Cholesterol-mediated anchoring of enzyme-loaded liposomes within disulfide-stabilized polymer carrier capsules Rona Chandrawati, Brigitte Sta ¨dler, Almar Postma, Luke A. Connal, Siow-Feng Chong, Alexander N. Zelikin, Frank Caruso * Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia article info Article history: Received 27 April 2009 Accepted 22 July 2009 Available online 15 August 2009 Keywords: Polymer capsules Liposome Cholesterol Enzyme abstract Polymer capsules containing multiple liposomes, termed capsosomes, are a promising new concept toward the design of artificial cells. Herein, we report on the fundamental aspects underpinning the assembly of capsosomes. A stable and high loading of intact liposomal cargo into a polymer film was achieved by non-covalently sandwiching the liposomes between a tailor-made cholesterol-modified poly(L-lysine) (PLL c ) precursor layer and a poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA c ) capping layer. The film assembly, optimized on planar surfaces, was successfully transferred onto colloidal substrates, and a polymer membrane was subsequently assembled by the alternating adsorp- tion of poly(N-vinyl pyrrolidone) (PVP) and thiol-modified poly(methacrylic acid) (PMA SH ) onto the pre- adsorbed layer of liposomes. Upon removal of the silica template, stable capsosomes encapsulating the enzyme luciferase or b-lactamase within their liposomal sub-compartments were obtained at both assembly (pH 4) and physiological conditions (pH 7.4). Excellent retention of the liposomes and the enzymatic cargo within the polymer carrier capsules was observed for up to 14 days. These engineered capsosomes are particularly attractive as autonomous microreactors, which can be utilized to repetitively add smaller reactants to cause successive distinct reactions within the capsosomes and simultaneously release the products to the surrounding environment, bringing these systems one step closer toward constructing artificial cells. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Biological cells, which separate their interior from the exterior media by a lipid bilayer, operate by performing multiple enzymatic cascade reactions within predefined sub-compartments, the cell organelles. The lipid barrier is equipped with various biomolecules, including ion channels and transmembrane proteins, which serve as specific gates. Artificial cells [1,2], on the other hand, do not require the complex multifunctionality of their biological counterparts, but rather can be more simply designed to perform a specific activity. However, certain prerequisites have to be fulfilled. Among them is the need of a micron-sized vessel with specific permeability that provides the structural scaffold and the encapsulated machinery that enables confined specific reactions to be conducted. Polymer [3–5] or lipid [6,7] vesicles and polymer capsules [8–13] are suitable platforms that are being explored as microreactors to conduct encapsulated reac- tions and as advanced therapeutic delivery vehicles. Recently, we reported the construction of a new class of colloidal carriers/microreactors, termed capsosomes [14]. These are obtained by incorporating intact liposomes into polymer capsules, assembled by the layer-by-layer (LbL) technique [15,16]. The first generation of capsosomes we reported consist of intact, unsatu- rated, zwitterionic 50 nm liposomes incorporated in a non-biode- gradable polyelectrolyte film assembled from poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) [14]. We confirmed the functionality of the capsosomes by performing a triggered quantitative enzymatic reaction using b-lactamase encapsulated within the liposomal sub-compartments [17]. These capsosomes are of interest because the polymer capsule provides a structural scaffold with controllable permeability and the lipo- somes divide the interior into sub-compartments, thus potentially allowing parallel encapsulated enzymatic cascade reactions within confined systems. The liposomes are well-suited to encapsulate small hydrophobic and hydrophilic drugs or fragile biomolecules. Furthermore, these loaded liposomes can be incorporated in the polymer film at different regions, giving access to multi-strata films that are expected to be useful for the co-administration of complementary drugs. * Corresponding author. Tel.: þ61 3 8344 3461; fax: þ61 3 8344 4153. E-mail address: fcaruso@unimelb.edu.au (F. Caruso). Contents lists available at ScienceDirect Biomaterials journal homepage: www.elsevier.com/locate/biomaterials 0142-9612/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biomaterials.2009.07.040 Biomaterials 30 (2009) 5988–5998