Synthesis of Bilayer-Coated Nanogels by Selective Cross-Linking of Monomers inside Liposomes Joris P. Schillemans, Frits M. Flesch, Wim E. Hennink, and Cornelus F. van Nostrum* Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht UniVersity, P.O. Box 80.082, 3508 TB Utrecht, The Netherlands ReceiVed March 31, 2006; ReVised Manuscript ReceiVed June 26, 2006 ABSTRACT: In this study, bilayer-coated polyacrylamide hydrogel nanoparticles were prepared by photoinitiated polymerization of acrylamide (AA) and bis(acrylamide) (BA) in the inner compartment of liposomes. The liposomes were formed in AA/BA solutions from lipid/Triton X-100 (TX100) mixed micelles by adsorption of TX100 to Bio-Beads SM2 and were studied by dynamic light scattering and transmission electron microscopy. The hydrodynamic diameters of the liposomes were ∼100 nm with low polydispersity. Addition of ascorbic acid before photopolymerization prevented macroscopic hydrogel formation by inhibition of free-radical polymerization of nonencapsulated monomers. Bare nanogel particles were finally obtained by removal of the lipid bilayer. As opposed to the commonly used dilution method, this convenient and versatile method of nanogel synthesis will allow incorporation of membrane proteins in the bilayer and the use of monomers that readily pass the lipid membrane. Introduction In recent years hydrogel micro- and nanoparticles have been identified as valuable materials for a number of applications, including drug delivery and targeting, chromatography, etc. 1-5 Synthesis of these hydrogel particles can be accomplished in several ways, each with their own advantages and drawbacks. Emulsion polymerization is often used for the synthesis of both micro- and nanoparticles. Using this method, the size of the particles can be controlled by the size of the droplets in the w/o emulsions. 6,7 However, such systems are generally incom- patible with biological macromolecules like proteins. To ensure compatibility with proteins, water-in-water emulsions can be used for synthesis of microparticles, but control of the particle size down to the nanosize has not been accomplished thus far. 8,9 Another method that is commonly used for both micro- and nanoparticle synthesis is dispersion polymerization. 10,11 Upon initiation by a suitable initiator, dilute systems of monomer and cross-linker form polymer chains in solution, which collapse to form a precursor particle when reaching a critical chain length. Precursor particles continue to increase in size by additional chain growth and aggregation until a colloidally stable particle is formed. 12 The major drawback of this method is that it does not provide straightforward control over particle size, since the colloidal stability is dependent on monomer composition, initiator, and temperature. 13 An alternative, less commonly used approach is available for hydrogel particle synthesis. Mon- shipouri et al. reported on a method that used the internal compartment of lipid vesicles (liposomes) for the preparation of hydrogel particles, which allows good control over particle size and size distribution, and is compatible with biological macromolecules. 14 Using this method, Kazakov et al. 15,16 described the synthesis of thermo- and pH-sensitive hydrogel nanoparticles, Van Thienen et al. 17 synthesized biodegradable dextran nanogels, and Patton and Palmer synthesized hemoglobin- entrapped nanogels. 18-20 In addition to providing an alternative method for hydrogel nanoparticle synthesis, bilayer-coated nanogels as such could also have numerous applications. Combining the properties of both hydrogels and liposomes, they could be used as, for example, controlled release devices, artificial cell analogues, and biomimetic sensory systems. 15 Especially for the latter two, the incorporation of functional membrane proteins will be important. The method most commonly used for functional incorporation of membrane proteins in liposomes is detergent dilution. 21,22 The liposomal reactors used for nanoparticle synthesis described thus far, however, were prepared by freeze- thawing, sonication, and extrusion, which are less suitable for functional protein incorporation. Using the detergent dilution method, a dispersion of lipid-containing mixed micelles is diluted below the critical micelle concentration (cmc) of the detergent, which leads to the formation of liposomes. Several detergents can be used for this purpose, e.g., octylglucoside, sodium cholate, C8E12, and Triton X-100 (TX100). Addition- ally, several methods of detergent dilution are available such as dialysis, rapid addition of solvent, and adsorption by Bio- Beads SM-2. 23-25 Removal of TX100 by Bio-Beads has been described in the literature extensively. 26-29 It has been proven to be an effective, simple, and inexpensive way of incorporating functionally active membrane proteins in liposomes. Neverthe- less, up until now this method has not been used to form liposomes in solutions of hydrogel-forming monomers. Liposomes formed in monomer solutions have monomers both on the inside and on the outside. Therefore, prevention of polymerization on the outside of the liposomes is required for controlled nanoparticle synthesis. This was accomplished previ- ously by dilution of the exterior compartment to a concentration of monomers too low for hydrogel formation. 15-20 However, dilution causes a steep concentration gradient across the lipid membrane and is only applicable when the monomers cannot diffuse through the liposomal bilayer. When relatively lipophilic monomers are used, diffusion could occur. Additionally, incorporation of membrane proteins can lead to increased permeability of the bilayer. 30 Therefore, in the current study we developed a method for nanoparticles synthesis that will be suitable for the functional incorporation of membrane proteins. First, the liposomes are prepared by detergent removal in order * Corresponding author: Tel 0031-(0)30 2536970; e-mail c.f.vannostrum@ pharm.uu.nl. 5885 Macromolecules 2006, 39, 5885-5890 10.1021/ma060727t CCC: $33.50 © 2006 American Chemical Society Published on Web 07/26/2006