Colloids and Surfaces B: Biointerfaces 113 (2014) 338–345 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces jou rn al hom epage: www.elsevier.com/locate/colsurfb Solid-phase PEGylation of an immobilized protein cage on polyelectrolyte multilayer Koichiro Uto a,b , Kazuya Yamamoto a , Kenji Iwahori c , Takao Aoyagi a,b,∗ , Ichiro Yamashita d,e,f,∗∗ a Department of Nanostructure and Advanced Materials, Graduate School of Science and Engineering, Kagoshima University, 1-21-40 Korimoto, Kagoshima 890-0065, Japan b Biomaterials Unit, International Research Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba, Ibaraki 305-0044, Japan c PRESTO, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan d Nara Institute of Science and Technology, Materials Science, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan e CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan f Advanced Technology Research Laboratories, Matsushita Electric Industrial Co. Ltd., Seika, Kyoto 619-0237, Japan a r t i c l e i n f o Article history: Received 7 March 2013 Received in revised form 2 August 2013 Accepted 6 September 2013 Available online 14 September 2013 Keywords: Protein cage Apoferritin Quartz crystal microbalance Polyelectrolyte multilayer PEGylation Solid-phase reaction a b s t r a c t We used a quartz crystal microbalance (QCM) to quantitatively characterize solid-phase poly(ethylene glycol) modification (PEGylation) of apoferritin that was electrostatically immobilized on the surface of a polyelectrolyte multilayer. The solid-phase PEGylation processes were monitored by analyzing QCM frequency shifts, which showed that the PEG chains were covalently introduced onto the surface of the immobilized apoferritin. We investigated the effect of PEG concentration, PEG molecular weight, and two- dimensional coverage of the immobilized apoferritin on the solid-phase PEGylation process in addition to the surface properties of the PEGylated apoferritin film, such as wettability and protein adsorption capacity. Since the reaction field is more spatially restricted in solid-phase PEGylation than in traditional aqueous-phase PEGylation, this study shows that a ferritin protein cage is potentially useful as a tailored building block, one that has well-defined structures different from the PEGylated ferritin prepared by an aqueous-phase approach. © 2013 Elsevier B.V. All rights reserved. 1. Introduction Protein cages such as ferritins and viral capsids are useful nanoscale building blocks because they can accommodate a vari- ety of functional nanomaterials into their interior spaces [1]. In addition, because protein cages are highly symmetric, they readily form isotropic hierarchical structures, such as two- or three- dimensional protein cage crystals. These unique features of protein cages make them attractive components for fabricating hybrid bionanocomposites and bionanostructures. Ferritin is an iron stor- age protein that has a spherical hollow shell composed of 24 ∗ Corresponding author at: Biomaterials Unit, International Research Center for Materials Nanoarchitectonics (WPI-MANA), National Institute for Materials Science (NIMS), 1-1 Namiki, Tsukuba 305-0044, Japan. Tel.: +81 29 860 4775; fax: +81 29 860 4708. ∗∗ Corresponding author at: Nara Institute of Science and Technology, Materials Science, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: +81 743 72 6135; fax: +81 743 72 6196. E-mail addresses: Aoyagi.Takao@nims.go.jp (T. Aoyagi), ichiro@ms.naist.jp (I. Yamashita). subunits and a ferrihydrite core, whereas apoferritin lacks this core. The inner and outer diameters of the ferritin shell are 7 and 12 nm, respectively. Various kinds of inorganic nanoparticles (including transition metal oxides, magnetic materials, and com- pound semiconductors) have been artificially synthesized in vitro within a ferritin cavity [2–10]. The nanoparticles’ size and shape are monodisperse because the protein shell acts as a restricted nanoscale reaction field. The exterior surfaces of such protein cage architectures also provide a diverse platform for chemical and genetic functionaliza- tion. For chemical modification, the endogenous available amino acid functionalities on the surface of the ferritin protein cage are amenable to conventional protein modification chemistry. Wetz and Crichton first reported a general reactivity screening to tar- get amines, carboxylic acids, and thiols derived from the amino acid residues on the ferritin surface [11]. These precisely positioned reactive sites have been functionalized with small molecules such as alkyl chains and fluorescent dyes as well as macromolecules such as polymers and viral glycoproteins [12–16]. Among such protocols, polymer-ferritin conjugation is a versatile means of endowing new functionalities to protein cages by using ‘grafting-to’ and ‘grafting- from’ approaches. Polymer-ferritin conjugates exhibit a unique 0927-7765/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2013.09.013