Nanoscale PAPER Cite this: Nanoscale, 2016, 8, 7501 Received 2nd November 2015, Accepted 5th February 2016 DOI: 10.1039/c5nr07665j www.rsc.org/nanoscale Biocompatible multilayer capsules engineered with a graphene oxide derivative: synthesis, characterization and cellular uptake† Loretta L. del Mercato,* a Flora Guerra, b Gianpiero Lazzari, c Concetta Nobile, a Cecilia Bucci b and Rosaria Rinaldi c,d Graphene-based capsules have strong potential for a number of applications, including drug/gene deli- very, tissue engineering, sensors, catalysis and reactors. The ability to integrate graphene into carrier systems with three-dimensional (3D) geometry may open new perspectives both for fundamental tests of graphene mechanics and for novel (bio)technological applications. However, the assembly of 3D com- plexes from graphene or its derivatives is challenging because of its poor stability under biological con- ditions. In this work, we attempted to integrate a layer of graphene oxide derivative into the shell of biodegradable capsules by exploiting a facile layer-by-layer (LbL) protocol. As a first step we optimized the LbL protocol to obtain colloidal suspensions of isolated capsules embedding the graphene oxide derivative. As a following step, we investigated in detail the morphological properties of the hybrid cap- sules, and how the graphene oxide derivative layer influences the porosity and the robustness of the multilayer composite shells. Finally, we verified the uptake of the capsules modified with the GO derivative by two cell lines and studied their intracellular localization and biocompatibility. As compared to pristine capsules, the graphene-modified capsules possess reduced porosity, reduced shell thickness and a higher stability against osmotic pressure. They show remarkable biocompatibility towards the tested cells and long-term colloidal stability and dispersion. By combining the excellent mechanical properties of a graphene oxide derivative with the high versatility of the LbL method, robust and flexible biocompatible polymeric capsules with novel characteristics have been fabricated. 1 Introduction Graphene consists of a single layer of sp 2 -hybridized carbon atoms that are tightly bonded together in a continuous pattern of hexagons. 1 This one-atom-thick fabric of carbon has attracted considerable interest in recent years, owing to its extraordinary electrical, optical, thermal and mechanical properties. 1–9 Currently, there are many methods for generat- ing colloidal suspensions of graphene and its derivatives, such as graphene oxide (GO) and reduced graphene oxide (rGO), which have been extensively summarised in numerous review articles. 10–12 The scaled-up and reliable production of GO and rGO has promoted the synthesis and characterization of a wide range of graphene-based composite materials for various applications in energy, electronics, catalysis, sensing, tissue engineering and drug/gene delivery. 13–21 GO is the highly oxidized form of graphene containing a carboxyl group (–COOH) at the edges and a hydroxyl group (–OH) and an epoxy group (–O–) in the basal planes, which makes GO relatively hydrophilic. 22 Recent studies by Rourke 23 and Thomas 24 showed that, independent of the method of syn- thesis used, the actual structure of exfoliated GO is composed of oxygenated functionalized graphene-like sheets with non- covalently attached oxidative debris (OD). This OD acts as a sur- factant, helps in stabilizing aqueous GO suspensions, and plays an important role in the noncovalent interaction with aromatic molecules such as 1-nitropyrene. 25 On the other hand, rGO is obtained upon reduction of GO using various reducing agents. It reduces the oxygen content of GO, makes it more hydro- phobic, introduces defects in the lattice and also partially restores electrical conductivity. 26 However, for biomedical † Electronic supplementary information (ESI) available. See DOI: 10.1039/ c5nr07665j a CNR NANOTEC - Institute of Nanotechnology c/o Campus Ecotekne, Via Monteroni, 73100 Lecce, Italy. E-mail: loretta.delmercato@nanotec.cnr.it b Dipartimento di Scienze e Tecnologie Biologiche ed Ambientali (DiSTeBA), Università del Salento, Via Monteroni, 73100 Lecce, Italy c Istituto Nanoscienze-CNR, Euromediterranean Center for Nanomaterial Modelling and Technology (ECMT), via Arnesano, 73100 Lecce, Italy d Dipartimento di Matematica e Fisica “Ennio De Giorgi”, Università del Salento, Campus Universitario Ecotekne, Via Lecce-Monteroni, 73047 Monteroni di Lecce, Italy This journal is © The Royal Society of Chemistry 2016 Nanoscale, 2016, 8, 7501–7512 | 7501