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RESEARCH

Open Access

Mechanism of cellular uptake of genotoxic silica nanoparticles Qingshan Mu1,2,3, Nicole S Hondow4, Łukasz Krzemiński1,2, Andy P Brown1,4*, Lars JC Jeuken1,2* and Michael N Routledge1,3*

Abstract Mechanisms for cellular uptake of nanoparticles have important implications for nanoparticulate drug delivery and toxicity. We have explored the mechanism of uptake of amorphous silica nanoparticles of 14 nm diameter, which agglomerate in culture medium to hydrodynamic diameters around 500 nm. In HT29, HaCat and A549 cells, cytotoxicity was observed at nanoparticle concentrations ≥ 1 μg/ml, but DNA damage was evident at 0.1 μg/ml and above. Transmission electron microscopy (TEM) combined with energy-dispersive X-ray spectroscopy confirmed entry of the silica particles into A549 cells exposed to 10 μg/ml of nanoparticles. The particles were observed in the cytoplasm but not within membrane bound vesicles or in the nucleus. TEM of cells exposed to nanoparticles at 4°C for 30 minutes showed particles enter cells when activity is low, suggesting a passive mode of entry. Plasma lipid membrane models identified physical interactions between the membrane and the silica NPs. Quartz crystal microbalance experiments on tethered bilayer lipid membrane systems show that the nanoparticles strongly bind to lipid membranes, forming an adherent monolayer on the membrane. Leakage assays on large unilamellar vesicles (400 nm diameter) indicate that binding of the silica NPs transiently disrupts the vesicles which rapidly self-seal. We suggest that an adhesive interaction between silica nanoparticles and lipid membranes could cause passive cellular uptake of the particles. Keywords: Nanoparticles, Silica, Genotoxicity, Electron microscopy, Model membrane, Non-endocytotic uptake

Background The unique physicochemical properties of nanoparticles (NPs) that have given rise to applications in many fields, including drug delivery [1], cancer therapy [2], biosensors [3], food additives and cosmetics [4], may also increase the risk of toxicity to humans or the environment [5]. Many in vitro studies have demonstrated that certain NPs are cytotoxic and can cause oxidative stress and DNA damage, which has raised human health concerns [5-10]. As more NPs and NP-containing products are developed and brought into commercial use, it is generally assumed that NPs will enter the environment [11]. * Correspondence: [email protected]; [email protected]; [email protected] 1 Centre for Molecular NanoScience (CMNS), University of Leeds, Leeds LS2 9JT, UK 2 Institute for Membrane and Systems Biology, University of Leeds, Leeds LS2 9JT, UK 3 Leeds Institute of Genetics, Health and Therapeutics, School of Medicine, University of Leeds, Leeds LS2 9JT, UK Full list of author information is available at the end of the article

Industrial production of NPs is increasing in scale and diversity, raising additional concerns of environmental exposure to nanomaterials. The potential for human and ecological toxicity associated with