Noncovalent Liposome Linkage and Miniaturization of Capsosomes for Drug Delivery Leticia Hosta-Rigau, †,‡ Rona Chandrawati, †,‡ Elli Saveriades, ‡ Pascal D. Odermatt, ‡ Almar Postma, § Francesca Ercole, § Kerry Breheney, | Kim L. Wark, | Brigitte Sta ¨ dler, ‡,⊥ and Frank Caruso* ,‡ Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia, CSIRO, Molecular and Health Technologies, Clayton, Victoria 3168, Australia, CSIRO, Molecular and Health Technologies, Parkville, Victoria 3052, Australia, and Interdisciplinary Nanoscience Center, Aarhus University, Aarhus 8000, Denmark Received August 31, 2010; Revised Manuscript Received October 25, 2010 We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA c ) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ∼1 μm diameter PMA c -based capsosomes, polymer hydrogel capsules that contain up to ∼2000 liposomal subcompart- ments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles. Introduction Liposomes, supramolecular assemblies of amphiphilic lipids, are a well established and versatile platform for diverse biomedical applications, including drug delivery, biosensing, and (encapsulated) catalysis. In drug delivery, liposomes are one of the few intravenously administrated carriers that have progressed to the clinic. 1-3 In recent years, polymer multilayer capsules, fabricated via the layer-by-layer (LbL) technique, 4-8 have emerged as par- ticularly promising scaffolds for the assembly of multifunctional carrier systems. Drug delivery is considered as one of the envisioned core applications of these capsules, largely because these carriers offer the potential of high payloads, stimuli- controlled degradation and drug release, and versatile surface functionalization with poly(ethylene glycol) 9-11 for reduced fouling or specific biological moieties 12,13 for targeting applications. So far only few reports have demonstrated the in vitro and in vivo delivery of active compounds using LbL-assembled polymer capsules. In the area of vaccination, an encapsulation strategy for oligopeptide antigens into disulfide poly(methacrylic acid) capsules via their conjugation to a carrier polymer, including the successful stimulation of the corresponding cells, has been reported. 14 These capsules were internalized by antigen presenting cells 15 and employed to deliver the full protein ovalbumin (OVA) and an immunogenic OVA-derived peptide sequence. Effective stimulation of the OVA-specific CD4 and CD8 T-cells was demonstrated in vitro and in vivo. 16 In another study, de Koker et al. used dextran sulfate/poly-L-arginine capsules to deliver OVA in vitro 17 and in vivo, 18 in both cases successfully stimulating the expected proliferation. In drug delivery, the controlled encapsulation of small, hydrophobic drugs into LbL polymer capsules has been examined using a range of approaches. The physical entrapment of small antitumoral compounds, 19-21 the covalent conjugation of the drug doxorubicin to a polymer that is used as a membrane building block, 22 the loading of the polymeric capsules with an emulsion containing the lipophilic compounds, 23,24 and the entrapment of the drug into mesoporous silica particles 25 have been considered. In all of these reports, the cargo was delivered to cells and its activity was assessed by measuring the viability of the cells. Some of these approaches exploit the increase in capsule/cell ratio to control the delivered payload. In this study, we explore the possibility of varying the amount of functional payload per carrier capsule to examine the dose response while keeping the capsule/cell ratio constant. Controlling the amount of loaded cargo per capsule, instead of varying the number of delivered capsules, may be beneficial in terms of delivering lower amounts of carrier materials (e.g., polymer building blocks or targeting moieties) and higher dosages of therapeutics. We recently introduced a new class of carriers, termed capsosomes. 26 These are nanoengineered liposome-loaded poly- mer hydrogel capsules assembled via the LbL technique. We have demonstrated their potential for the creation of synthetic microreactors 27,28 en route toward artificial cells and also for intracellular drug delivery. 29 Our previous reports have utilized ∼3 μm diameter hydrogel carrier capsules. To prepare such capsosomes, we introduced a novel noncovalent anchoring concept by using cholesterol-modified polymers to stably attach liposomes to polymeric films. 27,30 Cholesterol-functionalized polymers, for example, cholesterol-modified poly(L-lysine) (PLL c ) and poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA c ) have been successfully used as precursor, separation, and capping layers between liposomes and polymer films, allowing one to eight liposome deposition steps in a single * To whom correspondence should be addressed. E-mail: fcaruso@ unimelb.edu.au. † These authors contributed equally to this work. ‡ The University of Melbourne. § CSIRO, Clayton. | CSIRO, Parkville. ⊥ Aarhus University. Biomacromolecules 2010, 11, 3548–3555 3548 10.1021/bm101020e  2010 American Chemical Society Published on Web 11/19/2010