Colloids and Surfaces B: Biointerfaces 153 (2017) 263–271 Contents lists available at ScienceDirect Colloids and Surfaces B: Biointerfaces j o ur nal ho me pa ge: www.elsevier.com/locate/colsurfb Protocols Influence of dynamic flow environment on nanoparticle-protein corona: From protein patterns to uptake in cancer cells Sara Palchetti a,b,1 , Daniela Pozzi a,b,1 , Anna Laura Capriotti c , Giorgia La Barbera c , Riccardo Zenezini Chiozzi c , Luca Digiacomo d , Giovanna Peruzzi e , Giulio Caracciolo a,* , Aldo Laganà c a Department of Molecular Medicine, “Sapienza” University of Rome, Viale Regina Elena 291, 00161, Rome, Italy b Istituti Fisioterapici Ospitalieri, Istituto Regina Elena,Via Elio Chianesi 53, 00144 Rome, Italy c Department of Chemistry, “Sapienza” University of Rome, P.le A. Moro 5, 00185 Rome, Italy d Department of Bioscience and Biotechnology, University of Camerino, Via Gentile III da Varano, 62032 Camerino, MC, Italy e Istituto Italiano di Tecnologia, Center for Life Nano Science@Sapienza, Viale Regina Elena 291, 00161, Rome, Italy a r t i c l e i n f o Article history: Received 9 November 2016 Received in revised form 24 February 2017 Accepted 27 February 2017 Available online 28 February 2017 Keywords: Nanoparticles Physiological environments Protein corona Dynamic flow environment Cancer cells a b s t r a c t The fast growing use of nanoparticles (NPs) in biotechnology and biomedicine raises concerns about human health and the environment. When introduced in physiological milieus, NPs adsorb biomolecules (especially proteins) forming the so-called protein corona (PC). As it is the PC that mostly interacts with biological systems, it represents a major element of the NPs’ biological identity with impact on nanotox- icology, nanosafety and targeted delivery of nanomedicines. To date, NP-protein interactions have been largely investigated in vitro, but this condition is far from mimicking the dynamic nature of physiological environments. Here we investigate the effect of shear stress on PC by exposing lipid NPs with different surface chemistry (either unmodified and PEGylated) to circulating fetal bovine serum (FBS). PC formed upon in vitro incubation was used as a reference. We demonstrate that PC is significantly influenced by exposure to dynamic flow and that changes in PC composition are dependent on both exposure time and NP’s surface chemistry. Notably, alterations induced by dynamic flow affected cellular uptake of lipid NPs in both human cervical cancer (HeLa) and human breast adenocarcinoma (MCF7) cell lines. © 2017 Elsevier B.V. All rights reserved. 1. Introduction Recent years have seen rapid progresses in nanoparticle (NP) biotechnology, propelled by many promising applications in biomedicine [1–3]. When designed for biomedical applications, such as drug delivery, pristine NPs come into contact with bio- logical environments where they adsorb biomolecules forming the so-called protein corona (PC) [4–11]. PC is the bio-interface of NPs that is ‘seen’ by living organisms, while the primary particle surface remains buried and largely inaccessible [12–14]. Proteins involved in physiological [15,16] as well as toxicological relevant processes in the blood system, such as complement activation and coagulation, are main components of the coronas of various NPs. Thus, PC is now believed to regulate nanotoxicology [17,18], * Corresponding author. E-mail address: giulio.caracciolo@uniroma1.it (G. Caracciolo). 1 Equal contribution. macrophage recognition [19–22] and opsonization [23] of NPs. Fur- thermore, as the interaction between NPs and target cells is (at least in part) controlled by the ‘corona proteins’, PC is emerging as an important cofactor for targeted drug delivery [11,17,24–29]. In particular, understanding correlations between PC ‘fingerprints’ (PCFs) and cellular uptake is critical for clinical translation of NPs [30–34]. To date, almost the totality of these explorations has been carried out in vitro, while few studies on the PC have been performed in vivo [35,36], mainly because of the difficulty of recov- ering NPs after administration. Unfortunately, in vitro incubation is far from accurately representing the dynamic nature of phys- iological environments. When injected in the human body, NPs experience speeds from a few micrometers s -1 (in the peripheral blood vessels) up to ≈ 60 cm s -1 (in the aorta). A circulating flow could affect NP-protein interactions by creating shear stress on NPs and providing an incessant source of biomolecules [37,38]. To address this issue lipid NPs [39] with different surface chemistry (either unmodified and PEGylated) were injected into fetal bovine serum (FBS) propelled by a peristaltic pump furnished with silicon http://dx.doi.org/10.1016/j.colsurfb.2017.02.037 0927-7765/© 2017 Elsevier B.V. All rights reserved.