SHIMONI ET AL. VOL. 6 ’ NO. 2 ’ 1463–1472 ’ 2012 www.acsnano.org 1463 January 19, 2012 C 2012 American Chemical Society Macromolecule Functionalization of Disulfide-Bonded Polymer Hydrogel Capsules and Cancer Cell Targeting Olga Shimoni, † Almar Postma, †,‡ Yan Yan, † Andrew M. Scott, § Joan K. Heath, ^ Edouard C. Nice, ^, ) Alexander N. Zelikin, z and Frank Caruso †, * † Department of Chemical and Biomolecular Engineering, The University of Melbourne, Parkville, Victoria 3010, Australia, ‡ CSIRO Materials Science and Engineering, Clayton, Victoria 3168, Australia, § Ludwig Institute for Cancer Research, Melbourne, Austin Branch, Heidelberg, Victoria 3084, Australia, ^ Ludwig Institute for Cancer Research, Melbourne, Parkville Branch, Parkville, Victoria 3050, Australia, ) Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3800, Australia, and z Department of Chemistry and Interdisciplinary Nanoscience Centre (iNano), Aarhus University, Aarhus C 8000, Denmark F or more than a decade, multilayered polymer capsules have attracted inter- est as versatile candidates for a diverse range of biomedical applications, including drug delivery vehicles, 13 microreactors, 4 and as hierarchical components of artificial cells. 5,6 Significant effort has focused on engineering multilayered, responsive, and functional capsules with control over their physicochemical properties, such as size, permeability, and stability. Recently, we devel- oped a redox-responsive multilayered hy- drogel capsule system based on thiolated poly(methacrylic acid) (PMA SH ). 7 An approach to stabilize such capsules is based on rever- sible disulfide linkages, as these bonds are stable in the extracellular milieu, but then can degrade in simulated intracellular conditions. 8 These PMA hydrogel capsules (HCs) are pre- pared by the sequential adsorption of PMA SH and poly(N-vinyl pyrrolidone) (PVPON) on silica particles, mediated through hydrogen bonding. Following assembly, the PMA SH multilayers are stabilized by oxidation of the thiol groups to form disulfide bonds. The silica templates are then dissolved, and the PVPON is released from the multilayers, resulting in PMA HCs. Several studies have shown that this assembly procedure has the potential to controllably tailor key proper- ties of the PMA HCs, including size, perme- ability, 9 cargo loading, 10 and triggered re- lease. 11 Various therapeutics, including nucleic acids, 12 peptide vaccines, 10 and hy- drophobic chemotherapeutic drugs, 13,14 have been encapsulated in these capsules and effectively delivered to human cells in vitro and in vivo. 15,16 More recently, subcompartmentalized PMA HCs contain- ing intact liposomes 17,18 or smaller PMA HCs 19 have been reported, suggesting the potential of PMA HCs for microencapsulated catalysis and advanced drug delivery. 20 Given the complexity of biological envi- ronments, it is often desirable to functiona- lize capsules with biomolecular binding partners to achieve specific interactions. 21 For example, cells can be specifically tar- geted by functionalizing the surface of capsules with appropriate antibodies, or speci- fic cell signaling pathways can be activated * Address correspondence to fcaruso@unimelb.edu.au. Received for review November 7, 2011 and accepted January 3, 2012. Published online 10.1021/nn204319b ABSTRACT We present a generic and versatile method for functionalization of disulfide-stabilized PMA hydrogel capsules (HCs) with macromolecules, including a number of specific antibodies to cancer cells. Functionalization was achieved by reversible additionfragmentation chain transfer (RAFT) polymerization of poly(N-vinyl pyrrolidone) (PVPON), which introduced biorelevant heterotelechelic end groups (thiol and amine) to the polymer chain. The PVPON with heterotelechelic end groups was conjugated to the outermost layer of PMA HCs through the thiol groups and reacted with biotin via the amine groups to generate PMA/PVPON biotin HCs. On the basis of the high specific interaction and high affinity between biotin and avidin, and its derivates, such as NeutrAvidin (NAv), we functionalized the PMA HCs with biotinylated antibodies. We demonstrate significantly enhanced cellular binding and internalization of the antibody (Ab)-functionalized capsules compared with control human immunoglobulin (IgG)- functionalized capsules, suggesting these capsules can specifically interact with cells through antibody/antigen recognition. We anticipate that the versatility of the functionalization approach reported in this study will assist in targeted therapeutic delivery applications. KEYWORDS: PMA hydrogel capsules . biofunctionalization . RAFT polymerization . antibodies . biotinavidin interaction . targeting ARTICLE