Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression

Accepted Manuscript Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression

M.T. Ortega, J.E. Riviere, K. Choi, N.A. Monteiro-Riviere PII: DOI: Reference:

S0887-2333(17)30105-4 doi: 10.1016/j.tiv.2017.04.020 TIV 3984

To appear in:

Toxicology in Vitro

Received date: Revised date: Accepted date:

17 February 2017 30 March 2017 13 April 2017

Please cite this article as: M.T. Ortega, J.E. Riviere, K. Choi, N.A. Monteiro-Riviere , Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Tiv(2017), doi: 10.1016/ j.tiv.2017.04.020

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ACCEPTED MANUSCRIPT Biocorona formation on gold nanoparticles modulates human proximal tubule kidney cell uptake, cytotoxicity and gene expression.

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M.T. Ortega1, J.E. Riviere1, K. Choi1, N. A. Monteiro-Riviere1* 1

*Corresponding author:

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Nanotechnology Innovation Center of Kansas State (NICKS), Kansas State University, Manhattan, KS.

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Nancy A. Monteiro-Riviere, Ph.D., ATS Regents Distinguished Scholar & University Distinguished Professor of Toxicology Director, Nanotechnology Innovation Center of Kansas State (NICKS) Department of Anatomy and Physiology Kansas State University Manhattan, KS 66506-5802 [email protected]

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ACCEPTED MANUSCRIPT Abstract Gold nanoparticles (AuNP) adsorb macromolecules to form a protein corona (PC) after systemic delivery, to which the kidney as the primary excretory organ is constantly exposed. The role of the PC on AuNP cell uptake and toxicity was investigated in vitro in human proximal tubule

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cells (HPTC) using 40 and 80nm branched polyethylenimine (BPEI), lipoic acid (LA) and

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polyethylene glycol (PEG) coated AuNP with or without (bare) PCs composed of human plasma (HP) or human serum albumin (HSA) for 0.25 to 24h. Time-dependent intracellular uptake,

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assessed by ICP-MS showed PC modulated cell uptake and cytotoxicity; with bare 40nm BPEIAuNP showing the greatest responses. All AuNP showed minimal to no cytokine release. At the

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nontoxic dose, 40nm bare BPEI-AuNP significantly modified gene expression related to

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immunotoxicity, steatosis, and mitochondrial metabolism; while at the high dose, pathways of DNA damage and repair, apoptosis, fatty acid metabolism and heat shock response were

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modulated. HP corona BPEI-AuNP response was comparable to control. These studies clearly

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showed reduced uptake and cytotoxicity, as well as differentiated gene expression of AuNP with PCs, questioning the utility of in vitro studies using bare NP to assess in vivo effects.

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Significantly, only cationic bare BPEI-AuNP had HPTC uptake or cytotoxicity suggesting the

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relative safety of PEG and LA-AuNP as nanomedicine constructs.

Keywords: gold nanoparticles, human proximal tubule cells, protein corona, cellular uptake, gene expression, mechanisms of toxicity

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ACCEPTED MANUSCRIPT Introduction

Gold nanoparticles (AuNP) have been extensively studied in nanomedicine for their promising drug delivery applications (Karimi et al., 2016) due to their unique physicochemical and surface

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characteristics (Salata, 2004; Levy et al., 2010; Sasidharan and Monteiro-Riviere 2015). Upon

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entering a biological environment, biomolecules immediately attach to the NP surfaces to form

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a biocorona which imparts new properties to the NP (Lundqvist et al., 2008; Treuel and Nienhaus, 2012; Treuel et al., 2013; Walkey and Chan, 2012). The physicochemical properties of

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the corona may modulate the NP surface chemistry and affect cell uptake and toxicity

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(Monopoli et al., 2012; Monteiro-Riviere et al., 2013; Walkey et al., 2014; Cheng et al., 2015; Li and Monteiro-Riviere, 2016; Chandran et al., 2017; Choi et al., 2017). Thus, the corona

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determines the NP biological identity and subsequent biological responses (Lundqvist et al.,

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2011; Behzadi et al., 2014; Wolfram et al., 2014). The protein corona NP interactions may also

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alter protein conformation by modifying their binding interactions to cell receptors (Salvati et

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al., 2013).

The kidney is the primary organ of compound elimination from the body. Independent of the

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route of administration, AuNP biodistribution will include delivery to the kidneys because of its high blood flow and excretory processes. Relative to the excretory function, human proximal tubule cells (HPTC) are the most abundant, metabolically active and physiologically significant cells of the kidneys. HPTC play an essential role in metabolic waste and drug excretion, electrolyte homeostasis, acid-base balance, as well as having a role in endocrine and inherent immune responses (Tarloff and Lash, 2005; Nakhoul and Batuman, 2011). Nanomaterials in the

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ACCEPTED MANUSCRIPT systemic circulation may interact with these cells and cause adverse effects since HPTC could be exposed to NP either after filtration by the glomerulus in the glomerular filtrate, or after direct exposure to HPTC from the post-glomerular capillaries (Riviere, 2011). Non-phagocytic HepG2 liver and HEp-2 cells, and COS-1 renal cells showed AuNP toxicity whose severity was a function

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of their physicochemical properties such as size and surface coatings (Goodman et al., 2004;

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Boyoglu et al., 2013; Liu et al., 2013). AuNP exposure to rat kidneys altered the proximal tubule cells (PTC) rather than the distal tubules and resulted in renal tubular necrosis, swelling, and cell

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disassociation affecting cell adhesion (Abdelhalim and Jarrar, 2011). Studies with immortalized HPTC lines under hypoxic conditions showed that NP can induce ROS production, mitochondrial

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dysfunction, and cell death through autophagy and apoptosis (Ding et al., 2014).

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The objectives of this study were to determine AuNP interaction with HPTC with human plasma

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corona (HP) and human serum albumin (HSA) compared to bare (no protein corona) 40 and

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80nm AuNP with either cationic branched polyethylenimine (BPEI), anionic lipoic acid (LA) or neutrally charged polyethylene glycol (PEG) surface coatings. Cytotoxicity, cell uptake, and

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inflammatory cytokine responses were evaluated. In addition, gene expression profiling in HPTC

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was investigated to determine the underlying molecular mechanisms of toxicity at a nontoxic dose of 3.28μg/cm2 and a toxic dose of 22.43μg/cm2 with bare (no corona) or HP protein corona with 40nm BPEI-AuNP.

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ACCEPTED MANUSCRIPT Materials and methods AuNP Synthesis: Biopure™ 40 nm and 80 nm positive BPEI, negative LA and neutral coated PEG AuNP were

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custom synthesized in a large batch by nanoComposix (San Diego, CA). Briefly, AuNP were

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synthesized by the reduction of hydrogen tetrachloroaurate (III) hydrate in potassium

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carbonate followed by tangential flow filtration (TFF). PEG functionalization was accomplished

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by adding 5 kDa thiol-methoxy-terminated polyethylene glycol (PEG) in 1:2 ratio for each 1 mg of Au. LA was functionalized by adding reduced LA to a mass ratio of 0.2 mg of LA per mg of Au.

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BPEI surfaces were coupled with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC)

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coupling chemistry to link the carboxyl acid group of the LA to free the amines of BPEI followed

AuNP Characterization:

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remove any unbound BPEI.

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by TFF washing. After the surfaces were functionalized, the BPEI-AuNP was centrifuged to

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Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to

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determine particle size, surface properties, and hydrodynamic diameters and zeta potential of AuNP with the Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). All bare NP were conducted in deionized water (DI) and HP and HSA AuNP in phosphate buffered saline (PBS) at 0h at 25oC or epithelial cell medium (EpiCM) (ScienCell Research Laboratories, Inc., Carlsbad, CA) up to 24h at 37°C. EpiCM was supplemented with 5% fetal bovine serum (FBS), 2% epithelial growth supplement and 2% penicillin/streptomycin solution and then analyzed with the Zetasizer at 25oC. Each measurement was repeated five times with 11 sub-runs of 10 5

ACCEPTED MANUSCRIPT seconds each. Hydrodynamic diameters and zeta potentials were also obtained for AuNP dispersed in the EpiCM medium. TEM was used to characterize the AuNP morphology with and without HP and HSA. A drop of the AuNP solution was placed onto the formvar carbon-coated copper grids and allowed to air dry. Samples were visualized on a FEI Tecnai™ G2 Spirit BioTWIN

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(FEI Company, Hillsboro, OR) transmission electron microscope at an accelerating voltage of 120

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kV. The diameters of the AuNP were measured using the GATAN microscopy suite® (GATAN Inc., Pleasanton, CA). For TEM intracellular localization of AuNP in the HPTC, cells were seeded at 3.9

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x 104 cells/cm2 in 100mm plates and dosed with bare 40nm and 80 nm AuNP at 3.28 μg/cm2 and incubated at 37°C, 5% CO2 for 24 h. Cells were detached with trypsin and fixed in 4%

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formaldehyde and 1% glutaraldehyde and rinsed in 0.1M phosphate buffer and then post-fixed

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in 1% osmium tetroxide for 1h and dehydrated through graded ethanol series, cleared in

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acetone, infiltrated and embedded in Spurr’s resin (Polysciences, Inc., Warrington, PA). Thin sections of approximately 800 Å were cut on an ultramicrotome (Leica EM UC7, Wetzlar,

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Germany) and placed onto 200 mesh copper grids and viewed at 80kV on the FEI Tecnai™ G2

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Spirit BioTWIN with an Oxford detector for energy dispersive X-ray spectroscopy (EDX) for elemental analysis. The grids were not stained for the intracellular localization of AuNP in the

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HPTC to rule out any artifact due to staining with lead citrate.

Protein Corona Preparation: Pooled human blood plasma was obtained from five healthy volunteers (Biological Specialty Corporation, Colmar, PA). All AuNP were incubated in microcentrifuge tubes at physiological concentrations of HP 55% v/v and 40mg/ml (50:50, v/v) of HSA (Sigma-Aldrich, St. Louis, MO) for 1h at 37°C in an orbital shaker (MaxQ™ 4000 Orbital 6

ACCEPTED MANUSCRIPT Shaker, Thermo Fisher Scientific, Waltham, MA) at 250 rpm. After incubation, the soft protein corona (unbound and weakly associated proteins) were removed by washing 3 times with PBS and centrifuged at 20,000x g for 15min at 20°C. HP and HSA AuNP were dispersed in PBS for the

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characterization, cell uptake, toxicity, and gene profiling studies.

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Cell Cultures:

HPTC were obtained from three healthy individuals (Lonza, Walkersville, MD) and were

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cultured in EpiCM (ScienCell Research Laboratories, Inc., Carlsbad, CA) supplemented with 5% FBS, 2% epithelial growth supplement and 2% penicillin/streptomycin solution. HPTC were

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expanded to 80% confluency with media changes every 48h until detachment with 0.25%

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trypsin-ethylenediaminetetraacetic acid. Then the HPTC were seeded at 3.9 x 104 cells/cm2 in 6

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and 96 well-plates and T-12.5 cm2 flasks and grown overnight at 37°C with 5% CO2 in a

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humidified environment prior to experimental use.

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AuNP Cell Uptake and ICP-MS Analysis: HPTC were seeded 3.9 X 104 cells/cm2 in 6 well-plates and dosed with 40nm bare BPEI AuNP at

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the nontoxic low dose of 3.28 μg/cm2 for the time-dependent cell uptake study with 40 and 80nm bare, HP and HSA coated BPEI, LA, and PEG AuNP at 0.25, 0.5, 1, 3, 6, 12 and 24h. This dose is equivalent to 25µg/ml in a well of a 6-well plate having a 1ml media volume with a surface area of 9.5cm2 which is at the low end of the dose curve. HPTC were washed twice with PBS and subjected to an etching agent iodine (I2)/potassium iodine (KI) (0.34 mM/2.04 mM) for 3 min at 170 rpm (Cho et al., 2009) followed by two rinses with PBS. The adherent cells were 7

ACCEPTED MANUSCRIPT detached with 0.25% trypsin-EDTA then rinsed twice with PBS and stored at -20oC. The cells and PBS washes were dried using a HotBlock®(Environmental Express, Charleston, SC) at 90°C for 5h, following acid digestion at 95°C overnight with aqua regia digestion with 37% hydrochloric acid (HCL)/70% nitric acid (HNO3) aqueous solution

(1:3,v/v) at 94°C. After

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digestion, samples were diluted with 1% HNO3/2% HCl and then syringe filtered. All samples and Au standards (1, 10, 25, 50 and 100ng/ml) were analyzed using inductively coupled plasma

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mass spectrometry (ICP-MS) on the NexION™350X spectrometer (Perkin Elmer, Waltham, MA).

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Quantitation was based on a 5 point calibration curve from 1 to 100ng/ml. Experiments were conducted in triplicate and the results expressed as the number of AuNP per HPTC. The AuNP

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uptake by HPTC was estimated as Au atoms relative to the Au standard curve then converted to

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the number of AuNP and calculated with the following equation (Chithrani et al., 2010):

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Where the number of atoms in each volume of AuNP (U) is calculated by dividing the diameter

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of a AuNP sphere (D) by the length of the one side of the unit cell (α) (4.076 Å), with four Au atoms per unit cell due to the face-centered cubic structure. The number of NP per cell was

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determined by dividing the number of NP in the analyzed sample by the total number of cells for that sample. This calculation assumes a homogeneous intracellular distribution of AuNP in the cell population. Cytotoxicity Assessment: AuNP toxicity of HPTC were determined with the alamarBlue® viability assay as described by Monteiro-Riviere et al., 2009. HPTC were seeded in 96-well plates at 3.9 x 104 cells/cm2 and 8

ACCEPTED MANUSCRIPT dosed with 1:2 serial dilutions of all 40 and 80nm bare (no corona), HP or HSA coated BPEI, LA and PEG AuNP dispersed in EpiCM medium ranging from 0 to 62µg/cm2 and incubated at 37°C, 5% CO2 for 24h. Cytotoxicity was analyzed with 10% alamarBlue® reagent in HPTC medium. After 3h of incubation at 37°C, fluorescence (Ex555/Em585 nm) was quantified with the

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Synergy H1 hybrid multimode microplate reader (BioTek Instruments Inc. Winooski, VT).

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Fluorescence values were normalized by the controls by subtracting the background

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fluorescence and expressed as percent viability.

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Cytokine Production:

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The impact of AuNP with HP and HSA coronas on HPTC were evaluated for inflammatory responses. HPTC were seeded at 3.9 x 104 cells/cm2 in 96-well plates then dosed with bare, HP

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and HSA AuNP at the low dose of 3.28μg/cm2 and the high dose of 22.43μg/cm2 (based on the

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cytotoxicity results). Supernatants were collected at 3, 6, 12 and 24h and stored at -20°C until

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needed. Samples were thawed and centrifuged at 18,000rpm for 20min to remove cell debris. Quantitative enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Inc. Minneapolis, MN)

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was used to analyze interleukin (IL)-6 (9.38 pg/ml), and the kidney injury marker molecule (KIM1) (15.6pg/ml); IL-1β (0.8pg/ml), IL-8 (0.4pg/ml), and TNF-α (0.7pg/ml) were conducted using the MILLIPLEX® Multiplex Assays (Merck Millipore, Billerica, MA). Fluorescence was measured at 450nm with a wavelength correction at 540nm on a Synergy H1 the hybrid multimode microplate reader (BioTek Instruments Inc. Winooski, VT). For positive controls, HPTC were dosed with lipopolysaccharide (LPS) at 10μg/ml for 12 and 24h.

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ACCEPTED MANUSCRIPT Human Molecular Toxicology Pathway RT2 PCR array: The Human Molecular Toxicology Pathway Finder RT2 Profiler™ PCR Array (Qiagen Inc., Valencia, CA) was used for gene profiling. HPTC were seeded at 3.9 x 104 cells/cm2 in T-12.5cm2 flasks and dosed with the 3.28μg/cm2 or 22.43μg/cm2 of 40 nm bare and HP BPEI AuNP. We

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selected the 40 nm bare BPEI-AuNP because it showed the highest HPTC uptake and

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cytotoxicity and compared the response to the HP corona which eliminated both cell uptake and cytotoxicity. RNA isolation was performed by HPTC homogenization with Ribozol™ RNA

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extraction agent (Amresco, Solon, OH) continuing with chloroform extraction and isopropanol precipitation. RNA was purified with the E.Z.N.A.® Total RNA Kit (Omega BioTek, Norcross, GA).

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RNA quantity and the RNA integrity number (RIN) was determined on the 2100 Bioanalyzer

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(Agilent Technologies, Santa Clara, CA) ; RNA samples with RIN of 8 or higher were used for the

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RT PCR arrays. First strand cDNA synthesis and qPCR were described in Choi et al.,2017 and Chandran et al., 2017 using the RT2 First Strand kit (Qiagen, Inc., Valencia, CA) in the

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QuantStudio™ 7 Flex Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA). The

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obtained cDNA was mixed with the RT2 SYBR®Green master mix and applied to the human molecular toxicology pathway Finder RT2 Profiler™ PCR Array. The qPCR cycling conditions of

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the QuantStudio™ 7 Flex were 95°C for 10min, followed by 40 cycles consisting of denaturation at 95°C for 15sec, amplification at 55°C for 40 sec and annealing at 72°C for 30 sec. Gene expression was normalized to the expression of β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH ) and compared to the data obtained with the untreated control with the cyclic threshold exported from QuantStudio Real time PCR software v1.1 to calculate the change in gene expression using the RT2 profiler PCR array data analysis web portal 10

ACCEPTED MANUSCRIPT (http://www.qiagen.com/us/shop/genes-and-pathways/data-analysis-center-overview-page/) (Pfaffl, 2001). Differentially expressed genes relative to controls were set at a 2-fold change cutoff (±2, p<0.05). In order to validate the PCR array data, gene regulation for the 40nm bare and HP BPEI-AuNP

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on HPTC was confirmed by monitoring the expression of DDIT3, CASP1 and GAPDH as the

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housekeeping gene using qPCR with the following primers DDIT3 FWD 5’-

GTCTAAGGCACTGAGCGTATC -3’ and DDIT3 RV 5’- CAGGTGTGGTGATGTATGAAGA -3’; CASP1 FWD

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5’- AGCTGAGGTTGACATCACAGG -3’ and CASP1 RV 5’- GTCAGAGGTCTTGTGCTCTGG -3’; GAPDH FWD 5’- CAAGAGCACAAGAGGAAGAGAG -3’ and GAPDH RV 5’- CTACATGGCAACTGTGAGGAG -3’.

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The first cDNA strand synthesis and qPCR were carried out according to the manufacturer’s 2

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protocol using the RT SYBR Green qPCR Master Mix in the QuantStudio 7 as described above.

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The analysis of gene expression based on qPCR results was conducted as described above. All

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Statistical Analysis:

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PCR reactions were conducted in triplicate.

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LC50 value (22.43µg/cm2) for AuNP were calculated using Origin Pro version 8.0 software (OriginLab Corporation, Northhampton, MA). A one-way analysis of variance (ANOVA) was conducted using SAS 9.4 (SAS Institute, Cary, NC) to assess the effects of AuNP types and time for IL-6 and KIM release at each time-point. The means comparison was conducted with Tukey’s honest significant difference (HSD) test at p  0.05.

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ACCEPTED MANUSCRIPT Physicochemical Characterization of AuNP and with HP and HSA coronas: All AuNP were homogeneous, monodispersed and spherical as confirmed by TEM (Figure 1 AC). The inset in each figure shows the homogeneous biodistribution of AuNP except for the 40

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and 80nm HSA BPEI-AuNP. TEM characterized them as spherical and 40nm bare BPEI-AuNP as

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39.7 ± 4.4nm, lipoic-AuNP as 40.2 ± 4.3nm, and PEG-AuNP as 41.5 ± 3.5nm. For 80nm AuNP,

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TEM showed BPEI-AuNP 82.3 ± 10nm, LA 81.8 ± 8.7nm and PEG 81.7 ± 11nm. Comparable dimensions using DLS in DIwater at 0 h were: bare BPEI-AuNP 52.6± 0.6, LA 50.1 ± 0.6 and PEG

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68.8 ± 1.7 nm for the 40nm AuNP; and for the 80nm AuNP 106.7 ± 0.8, 92.1 ± 0.6 and 112.2 ±

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1.2 nm respectively (Table 1). Zeta potentials for all AuNP in DI water or PBS were negative

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except for the 40 and 80nm bare BPEI-AuNP in DI water which were highly positive. The hydrodynamic diameters and zeta potentials of 40 and 80nm bare, HP and HSA coated BPEI

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AuNP, LA AuNP and PEG AuNP dispersed in the HPTC EpiCM growth medium are shown in

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Supplement Table 1. These data are more complex and show different trends based on particle

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size, surface coating and corona composition, reflecting differential stability in the media as a function of these characteristics. In all cases, incubation in the EpiCM media altered the

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characteristics compared to incubation in PBS or DI water. The zeta potential for all 40nm and 80nm bare AuNP with and without HP or HSA were generally negative except for the 80nm HP BPEI at 0h. For the polydispersity index (PDI), a metric of particle size heterogeneity for the 40 and 80nm bare BPEI AuNP, LA and PEG dispersed in DI water or EpiCM at 0h were relatively homogeneous (Table 1 and Supplement Table 2) in contrast to 40nm bare LA (0.25) and 40nm bare PEG (0.26) upon initial exposure at 0h. Subsequently, polydispersity patterns were

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ACCEPTED MANUSCRIPT complex across all NP types, changed as a function of the biocorona present, and varied over time. These data reflect differential levels of AuNP stability that evolved in contact with the culture media, yet did not show direct correlations to cell uptake or cytotoxicity profiles.

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Intracellular Uptake of AuNP in HPTC:

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Time-dependent HPTC intracellular uptake up to 24h with all AuNP at the non-toxic dose of

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3.28μg/cm2 with and without HP and HSA corona is shown in Figure 2. The 40nm bare BPEI

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AuNP showed intracellular uptake in HPTC as early as 3h which continued to increase up to 24h showing the greatest uptake. Figure 3A ( control, no AuNP), 3B and 3D shows the ultrastructure

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of 40 and 80 nm bare BPEI AuNP in HPTC depicting the precise intracellular location and

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distribution of AuNP within cytoplasmic vacuoles at 24h. Figures 3C depicts a higher magnification of AuNP shown in Figure 3B by the arrow. Figure 3E shows a higher magnification

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of the AuNP in Figure 3D depicted by the arrow. Figure 3F is a representative energy dispersive

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X-ray (EDX) spectrum confirming that AuNP in Figure 3B contains the element of Au within

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HPTC.

Figure 2A depicts all the other 40 and 80nm bare AuNP had minimal uptake except for the

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40nm LA AuNP at 24h and the 40nm and 80nm BPEI at 12 and 24h. AuNP uptake with a HP corona (Figure 2B) showed minimal uptake across all AuNP except for 40nm BPEI which showed the greatest, but was much less than the 40nm bare BPEI. HSA corona 40nm BPEI-AuNP (Figure 2C) also had the greatest uptake at 12h but slightly decreased at 24h compared to the other AuNP except for the 40nm HSA LA AuNP which showed an increase at 12 and 24h but was greater than the bare 40nm LA AuNP. These data show two patterns; first that BPEI surface

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ACCEPTED MANUSCRIPT coating seems to be associated with an increase in cell uptake for both AuNP sizes, especially for at the 40nm BPEI, even when the HSA corona was present. Also, PEG AuNP with HP and HSA coronas blocked cell uptake across all NP types but the HSA 40nm PEG caused a slight increase,

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while the LA corona with HSA resulted in a greater increase than the bare..

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HPTC Viability with All AuNP with and without Coronas:

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The cytotoxicity of 40 and 80nm bare BPEI, LA, and PEG-AuNP with and without HP and HSA

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coronas was determined in HPTC using the alamarBlue viability assay (Figure 4). All bare AuNP were nontoxic except for the 40nm BPEI-AuNP which had an LC50 of 22.43μg/cm2 (Figure 4A).

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HPTC were all viable after 24h of exposure to 40 or 80 mm AuNP from 0 to 62.5μg/cm2

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regardless of the NP functionalized coating or the presence of HP (Figure 4B) or HSA (Figure 4C)

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coronas. HPTC Effects with Cytokines Release after Exposure to AuNP: Cytokine release was assessed to probe more subtle biological effects after AuNP exposure.

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Based on the above cytotoxicity data, we operationally defined “low” and “high” doses based

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on the BPEI dose-response toxicity study since the other AuNP types demonstrated minimal effects. The HPTC was exposed to 40 and 80nm bare BPEI, LA, and PEG-AuNP with HP and HSA

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coronas at the low dose of 3.28 and the high dose of 22.43µg/cm2 for 3, 6, 12 and 24h. The expression of IL-1β,IL-8, and TNF-α for all NP was not detectable at their detection limit. Only IL6 release increased over time for 40 and 80nm BPEI, LA and PEG-AuNP compared to the controls (Supplement Figure 1A), although at levels far less than the positive lipopolysaccharide (LPS) controls with (Supplement Figure 3). These data showed some surface chemistry and size-dependent differences (e.g., PEG less than BPEI); however, the overall level and pattern of 14

ACCEPTED MANUSCRIPT cytokine production was not significant. Similarly, KIM-1 release only showed significant increases (2-3X) at 24hr across all NP types (Supplement Figure 2), but these levels were dramatically lower than the LPS positive control (Supplement Figure 3).

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AuNP Induced Gene Expression in HPTC:

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Since the bare 40 nm BPEI-AuNP showed both intracellular uptake and cytotoxicity in the HPTC

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and HP showed no toxicity, they were selected to investigate the potential mechanisms of

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toxicity utilizing the pathway focused Human Molecular Toxicology PathwayFinder RT2 Profiler™ PCR array system. This allows for sensitive transcriptional analysis with 370 different genes

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grouped into 13 biological pathways which are involved with apoptosis, necrosis, DNA damage

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and repair, mitochondrial energy metabolism, fatty acid metabolism, oxidative stress and antioxidant response, heat shock response, ER stress and unfolded protein response,

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cytochrome P450s and Phase I metabolism, steatosis, cholestasis, phospholipidosis and

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immunotoxicity. HPTC exposed to bare 40nm BPEI-AuNP at the nontoxic dose of 3.28µg/cm2

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induced the regulation of 24 genes (12↑, 12↓) and the toxic dose (LC50) 22.43 µg/cm2 induced the regulation of 65 genes (14↑, 51↓). However, with a HP corona the 40nm BPEI-AuNP at the

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low dose of 3.28µg/cm2 showed only one gene (ACO2) was upregulated that was involved with mitochondrial energy metabolism but the high dose LC50 22.43 µg/cm2 showed 11 genes were regulated (3↑, 8↓) out of 75 modified genes of all four AuNP treated HPTC from a total of 370 genes of interest in the array when using a two-fold difference in relative gene expression as the cut-off. The specific pathways involved with bare (no corona) 40nm BPEI-AuNP at the low dose of 3.28 µg/cm2 were immunotoxicity (2↑, 2↓), mitochondrial energy metabolism (3↑,

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ACCEPTED MANUSCRIPT 1↓), and steatosis (1↑, 2↓) pathways. Significantly, a greater number of genes were affected with the bare 40nm BPEI-AuNP at the LC50 22.43 µg/cm2 and the four pathways that were involved were DNA damage and repair (3↑, 5↓), apoptosis (1↑, 7↓), and heat shock response (2↑, 5↓) and fatty acid metabolism (0↑, 5↓) pathways. However, the molecular mechanisms

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of 40nm BPEI-AuNP with HP corona at the low dose were involved with mitochondrial energy

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metabolism (1↑, 0↓) but the HP corona high dose primarily showed fatty acid metabolism (βoxidation) (0↑, 6↓) pathways were involved Tables 2 and 3; Supplement Figure 4. The PCR

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array data of bare (no corona) 40nm AuNP and HP effects on gene regulation was discussed above and confirmed by monitoring the expression of DDIT3, CASP1 and GAPDH as the

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housekeeping gene using qPCR with the following primers stated in the materials and methods

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section (Tables 2 and 3).

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Discussion and Conclusion

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All NP in aqueous media were relatively homogeneous, monodispersed, spherical, and

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consistent with the stated sizes after synthesis. However, the size after exposure to complex cell culture medium varied, showing size and surface coating dependency, that changed over

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time as evidenced by the complex PDI profiles and size distribution. This is similar to what has been reported previously by our laboratory (Sasidharan et al., 2015; Li and Monteiro-Riviere, 2016; Choi et al., 2017; Chandran et al., 2017) and by other investigators (Maiorano et al., 2010). Such a high degree of variation and change in NP stability over time in a biomolecule-rich cell culture medium suggests that the nature of the NP actually exposed to cells changes over

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ACCEPTED MANUSCRIPT the course of an experiment making extrapolations between different NP types fraught with uncertainty. The toxicity data was clear-cut and demonstrated only the cationic bare 40nm BPEI AuNP were

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cytotoxic to the HPTC and the presence of HP or HSA coronas ameliorated the toxicity. All other

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coatings on the 40nm and all 80nm AuNP, irrespective of surface coatings or coronas, were not

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toxic. These data are consistent with the cell uptake data where 40 nm bare BPEI AuNP also showed the greatest cell uptake at 24h. However, HSA coated 40nm BPEI and 40nm and 80nm

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LA coated AuNP, also showed an increase in cell uptake but did not express overt cytotoxicity as

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was seen with the 40nm bare BPEI AuNP. However, the 40nm BPEI with a HP corona showed only slight uptake with no toxicity but gene expression data showed one gene at the low dose

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and eleven genes at the high dose were affected.

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These results imply that a cationic charge seems necessary for HPTC cytotoxicity and larger

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sizes may decrease toxicity even if the NP possesses a positive zeta potential. However, in the

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cell culture medium, even the “bare” 40nm BPEI AuNP were not cationic and hydrodynamic diameters were comparable to or even larger than bare 80 nm BPEI AuNP (Supplement Table

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1). Additionally, the hydrodynamic diameters of HP or HSA coated BPEI AUNP were actually less than the bare NP at all time points after incubation in the cell culture medium, though their PDI was greater, suggesting complex interactions between AuNP and the constituents of the cell culture medium or NP self-aggregation. These data also show an uncoupling of cellular uptake with cytotoxicity for the 40nm BPEI AuNP where HSA coronas showed minimal uptake but no cytotoxicity.

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ACCEPTED MANUSCRIPT In order to study the inflammatory potential and subtle effects of AuNP exposure to HPTC, interleukin (IL-1β, IL-6, IL-8, and TNF-α) production was investigated. IL-1β, IL-8, and TNF-α release were not detected with exposure to any AuNP. However, IL-6 showed a more complex pattern but at levels significantly less than seen with the positive control. Consistent with the

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cytotoxicity results, 40nm BPEI showed the greatest response. Interestingly, the 40nm LA coated AuNP showed some degree of cellular uptake, had the highest IL-6 release among bare

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AuNP at the 22.43 µg/cm2 dose. These data suggest that mild IL-6 release was not consistently

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related to cell uptake, NP corona composition, or AuNP size, but just to continuous exposure to AuNP. Proximal tubular IL-6 production has been influenced by cytokine and inflammatory

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mediators (IL-α, IL-β, TNF-α, IgA) (Gomez-Guerrero et al., 1994; Boswell et al., 1994; Leonard et

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al., 1999; Baer et al., 2004), the presence of apoptotic monocytes (Heidenreich et al., 1997),

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oxalate (Huang et al., 2005) and albumin (Pearson et al., 2008) exposure. In order to probe a different manifestation of AuNP effects on HPTC, the kidney injury

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biomarker KIM-1 was also assessed after exposure to all treatments. In these studies, a modest

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increase in KIM-1 release compared to control was only seen after prolonged AuNP exposure across most NP types, and significantly less than the positive toxin control.

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The gene expression studies confirmed that exposure to bare 40 nm BPEI NP caused biological effects in cells and the presence of a HP corona mitigated most of these effects. At the low dose of 3.28µg/cm2, significant modulation in immunotoxicity, mitochondrial energy metabolism and steatosis were still affected but only one gene (ACO2) was seen with mitochondrial energy metabolism with a HP corona. However, with the high dose 22.43µg/cm2 the primary pathways involved were apoptosis and DNA damage repair which is consistent with the cytotoxicity data 18

ACCEPTED MANUSCRIPT in Figure 4. Also, heat shock response and fatty acid metabolism pathways were involved but with a HP corona completely suppressed the expression of the DNA damage repair genes and decreased the pro-apoptotic genes but a slight increase was noted in the fatty acid metabolism pathways. These data highlights the complexity of the biological response seen after exposure

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to these NP based on both dose and the presence of a protein corona. This is similar to a recent

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study that showed 40nm bare BPEI-AuNP primarily modified genes in DNA damage and repair and heat shock response but with HP coronas, highly modified genes in phospholipidosis were

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expressed in human umbilical vein endothelial cells (HUVEC) (Chandran et al., 2017). In contrast, human hepatocytes, the predominant functional cells of the liver, were toxic to 40nm

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bare and HP coated BPEI AuNP whose transcriptional changes were predominantly involved in

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phase I metabolism, i.e., CYP2C9, CYP3A4 and phospholipidosis pathways. In addition, 40nm

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bare and HP coated BPEI AuNP inhibited the efflux/uptake transporter genes and CYP7A1 maintaining bile acid homeostasis which may cause a defect in homeostasis leading to potential

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pathogenesis of hepatobiliary disorders (Choi et al., 2017). Importantly, these comparative cell

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studies suggest that 40nm BPEI-AuNP caused cell type specific responses and molecular mechanisms of action in the presence or absence of protein coronas. Modulation of the

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expression of these genes in vivo could have dramatic effects on the energy-dependent transport and metabolism functions that are the hallmark of HPTC function (Tarloff and Lash, 2005; Nolin and Himmelfarb, 2010). As the dose increased, typical pathways associated with overt cytotoxicity including apoptosis, DNA damage repair, heat shock response and fatty acid metabolism were affected. Interestingly, after high exposure even to the HP coated BPEI AuNP, which had minimal cell uptake, there were genes associated with adverse cellular toxicity such 19

ACCEPTED MANUSCRIPT as apoptosis, fatty acid metabolism and mitochondrial energy metabolism expressed despite minimal overt cytotoxicity. AuNP-mediated cytotoxicity of 40nm bare BPEI-AuNP was reported in human hepatocytes at the LC50 dose of 52.9 µg/cm2 (Choi et al., 2017) and for HUVEC at the LC50 of 75.44µg/ml

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equivalent to 23.44 µg/cm2 (Chandran et al., 2017). Based on the LC50 data, HPTC were more

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sensitive to the effects of 40nm bare BPEI-AuNP at 22.43µg/cm2 compared to hepatocytes and similar to HUVEC. The cytotoxicity occurring via the proton sponge effect causing lysosomal

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swelling and rupture has been reported for cationic NP, these findings are also consistent with

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gene expression such as apoptosis Cellular apoptosis results from a potential decrease in ATP production due to Ca2+ regulated permeability transition pores in the mitochondria (Nel et al.,

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2009).

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What do these in vitro results suggest about the possibility of in vivo nephrotoxicity from AuNP?

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The first point is that AuNP exposure to a protein corona mitigates cellular uptake and the

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severity of nephrotoxic response as previously reported in other cell types (Monteiro-Riviere et al., 2013; Choi et al., 2017; Chandran et al., 2017). This observation questions the applicability

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of extrapolating in vitro bare NP data to the in vivo situation since after circulating through the blood, all AuNP would be expected to have protein coronas in vivo. Secondly, only the cationic BPEI AuNP caused any degree of overt cytotoxicity suggesting the relative safety of neutral and anionic AuNP to renal tubular cells as assessed by cytotoxicity and cytokine biomarkers. The interesting aspect of this conclusion is that in biological media, all AuNP are not cationic due to

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ACCEPTED MANUSCRIPT corona formation with media or added proteins however, the toxicity of the underlying NP surface still seems to be important. It is interesting to note that these findings are consistent with the fact that many chemical

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nephrotoxins are cationic molecules (e.g. aminoglycosides, cisplatin, polyamines, fibrates, and

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melamine) (Nolin and Himmelfarb, 2010; Tarloff and Lash, 2005). This may both modify

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propensity for cellular uptake and subsequent cellular toxicity. However, since all NP in the cell culture medium had some degree of biomolecular corona formation which neutralized the

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inherent BPEI cationic charge seen in PBS, a primary role for charge mediated cell uptake does

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not seem likely, unless the corona formed from the medium is weakly adhered to the AuNP

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allowing the native particle’s properties to dominate.

Additionally, corona formation is a dynamic process (Sahneh et al., 2013), as evident in the

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complex time-dependent patterns seen after incubation in media in Supplement Tables 1 and 2,

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and we do not know what events occur in the complex molecular environment at the interface

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of the NP biocorona with the cells own glycocalyx coating and membrane constituents, that could further modify these processes and expose the underlying NP surface properties.

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Although the common characteristic for activity of these AuNP noted was the BPEI coating (and hence positive charge) of the bare NP, size was also a major factor since the uptake was greater and cytotoxicity only occurred with the 40nm BPEI AuNP. Thus, it is possible that it is not the easily measured zeta potential of the bare NP that is common, but rather some other physical chemical property of the BPEI surface coating, especially on the small 40nm NP that

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ACCEPTED MANUSCRIPT modulates uptake and subsequent cytotoxicity. The complexity of this NP – cellular interfacial environment has been well recognized (Nel et al., 2009) and deserves further study. Extrapolating these findings to the in vivo setting is complex. Based on the limited studies

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conducted to date relative to NP nephrotoxicity, a few workers have reported kidney toxicity

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after exposure to NP in vitro (Goodman et al., 2004; L’Azou et al., 2008; Ding et al, 2014) and in

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vivo (Chen et al., 2006; Abdelhalim and Jarrar 2011). It is well-documented that NP will accumulate in the kidney after systemic administration as this organ shares the distinction,

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along with the liver and spleen, of being a primary site of NP deposition across most NP types

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(Riviere, 2009; Lin et al.; 2015). Small quantum dots NP less than 6 nm are able of being filtered through the renal glomerular basement membrane (Choi et al., 2007), while PEGylated AuNP

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between 50-100nm may become trapped in the mesangium of the kidney (Choi et al., 2011).

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One must stress that these are generalizations since extensive studies have not been conducted

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to determine the specific properties that dictate this behavior. For example, we have used a physiological based pharmacokinetic (PBPK) model both to confirm renal deposition of AuNP in

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mice, rats and pigs (Lin et al., 2016) and differential renal deposition of quantum dots

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depending on particle size (Lee et al., 2009). Polycation-siRNA NP has been reported to directly interact with the renal glomerular filtration membrane in vivo (Zuckerman et al., 2012). The one consistent finding across the literature is that systemically administered NP can distribute to the kidney, thus raising the question of their inherent toxicity to renal cells. It would be expected that all of the AuNP employed in this study would not be excreted into the urine due to their larger size, but a fraction would still be delivered to the kidney. As discussed

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ACCEPTED MANUSCRIPT above, the 40 and 80nm AuNP used in these studies would not clear the filtration barrier and could be trapped in the glomerular basement membrane. The consequences of this entrapment are most likely related to the inherent toxicity of the core NP. For example, the polycation siRNA particles that are electrostatically held together actually disintegrate within the

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glomerular basement membrane, releasing active siRNA (Zuckerman et al., 2012). The same

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would not occur with stable metallic NP. However, one could hypothesize that non-filtered protein bound NP (e.g. those possessing protein coronas) would remain in the post glomerular

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capillary filtrate to be then exposed to the HPTC. In this post-glomerular capillary environment where water and ions have been removed into the glomerular ultrafiltrate, protein and NP

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concentrations would be much higher than in normal plasma, potentially approaching the in

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vitro levels modelled here. Based on our HPTC uptake data, smaller 40nm BPEI AuNP would

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tend to have greater cellular uptake and cytotoxicity once inside the tubular cells compared to other particles, suggesting the possibility that nephrotoxicity could occur. In vivo studies would

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be required to test this hypothesis.

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In conclusion, these experiments clearly demonstrate the potential for the 40 nm cationic BPEI

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AuNP to adversely affect HPTC and suggest both the relative mitigation of this effect when AuNP area associated with HP coronas as well as the reduced nephrotoxic potential of the neutral and anionic AuNP. Non-cationic 40 and 80nm AuNP seem relatively safe based upon all biomarkers studied, a finding supporting their use as nanomedicine constructs.

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Conflict of Interest. The authors declare no competing financial interest. Acknowledgements. This research was supported by the Nanotechnology Innovation Center of Kansas State University research grant. The authors would like to thank Mr. Ravi Thakkar for TEM technical support and Dr. Hyun Joo for ICP-MS analysis.

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Figure Legends

Figure 1. Transmission electron microscopy (TEM) and dynamic size distribution of 40 and 80

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nm AuNP dispersed in PBS. TEM and size distribution profiles (insets) for BPEI, LA and PEG

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AuNP; (A) 40 and 80 nm bare (no corona) AuNP in DI water; (B) 40 and 80 nm with AuNP with HP corona in PBS; (C) 40 and 80 nm AuNP with HSA corona in PBS. PBS: phosphate buffered saline; AuNP: gold nanoparticles; BPEI: branched polyethylenimine; LA: lipoic acid; PEG: polyethylene glycol; HP: human plasma; HSA: human serum albumin. Figure 2. Cell uptake of 40nm and 80nm AuNP in the presence or absence of HP or HSA coronas. HPTC were exposed to 3.28μg/cm2 of AuNP in the presence or absence of HP or HSA for 0.25 to

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ACCEPTED MANUSCRIPT 24h. (A) Bare (no corona) BPEI, LA, and PEG-AuNP; (B) HP BPEI, LA, and PEG-AuNP; (C) HSABPEI, LA, and PEG-AuNP. Data represents mean ± SD (n=3 biological replicates /3 technical replicates). AuNP: gold nanoparticles; BPEI: branched polyethylenimine; LA: lipoic acid; PEG: polyethylene glycol; HP: human plasma; HSA: human serum albumin; HPTC: human proximal

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tubule cells.

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Figure 3. High contrast TEM micrographs of control and treated with the low dose 3.28 μg/cm

with bare 40 and 80nm BPEI-AuNP in HPTC for 24h. (A) control HPTC, (B) 40nm BPEI-AuNP, (C)

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higher magnification of AuNP in vacuoles in B, (D) 80 nm BPEI-AuNP depicting the precise

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location of BPEI AuNP within the cytoplasmic vacuoles, (E) higher magnification of AuNP in the vacuoles in D, and (F) representative EDX spectrum of AuNP within cytoplasmic vacuoles

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depicting Au as the major element. Black arrows depict AuNP. AuNP: gold nanoparticles; HPTC:

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human proximal tubule cells ; BPEI: branched polyethylenimine; EDX: energy dispersive x-ray

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spectroscopy spectrum.

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Figure 4. Cell viability of HPTC exposed to 40nm and 80nm BPEI, LA and PEG-AuNP with or without HP or HSA coronas for 24 h. (A) Bare (no corona); (B) HP corona; and (C) HSA corona

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AuNP. Data represents mean ± SD (n=3 biological replicates; n=6 technical replicates). NT: nontoxic; BPEI: branched polyethylenimine; LA: lipoic acid; PEG: polyethylene glycol; HPTC: human proximal tubule cells; HP: human plasma; HSA: human serum albumin; AuNP: gold nanoparticles;

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ACCEPTED MANUSCRIPT Table 1. Hydrodynamic diameters, zeta potentials and PDI of 40 and 80 nm bare BPEI, LA and PEG AuNP in deionized water and HP and HSA coronas in phosphate buffered saline at 0h.

LA

40

80

PEG

40

52.6 296.5 127.1 106.7 266.4 188.1 50.1 74.3 79.2 92.1 131.1 111.9 68.8 72.7 61.7 112.2 113.5 112.6

39.2 -14.0 -12.3 46.2 -9.2 -9.8 -44.0 -11.5 -15.3 -53.6 -12.1 -14.5 -4.6 -9.8 -6.3 -3.4 -9.7 -7.9

0.18 0.30 0.35 0.11 0.30 0.29 0.07 0.09 0.23 0.05 0.08 0.11 0.06 0.08 0.07 0.50 0.07 0.01

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CE

PT

BPEI, branched polyethylenimine; LA, lipoic acid; PEG, polyethylene glycol; HP, human plasma; HSA, human serum albumin; PDI, polydispersity index; AuNP, gold nanoparticles. Bare refers to no corona.

37

ACCEPTED MANUSCRIPT Table 2. Differential gene expression in HPTC after 24h exposed to 40nm BPEI-AuNP at the nontoxic dose of 3.28 μg/cm2 and the toxic dose of 22.43 μg/cm2. Fold changes

Symbol

GenBank

40 nm BPEI AuNP at the nontoxic dose

Bare

HP

40 nm BPEI AuNP at the toxic dose Bare HP

Description

NS

FAS

NM_000043

NS

NS

APAF1

NM_001160

NS

NS

TNFRSF10A

NM_003844

NS

NS

14.8 10.8 -6.0 -2.4

NM_000633

NS

NS

-2.4

TNFRSF1A

NM_001065

NS

NS

-2.2

BAX

NM_004324

NS

NS

-2.0

CASP3

NM_004346

10.2

NS

7.8

NUP210

NM_024923

NS

ABCC1

NM_004996

TGFB1

NM_000660

ED

M

AN

BCL2L1

Cholestasis

-2.6

NS

NS

-2.5

NS

NS

-2.4

PT

NS

CE

5.6 2.8

TNF receptor superfamily member 5 Fas (TNF receptor superfamily, member 6) Apoptotic Peptidase Activating Factor 1 Tumor necrosis factor receptor superfamily, member

IP

-12.4

CR

NM_001250

US

CD40

T

Apoptosis

NS NS NS NS NS

3.4

NS NS NS

10a

BCL2-like 1 Tumor necrosis factor receptor superfamily, member 1A BCL2-associated X protein Caspase 3

Nucleoporin 210kDa ATP-binding cassette, subfamily C (CFTR/MRP), member 1 Transforming growth factor, beta 1

Cytochrome P450s & Phase I Drug Metabolism

CYP1A1 FMO5

NM_000106

NS

NS

-3.1

NM_000780

-2.1

NS

NS

NM_001461

-2.0

NS

NS

AC

CYP2D6

NS NS NS

Cytochrome P450, family 2, subfamily D, polypeptide 6 Cytochrome P450, family 7, subfamily A, polypeptide 1 Flavin containing monooxygenase 5

DNA damage and repair OGG1

NM_002542

NS

NS

-5.4

CHEK1

NM_001274

NS

NS

-5.3

BRCA1

NM_007294

NS

NS

-4.5

BRCA2

BRCA2

NS

NS

-3.4

38

NS NS NS NS

8-oxoguanine DNA glycosylase CHK1 checkpoint homolog Breast cancer 1, early onset Breast cancer 2, early onset

ACCEPTED MANUSCRIPT LIG4

NM_002312

-2.9

NS

2.1

ERCC2

NM_000400

NS

NS

-2.7

XPA

NM_000380

NS

NS

2.1

DDIT3

NM_004083

6.2

NS

7.8

NS

Ligase IV, DNA, ATPdependent Excision repair crosscomplementing rodent repair deficiency Xeroderma pigmentosum, complementation group A DNA-damage-inducible transcript 3

NS

Synovial apoptosis inhibitor 1, synoviolin

NS NS NS

NS

NS

-2.5

NUCB1

NM_006184

NS

NS

-2.5

ATF4

NM_00167

-2.4

NS

NS

NS

EDEM3

NM_025191

NS

NS

OS9

NM_006812

NS

NS

SERP1

NM_014445

4.3

NS

NM_005157

NS

ABCB4

NM_000443

NS

ACADVL

NM_000018

NS

ACADS

NM_000017

CPT1A

NS

ED

ABL1

-2.2

M

Fatty Acid Metabolism

-2.4

US

NM_017921

NS

AN

NPLOC4

-2.2 4.2

NS NS

NS

13.6

NS

NS

-2.9

NM_001876

NS

NS

-2.3

GCDH

NM_000159

NS

NS

-2.2

CPT2

NM_000098

NS

NS

-2.1

CE

PT

NS

AC

IP

NM_172230

NS

CR

SYVN1

39

T

ER Stress & Unfolded Protein Response

NS NS NS NS NS

2.9 2.0 9.4 2.3 2.0 2.0 NS

Nucleobindin 1 Activating transcription factor 4 (tax-responsive enhancer element B67)

Nuclear protein localization 4 homolog ER degradation enhancer, mannosidase alpha-like 3 Osteosarcoma amplified 9, endoplasmic reticulum lectin Stress-associated endoplasmic reticulum protein 1 C-abl oncogene 1, nonreceptor tyrosine kinase ATP-binding cassette, subfamily B (MDR/TAP), member 4 Acyl-CoA dehydrogenase, very long chain Acyl-CoA dehydrogenase, C-2 to C-3 short chain Carnitine palmitoyltransferase 1A (liver) Glutaryl-CoA dehydrogenase Carnitine palmitoyltransferase 2

ACCEPTED MANUSCRIPT Table 3. Differential gene expression in HPTC for heat shock response, immunotoxicity, mitochondria energy metabolism, necrosis, oxidative stress and antioxidant response, phospholipidosis, steatosis after 24h exposed to 40nm BPEI-AuNP at the nontoxic dose of 3.28 μg/cm2 and the toxic dose of 22.43 μg/cm2. Fold changes

NS NS

NS NS

-7.0 -3.7

HSPA1A

NM_005345

NS

NS

HSF1

NM_005526

NS

NS

DNAJC5

NM_025219

NS

NS

DNAJA3

NM_006260

6.3

NS

HSPD1

NM_002156

19.3

NS

2.5

GPT

NM_005309

-49.2

EP300 C3

NM_001429 NM_000064

-2.8 NS

MKI67

NM_002417

ED

NM_033292

PT

CASP1

8.7

NS

-2.2

NS

US

-2.9

-2.1

NS

4.7

NS

15.9

NS

NS

2.6

NS

NS

-40

NS

NS NS

NS -2.1

NS NS

NS

5.2

NS

M

Immunotoxicity

NS NS

CR

40 nm BPEI AuNP at the nontoxic dose

AN

GenBank

Description

T

HP

Heat Shock Response HSPB1 NM_001540 HSPA1B NM_005346

Symbol

IP

Bare

40 nm BPEI AuNP at the toxic dose Bare HP

Heat shock 27kDa protein 1 Heat shock 70kDa protein 1B Heat shock 70kDa protein 1A Heat shock transcription factor 1 DnaJ (Hsp40) homolog, subfamily C, member 5 DnaJ (Hsp40) homolog, subfamily C, member 3 Heat shock 60kDa protein 1 (chaperonin) Caspase 1, apoptosis-related cysteine peptidase Glutamic-pyruvate transaminase E1A binding protein p300 Complement component 3 Antigen identified by monoclonal antibody Ki-67

CE

Mitochondrial Energy Metabolism NM_001039845

-2.6

NS

NS

NS

OGDH

NM_002541

NS

NS

-2.2

NS

NM_002197 NM_000143 NM_001098

NS 2.5 9.0

NS NS 3.6

2.2 2.3 5.1

NS NS 2.4

NM_174869

7.8

NS

6.8

4.1

SPATA2

NM_006038

NS

NS

-3.4

NS

TNFAIP8L1

NM_152362

NS

NS

-2.9

NS

PVR EIF5B

NM_006505 NM_015904

NS NS

NS NS

-2.7 -2.5

NS NS

ACO1 FH ACO2 IDH3G

AC

MDH1B

Malate dehydrogenase 1B, NAD (soluble) Oxoglutarate (alphaketoglutarate) dehydrogenase Aconitase 1, soluble Fumarate hydratase Aconitase 2, mitochondrial Isocitrate dehydrogenase 3 (NAD+) gamma

Necrosis

40

Spermatogenesis associated 2 Tumor necrosis factor, alpha-induced protein 8-like 1 Poliovirus receptor Eukaryotic translation

ACCEPTED MANUSCRIPT

NS

NS

NS NS

-2.8 -2.7

NS NS

DHCR24

NM_014762

NS

NS

-2.2

NS

AASS

NM_005763

-2.2

NS

NS

NS

PTGS2

NM_000963

NS

NS

-2.2

NS

DUOX2

DUOX2

NS

NS

-2.0

NS

NQO1

NM_000903

2.3

NS

NS

NS

NS

GSTM4

NM_000850

NS

NS

POR

NM_000941

NS

NS

Steatosis LMNA

NM_005572

NS

SREBF1

NM_004176

NS

MTTP

NM_000253

-2.9

LSS

NM_002340

NS

AQP4

NM_001650

-2.1

UCP2

NM_003355

-3.0

NS

Starch binding domain 1 Glutathione S-transferase mu 4 P450 (cytochrome) oxidoreductase

NS

NS

-3.3

NS

NS

-2.9

NS

NS

-2.7

NS

NS

-2.6

NS

oxidosqualene-lanosterol cyclase)

NS

-2.1

NS

NS

8.2

NS

Aquaporin 4 Uncoupling protein 2 (mitochondrial, proton carrier)

M ED

PT

NS

-2.1

AC

CE

9.4

-3.2

US

NM_003943

Thioredoxin reductase 2 Dual oxidase 1 24-dehydrocholesterol reductase Aminoadipate-semialdehyde synthase Prostaglandin-endoperoxide synthase 2 Dual oxidase 2 NAD(P)H dehydrogenase, quinone 1

AN

STBD1

NS

CR

Phospholipidosis

initiation factor 5B Defensin, beta 1

T

NS

IP

DEFB1 NM_005218 -2.4 Oxidative Stress & Antioxidant Response TXNRD2 NM_006440 NS DUOX1 NM_175940 NS

41

Lamin A/C Sterol regulatory element binding transcription factor 1 Microsomal triglyceride transfer protein Lanosterol synthase (2,3-

ACCEPTED MANUSCRIPT

T

IP

CR

US AN M ED PT CE AC

   

Highlights Cationic AuNP accumulate and exert toxicological effects in human kidney cells Presence of a protein corona eliminates these outcomes Gene expression profiles for BPEI particles were a function of applied dose Anionic and neutral AuNP exert no detectable cell uptake or toxicological effects

42