2.1.2 Vesicle formation methods
The basic principle for the formation of vesicles is the hydrophilic/hydrophobic interaction between surfactant-surfactant and surfactant-water molecules. Bilayers form spontaneously when the relevant surfactant disperses in water, due to the unfavorable interaction of water and surfactant.5, 14, 15 Therefore the purpose of the vesicle formation procedures is not to make surfactant self-assemble, as this happens spontaneously, but to disrupt spontaneously formed bilayers and multilamellar vesicles, by dissipation of energy to get the vesicles of required size and structure and to entrap material within them.15 Input of energy in the form of sonication, homogenization, shaking, heating etc4, 9, 16 results in the arrangement of the surfactant molecules, in the form of bilayer vesicles, to achieve a thermodynamic equilibrium in the aqueous phase. This can be done by using a variety of methods, for example, sonication9, thin-film hydration9, 15, solvent (mostly ethanol, ether or chloroform) injection6 and membrane extrusion of the amphiphile dispersion. However, all these methods involve at least three of the following stages: Making surfactant film by evaporating the solvent, dispersion of the surfactant in aqueous media, application of energy, purification and analysis
of the resultant vesicle dispersion. In this work we use sonication and membrane extrusion methods which are by far the simplest and widely applied methods to obtain vesicles of various size ranges.
Sonication. Sonication is primarily used for the preparation of small unilamellar vesicles
(SUVs) but can also be used for the subsequent sonication of large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) prepared by other methods such as extrusion or solvent injection.15 Sonication can either be a bath type or a probe type, normally performed under an inert atmosphere, usually nitrogen or argon. The sample containing surfactant or lipid hydrated dispersion is irradiated with pulsed, high frequency sound waves (sonic energy). Ultrasonic irradiation imparts energy at a high level to the lipid or surfactant suspension leading to their break-up into small unilamellar vesicles (SUVs) of diameter typically around 15 to 50 nanometers.15 Sonication is still the method of choice for producing the smallest vesicle sizes. For instance, in biomedical formulations, sonication is the most frequently used method as it serves the purpose of producing a homogenous dispersion of small vesicles capable of greater tissue penetration.15
Probe sonication is usually applied to produce small volumes of vesicle dispersions. An ultrasonic transducer probe is immersed into the surfactant dispersion, maintained at a certain temperature above the main gel-to-liquid crystalline phase transition temperature (Tm) of the
surfactant. However, as sonication produces a lot of heat, it is often necessary to maintain the temperature of the dispersion by using a cooling bath to avoid the evaporation of the solvent and more importantly, degradation of the lipid or surfactant.
In bath sonication, a flask or test tube containing surfactant dispersion is placed in the
water bath of the sonicator followed by sonication at a temperature above the main gel-to-liquid crystalline phase transition temperature (Tm) of the surfactant. Bath sonication is a
milder technique in comparison to probe sonication. Hence, it prevents the unwanted degradation of the surfactant and the ingredients to be encapsulated therein. Relatively large sample volumes can be handled, ultrasonic irradiation is more uniform and better reproducibility can be achieved provided the sample flask is positioned carefully at the same position each time. Besides, temperature control is relatively easy as the heat generated during ultrasonication can be removed by the water bath. The main drawback is the broad
size distribution of the resulting vesicle population. Moreover, in bath sonication, as the energy is dispersed over a much larger area, it is not possible to reach the minimum size limit for sonicated vesicles and probe sonication is still the method of choice to get smaller vesicles.
Membrane Extrusion. Extrusion represents a simple, gentler and widely used method for
the synthesis and size reduction of the vesicles.15 In extrusion, hydrated surfactant dispersion is heated above the main gel-to-liquid crystalline phase transition temperature (Tm) and then
repeatedly forced or extruded through a membrane filter of defined pore size using either pressurized gas or syringe-based plunger setups. This can be achieved at much lower pressures (<100 p.s.i) then are required for similar approaches such as the French press cell.15 Few stacks of track-etched filter membranes are normally used in extrusion. These membranes consist of thin continuous sheets of inert polymeric material such as polycarbonate. Several passes through the membrane are normally needed to get vesicles of reasonably narrow size distributions.15, 16 Extrusion reduces the size of the vesicle population to somewhat lower than the pore diameter of the membrane; however a small population of vesicles larger in size than the pore diameter can also squeeze through the pores.16, 17 Extrusion can be done using the apparatus normally employed for pressure filtration, hence the scaling up of the process is relatively easy. A variety of different lipids/surfactants can be
used to form stable vesicles by this method. A major advantage of extrusion over the other
procedures is the fast preparation time and the production of vesicles directly from the hydrated dispersion, thus eliminating the problems of removing the organic solvents and debris from the titanium tip associated with other techniques.15
2.2 Experimental
Section
Materials: Dimethyldioctadecyl ammonium bromide (DODAB, Acros, 99%) and dodecyltrimethylammonium bromide (DTAB, Aldrich, 99%) were used as received. Polycarbonate track-etched membrane filters (100 and 200 nm pore sizes) were obtained from Millipore.
Vesicle Preparation: Large unilamellar vesicles (LUVs) were prepared by membrane extrusion, probe sonication and bath sonication. Details of the procedures are given below:
Figure 2. Membrane extrusion set-up.
Extrusion. The experimental set-up for extrusion consists of a pressure vessel fitted with a
nitrogen line, a filter holder containing three stacked polycarbonate filters of appropriate pore size, and a collection vessel (Figure 2). The whole assembly was temperature controlled using a thermostated heating tape. The extruder was loaded with a 10 mM DODAB aqueous dispersion and heated to 65 °C, well above the main gel-to-liquid crystalline phase transition
temperature (Tm ~ 44 °C). Extrusion was accomplished by pressurizing the vessel with
nitrogen at 7 bar, which forced the dispersion to pass through the pores of the membrane filter. The filtrate was collected in the collection vessel and was stirred and maintained at 65 °C before being recycled for a second extrusion run. After every extrusion run, the particle diameter was monitored by DLS. Typically, 4 to 5 passes were found to be enough to get a constant particle diameter. After the completion of the extrusion, the resulting vesicle dispersion was maintained at 65 °C for more than 12 hours before cooling down to room temperature.
Sonication. Probe sonicated, small unilamellar vesicles (SUVs) were obtained by
sonication of a preheated DODAB dispersion using a Vibracell tip sonicator (at 50% amplitude). Sonication was accomplished in 2 minutes steps, with one minute cooling time to avoid evaporation, for a total of 10 minutes. Any debris from the sonication probe was removed by simple filtration. Bath sonicated vesicles were obtained using a Vibracell bath sonicator. The sample was contained in a 100 ml round bottom flask maintained at 65 °C and sonicated for 90 minutes. The vesicle dispersions obtained by probe and bath type sonications were maintained at 65 °C overnight before slowly cooling down to room temperature.
Characterizations: 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. Particle sizes were measured using the conditions optimized by the instrument (position in the sample and attenuation). The autocorrelation function was converted to particle size distribution using the standard software settings (“General purpose (normal distribution)”).
Cryogenic transmission electron microscopy (cryoTEM) measurements were performed on a FEI Tecnai 20, type Sphera TEM instrument (with a LaB6 filament, operating voltage =
200 kV). The sample vitrification procedure was performed using an automated vitrification robot (FEI Vitrobot Mark III). A 3 µl sample was applied to a Quantifoil grid (R 2/2, Quantifoil Micro Tools GmbH; freshly glow discharged for 40 seconds just prior to use) within the environmental chamber of the Vitrobot and the excess liquid was blotted away. The thin film thus formed was shot into melting ethane. The grid containing the vitrified film
was immediately transferred to a cryoholder (Gatan 626) and observed under low dose conditions at -170 °C.
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 recorded in the temperature range of 20 – 60 °C with 0.5 °C increments and allowing a thermal equilibration time of 2 minutes between measurements.
2.3 Results and Discussion
In this work we looked for a suitable method for the synthesis of a model vesicle template. Three different vesicle formatting procedures namely, membrane extrusion, probe sonication and bath sonication were compared to determine the influence of the method of preparation on the physical and morphological properties of the resulting vesicles.
2.3.1 Hydration behavior of DODAB
Vesicle formation starts with the hydration of the lamellar crystals which leads to the swelling of the repetitive water layer.5, 15 At room temperature, the hydration of DODAB crystals was found to be very slow as the surfactant remains suspended as a solid in the aqueous phase even after days of mixing. Increasing temperature increases the hydration of the amphiphile and transform the coarse dispersion of crystals into a more gel-like appearance, and the color of the dispersion becomes bluish translucent.
20 25 30 35 40 45 50 55 60 65 1.35 1.40 1.45 1.50 1.55 1.60 1.65 1.70 1.75
Ab
so
rb
an
ce
(A
U
)
Temperature °C
Figure 3. Absorbance as a function of temperature of 10 mM DODAB dispersion.
The hydrated dispersion probed by measuring the apparent absorbance (turbidity) as a function of temperature. As shown in Figure 3, the absorbance decreases with increasing temperature until the temperature reaches around 40 °C at which a steep change in the absorbance is observed. This is likely caused by a decrease in the bilayer density (hence decrease in the refractive index) as the membrane undergoes a transition from an ordered gel phase to the liquid crystalline phase (T > Tm). This transition is commonly known as the
main gel-to-liquid crystalline transition and is generally accompanied by a loosening of amphiphile packing and increasing the head group surface area.5, 15, 18, 19 As a result, the distance between the lamellar sheets further increases, accompanied by the increase mobility of the amphiphile molecules within the lamellar sheets, allowing the lamellar sheets to
