Importance of the Protein Corona - ACS Publications - American

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TiO Nanoparticle-Induced Oxidation of the Plasma Membrane: Importance of the Protein Corona Sabiha Runa, Melike Lakadamyali, Melissa L. Kemp, and Christine K. Payne J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b04208 • Publication Date (Web): 26 Aug 2017 Downloaded from http://pubs.acs.org on August 29, 2017

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The Journal of Physical Chemistry

TiO2 Nanoparticle-Induced Oxidation of the Plasma Membrane: Importance of the Protein Corona Sabiha Runa,† Melike Lakadamyali,¶ Melissa L. Kemp,‡,§ and Christine K. Payne†,§,* School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332, USA. ¶ICFO-Institut de Ciencies Fotoniques, The Barcelona Institute of Science and Technology, 08860 Castelldefels, Barcelona, Spain. ‡The Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA. § Parker H. Petit Institute for Bioengineering and Biosciences, Georgia Institute of Technology, Atlanta, GA 30332, USA. †

*e-mail: [email protected]

Present Address: ¶Perelman School of Medicine, Department of Physiology, University of Pennsylvania, Philadelphia, PA 19104, USA.

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Abstract Titanium dioxide (TiO2) nanoparticles, used as pigments and photocatalysts, are widely present in modern society. Inhalation or ingestion of these nanoparticles can lead to cellular-level interactions. We examined the very first step in this cellular interaction, the effect of TiO 2 nanoparticles on the lipids of the plasma membrane. Within 12 hours of TiO2 nanoparticle exposure, the lipids of the plasma membrane were oxidized, determined with a MDA assay. Lipid peroxidation was inhibited by surface passivation of the TiO2 nanoparticles, incubation with an anti-oxidant (Trolox), and the presence of serum proteins in solution. Subsequent experiments determined that serum proteins adsorbed on the surface of the TiO2 nanoparticles, forming a protein corona, inhibit lipid peroxidation. Super-resolution fluorescence microscopy showed that these serum proteins were clustered on the nanoparticle surface. These protein clusters slow lipid peroxidation, but by 24 hours the level of lipid peroxidation is similar independent of protein corona or free serum proteins. Additionally, over 24 hours, this corona of proteins was displaced from the nanoparticle surface by free proteins in solution. Overall, these experiments provide the first mechanistic investigation of plasma membrane oxidation by TiO2 nanoparticles, in the absence of UV-light and as a function of the protein corona, approximating a physiological environment.


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Introduction Titanium dioxide nanoparticles (TiO2 NPs) are estimated to be produced at levels of >200,000 metric tons each year for use as pigments and photocatalysts.1-3 This high level of production has raised concerns about human exposure, both the workers responsible for processing of TiO2 NPcontaining materials and consumers who are routinely exposed to the white TiO2 NP-based pigments in food, cosmetics, and paint.4-9 Previous work from our lab, and others, have shown that TiO2 NPs lead to oxidative stress in cells, even in the absence of UV light. 10-12 Experiments in the absence of UV light are important as the major exposure pathways, inhalation and ingestion, preclude exposure to UV light. TiO2 NPs used in sunscreens are coated with alumina, silica, or silicone dioxide, or doped with metals to reduce the generation of reactive oxygen species and increase photostability.13-14

Recent work from our lab has shown that TiO2 NP-induced oxidative stress is due to oxidation of the protein “corona,”15 the layer of non-specifically adsorbed serum proteins that form an interface between the cell and the NP.10,16-21 Oxidation of the protein corona leads to an oxidative stress response,10,15 characterized by changes in expression of the peroxiredoxin family of antioxidant enzymes.22-26 In the course of this previous research, we observed that “bare” TiO2 NPs, lacking a protein corona, were more cytotoxic than TiO2 NPs in the presence of serum proteins. Having confirmed that TiO2 NPs oxidize proteins,15 we hypothesized that a direct oxidation of the plasma membrane lipids could be responsible for the greater cytotoxicity of bare TiO2 NPs.

Our current research examines the TiO2 NP-induced oxidation of the plasma membrane. We find that bare TiO2 NPs lead to lipid peroxidation, quantified with a malondialdehyde (MDA) assay, and that peroxidation can be inhibited by passivation of the NP surface with an alumina-silica shell or the presence of Trolox, an antioxidant. Importantly, a protein corona has a similar inhibitory effect, slowing lipid peroxidation. We used super-resolution fluorescence microscopy to image the 3 ACS Paragon Plus Environment


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protein corona formed on the surface of TiO2 NPs and observed small clusters of proteins, rather than a diffuse layer. The protein corona is displaced by free proteins over a 24 hr period. The clustering and dynamic nature of the protein corona suggests that inhibition of lipid peroxidation will be incomplete. This is in agreement with our observation that lipid peroxidation is similar to that of bare TiO2 NPs at 24 hours. Interestingly, lipid peroxidation does not correlate with cytotoxicity, pointing towards a more complex pathway for cell death.

Experimental Methods Nanoparticles (NPs) Titanium dioxide nanopowder (21 nm, #718467, Sigma-Aldrich, St. Louis, MO) was used for all experiments. For experiments in 25 cm2 cell culture flasks (MDA assays), TiO2 NPs were used at a concentration of 400 g/mL for experiments with HeLa cells. For experiments in 12 well plates (MTT assay) the concentration of NPs (270 g/mL) was scaled down based on the number of cells forming a monolayer on the surface of the culture dish to keep the ratio of NPs to cells constant. NP concentration was similarly scaled based on cell number for experiments using 3.5 cm optical dishes (#P35G-1.5-14-C, MatTek, Ashland, MA), with TiO2 NPs used at a concentration of 124 g/mL. All experiments were carried out in the dark. The surface passivation of TiO2 NPs with a combination of alumina (sodium aluminate, #11138491, Sigma-Aldrich) and silica (#13870285, Sigma-Aldrich) has been described previously.27 Passivation was confirmed with X-ray photoelectron spectroscopy (XPS, K-Alpha, Thermo Scientific, Waltham, MA).15

Cell Culture HeLa cervical epithelial carcinoma cells (CRM-CCL-2, ATCC, Manassas, VA) and A549 lung epithelial carcinoma cells (CCL-185, ATCC) were cultured in Minimum Essential Medium (MEM,


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#61100061, Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS, #10437028, Invitrogen) at 37oC and 5% carbon dioxide. Cells were passaged every five days.

Lipid Peroxidation Assay A malondialdehyde (MDA) lipid peroxidation assay was carried out according to the manufacturer’s protocol (#K739-100, BioVision, Milpitas, CA) for cells grown in 25 cm2 flasks. Conditions for specific experiments are described in Results and Discussion. Following incubation with thiobarbituric acid (1 hr, 95 °C), the MDA-TBA adduct absorbance was measured at 532 nm with a spectrophotometer (DU 800, Beckman Coulter). Protein concentration was measured at 280 nm. Experiments were carried out in triplicate and significance was determined by a twotailed student’s t-test.

Microscopy and Image Analysis Conventional fluorescence microscopy: Measurement of corona displacement FBS used for imaging experiments was labelled with AlexaFluor647 (#A37566, ThermoFisher, Carlsbad, CA) and purified according to the manufacturer’s instructions. TiO2 NPs were incubated with labeled FBS (AF647-FBS) for 30 min at room temperature to allow a protein corona to form on the surface of the TiO2 NPs. Excess FBS was removed by repeated centrifugation and resuspension in water (8,000 rcf, 15 min, x3; Figure S1). The final TiO2 NP concentration after this washing process was determined by UV-visible spectroscopy (max=330 nm). HeLa cells were then incubated with the AF647-FBS-TiO2 NP complexes (124 g/mL) with conditions described in Results and Discussion.

Epifluorescence microscopy was performed with an inverted microscope (Olympus IX71, Tokyo, Japan) using a 60x, 1.20 N.A. objective (Olympus). A xenon lamp was used as a light source and



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images were acquired (5 ms) with a Cy5 filter cube and EMCCD camera (#DU-897, Andor, South Windsor, CT). Brightfield microscopy using the same microscope system was used to image the TiO2 NPs. Image analysis was carried out in ImageJ.28 Brightness and contrast settings for all epifluorescence images were set equal. Mean intensities were measured for 30 NPs, or NP aggregates, from 3 separate images from 2 distinct experiments for each experimental condition.

Super-resolution fluorescence microscopy: Protein clustering analysis Bovine serum albumin (BSA, #12657, Merck, Darmstadt, Germany) was labelled with AlexaFluor 647 (#A37566, ThermoFisher) and purified according to the manufacturer’s instructions. TiO2 NPs were incubated with labelled BSA (AF647-BSA) at mass ratios of 0.01-10 mg of BSA/mg TiO2, as noted in the text, for 30 minutes with constant vortexing in the dark. Following five wash steps (14,000 rcf, 10 minutes, 5 times) to remove residual AF647-BSA, the AF647-BSA-TiO2 NPs were transferred to a glass coverslip for imaging. An imaging buffer of 5% glucose, 1% glucose oxidase/catalase, and 10% ethanolamine was added to samples prior to experiments. The experimental setup used for the super-resolution stochastic optical reconstruction microscopy (STORM, 100x objective, 1.49 NA, Nikon Instruments, Tokyo, Japan) experiments has been described previously.29 In brief, stochastic imaging used activation at 405 nm followed by three collection frames at 647 nm excitation. Emission from the AF647-BSA was collected by an EMCCD camera with an exposure time of 20 ms per frame. The resulting images were reconstructed using custom software (Insight3) provided by Bo Huang, University of California, San Francisco.

Cellular Health Assays Mitochondrial activity was quantified using the Vybrant MTT Cell Proliferation Assay Kit (#V13154, Invitrogen). Cells were cultured in 12 well plates (#62406-165, VWR). MTT absorbance was


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measured with a SpectraMax M2e plate reader (Molecular Devices, Sunnyvale, CA) at 540 nm and normalized to cells in the absence of NPs. Each assay was carried out on three separate samples and significance was determined by a two-tailed student’s t-test.

Results and Discussion

TiO2 NP Oxidize Plasma Membrane Lipids HeLa cells incubated with TiO2 NPs (124 g/mL, 12 hrs), in the absence of serum proteins, show the majority of NPs bound to the cell surface with some possibly internalized into the cells (Figure 1a). Based on our previous work examining TiO2 NP-induced protein oxidation,15 we hypothesized that TiO2 NP-induced lipid peroxidation of the plasma membrane could result from these TiO2 NPs lacking a protein corona. A MDA assay was used to measure lipid peroxidation following incubation of HeLa cells with TiO2 NPs (400 g/mL, scaled for consistent NP:cell ratio) at 2 hours, 4 hours, 6 hours, and 12 hours. MDA is a cellular product of lipid peroxidation.30-32 The reaction of MDA with thiobarbituric acid leads to a colored product (Abs: 540 nm) that allows quantification of lipid peroxidation normalized against protein concentration (Abs: 280 nm). The lipid peroxidation of cells incubated with bare TiO2 NPs was compared to cells cultured in only minimum essential medium (MEM). Previous work from our lab showed that the lack of serum proteins in the cell culture media for this period of time did not affect cell health (86% viability compared to 100% with FBS present, no significant difference).10 Following incubation of HeLa cells with TiO2 NPs for up to 4 hours, no significant difference was observed in MDA concentration (Figure 1b). After a 6 hour incubation, the MDA concentration increased to 0.27  0.01 nmol MDA/mg protein, compared to 0.15  0.03 nmol MDA/mg protein, demonstrating TiO2 NP-induced lipid peroxidation of the plasma membrane. At 12 hours, lipid peroxidation was similar, 0.28  0.05 nmol MDA/mg protein, compared to 0.18  0.03 nmol MDA/mg protein.



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Figure 1. Cellular response to TiO2 NPs. (a) Brightfield microscopy image of HeLa cells incubated with TiO2 NPs (124 g/mL, 12 hrs, MEM). (b) Lipid peroxidation following the incubation of HeLa cells with TiO2 NPs (400 g/mL, concentration scaled for consistent NP:cell ratio, 12 hrs; gray) was quantified by a MDA assay. Error bars show standard deviation for n=3 distinct experiments. **p

Importance of the Protein Corona - ACS Publications - American

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