Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb Supramolecular functionalization of carbon nano-onions with hyaluronic acid-phospholipid conjugates for selective targeting of cancer cells Marta d’Amora a , Adalberto Camisasca b , Alice Boarino a,c , Silvia Arpicco d , Silvia Giordani a,b,c, * a Nano Carbon Materials, Istituto Italiano di Tecnologia (IIT), via Livorno 60, 10144, Torino, Italy b School of Chemical Sciences, Dublin City University (DCU), Glasnevin, Dublin 9, Ireland c Department of Chemistry, University of Turin, via Giuria 7, 10125, Torino, Italy d Department of Drug Science and Technology, University of Turin, via Giuria 9, 10125, Torino, Italy ARTICLEINFO Keywords: Carbon nano-onions Hyaluronic acid Targeted drug delivery Cancer cells Toxicity Zebrafish ABSTRACT Carbon nano-onions (CNOs) are promising materials for biomedical applications due to their low cytotoxicity and excellent biocompatibility. Supramolecular functionalization with biocompatible polymers is an effective strategytodevelopengineereddrugcarriersfortargeteddeliveryapplications.Inthisstudy,wereporttheuseof a hyaluronic acid-phospholipid (HA-DMPE) conjugate to target CD44 overexpressing cancer cells, while en- hancing solubility of the nanoconstruct. Non-covalently functionalized CNOs with HA-DMPE show excellent in vitro cell viability in human breast carcinoma cells overexpressing CD44 and are uptaken to a greater extent compared to human ovarian carcinoma cells with an undetectable amount of CD44. In addition, they possess high in vivo biocompatibility in zebrafish (Danio Rerio) during the different stages of development and they prevalently localize in the digestive tract of the zebrafish larvae. 1. Introduction CNOs are a fascinating member of the carbon family [1]. They consist of a multi-shell fullerene structure with a size ranging from 2 to 100 nm, depending on the method of synthesis [2]. The major applicative limitation of pristine CNOs (p-CNOs) is re- lated to their poor solubility in biological fluids, due to the marked tendencytoaggregatepromotedbyintermolecularinteractions(i.e.van der Waals forces). To address this drawback, appropriate surface functionalization strategies have been developed, including covalent and non-covalent approaches [3], allowing for the successful use of CNOs in the biomedical field due to their high surface area to volume ratio and biocompatibility [4]. In particular, we have reported that functionalized CNOs (f-CNOs) exhibited notable dispersing abilities and stability in aqueous and biological media as well as low toxicity in several normal and cancer cell lines [5–8]. Furthermore, we confirmed their high in vivo biocompatibility in Hydra vulgaris [9] and zebrafish during the development [10,11]. Non-covalent functionalization is an efficient route to increase the water solubility of a material while preserving its intrinsic properties as the adsorption of aromatic compounds or macromolecules such as polymers onto the material surface via π-π or hydrophobic interactions may lead to the formation of a stable suspension. Hyaluronic acid (HA) isanaturallyoccurringbiopolymerbelongingtotheglycosaminoglycan group and a major component of the extracellular matrix. HA is bio- degradable, biocompatible and non-toxic and, when it is administered in an organism, it is not recognized as foreign by the immune system. Moreover, the HA receptor CD44 is overexpressed in several cancer cell lines [12]. The interaction between HA and CD44 has been deeply in- vestigated and it is well known that HA represents an optimal tool in drug delivery for increasing the uptake of different platforms, including polymeric nanoparticles [13,14] and liposomes [15,16]. Herein, we report the non-covalent functionalization of p-CNOs with a HA-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (HA- DMPE) conjugate to target CD44 overexpressing cancer cells while enhancing the dispersibility of the nanoconstruct. In this study, high molecular weight (i.e. 200 kDa) HA was selected and two different CNO:conjugate ratios have been investigated. The non-covalent func- tionalization is promoted by hydrophobic interactions between the phospholipid chains of the conjugate and the CNO surface, while the hydrophilic HA chains, remaining exposed to the outside, promote the solubilisation of the material in aqueous solution. The functionalized CNO:HA-DMPE have been characterized by UV–vis absorbance spec- troscopy, thermogravimetric analysis (TGA), dynamic light scattering (DLS), zeta potential and high resolution transmission electron micro- scopy (HRTEM), confirming that the proposed functionalization https://doi.org/10.1016/j.colsurfb.2020.110779 Received 14 October 2019; Received in revised form 17 December 2019; Accepted 4 January 2020 ⁎ Corresponding author at: Nano Carbon Materials, Istituto Italiano di Tecnologia (IIT), via Livorno 60, 10144, Torino, Italy. E-mail address: silvia.giordani@dcu.ie (S. Giordani). Colloids and Surfaces B: Biointerfaces 188 (2020) 110779 Available online 08 January 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved. T