Clathrin-mediated Endocytosis of Gold Nanoparticles In Vitro

CHENG TENG NG, 1 FLORENCE MEI AI TANG, 1 JASMINE JIA’EN LI, CYNTHIA ONG, 1 LANRY LIN YUE YUNG, 2* AND BOON HUAT BAY 1*

1

1

Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 2

Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, Singapore

Grant sponsor: Singapore National Research Foundation

* Correspondence to: Boon Huat Bay, Department of Anatomy, Yong Loo Lin School of Medicine, National University of Singapore, 4 Medical Drive, MD10, Singapore 117594. Fax: +65-67787643. E-mail: [email protected]; or Lin Yue Lanry Yung, Department of Chemical and Biomolecular Engineering, Faculty of Engineering, National University of Singapore, 4 Engineering Drive 4, E5-02-09, Singapore 117576, Fax: +65-67791936; E-mail: [email protected]

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an ‘Accepted Article’, doi: 10.1002/ar.23051

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ABSTRACT Gold nanoparticles (AuNPs) have potential biomedical and scientific applications. In this study, we evaluated the uptake and internalization of FBS-coated 20 nm AuNPs into lung fibroblasts and liver cells by different microscopy techniques. AuNP aggregates were observed inside MRC5 lung fibroblasts and Chang liver cells under light microscopy, especially after enhancement with automegallography. Clusters of AuNPs were observed to be adsorbed on the cell surface by scanning electron microscopy. Ultrathin sections showed that AuNPs were mainly enclosed within cytoplasmic vesicles when viewed under transmission electron microscopy. We also investigated the mechanism of uptake for AuNPs, using endocytosis inhibitors and quantification of Au with inductively coupled plasma mass spectrometry. Cells treated with concanavalin A and chloropromazine showed significant decrease of Au uptake in MRC5 lung fibroblasts and Chang liver cells respectively, implying that the uptake of AuNPs was facilitated by clathrin-mediated endocytosis. It would therefore appear that uptake of 20 nm AuNPs in both cell types with different tissues of origin, was dependent upon clathrin-mediated endocytosis.

Key Words: Gold nanoparticles; autometallography; transmission microscopy; endocytosis inhibitors; clathrin-mediated endocytosis

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INTRODUCTION The potential of nanoparticles (NPs) for medical and scientific applications are far reaching. The particles, which are in the range of 1-100 nm (Alkilany and Murphy, 2010), have been employed in applications such as targeted therapy, drug delivery, bioimaging and diagnostics (Caruthers et al., 2007; Liu et al., 2007; Murphy et al., 2008; Boisselier and Astruc, 2009). Gold nanoparticles (AuNPs) are of particular interest, as they are easy to synthesize, exhibit chemical stability, and can support different types of surface modifications (Pissuwan et al., 2006; Boisselier and Astruc, 2009). For example, precise drug delivery to tumors achieved through conjugating NP surface with different ligands, has led to enhanced drug uptake by hepatocellular carcinoma cells (Ashley et al., 2011). CYT-6091, which is AuNPs conjugated with recombinant human tumor necrosis factor alpha (rhTNF) and thiolated polyethylene glycol, has completed a Phase I clinical trial for cancer treatment (Libutti et al., 2010). In view of the promising results observed in cancer treatment, there is a need to address the mechanism(s) of entry of NPs into tumor cells. The prospective value of AuNPs in biomedicine requires a sound understanding of pathway(s) mediating its cellular uptake in order to enhance targeting of the cancer cells (Pirollo and Chang, 2008; Brandenberger et al., 2010; Chithrani, 2010; Wang et al., 2010; dos Santos et al., 2011). Receptor-mediated endocytosis (RME) is known to be a major uptake pathway for AuNPs (Mukherjee et al., 1997; Jin et al., 2009), and occurs when surface proteins adsorb on AuNPs, interact with receptors on the cell membrane (Chithrani, 2010). RME may involve the participation of clathrin-coated vesicles, caveolae internalization, or other lesser-known mechanisms (Wileman et al., 1985; Rejman et al., 2004). The size and shape of NPs have been found to correlate with the rate and quantity of AuNPs internalized (Chithrani et al., 2006; Jiang et al., 2008; Wang et al., 2010). Chithrani and Chan (2007) showed that transferrin-coated 2 John Wiley & Sons, Inc.

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AuNPs (with sizes 14 nm, 50 nm and 74 nm) were taken up solely by clathrin-mediated endocytosis in STO fibroblasts, HeLa cervical cancer and SNB19 brain tumor cell lines. Nativo et al. (2008) observed the involvement of clathrin- and caveolae-mediated endocytosis in the uptake of PEG-modified 16 nm AuNPs in HeLa cells, while another group of investigators found that smaller size AuNPs (4.5 nm) were taken up by the caveolae-mediated endocytic pathway (Hao et al., 2012). Uptake of gold nanostructures has also been reported to be affected by the functional groups present on their surfaces (Cho et al., 2010). We have previously shown that internalization of 20 nm AuNPs in lung fibroblasts can induce phenotypic effects (Li et al., 2008; Li et al., 2010; Ng et al., 2011). In this study, we investigated the endocytic pathway by which fetal bovine serum (FBS)-coated 20 nm AuNPs were taken up into MRC5 lung fibroblasts. Internalization of the FBS-coated AuNPs was verified by both light microscopy (LM) and transmission electron microscopy (TEM). Clusters of AuNPs were also observed on the surface of lung fibroblasts by scanning electron microscopy (SEM). Quantitative cellular uptake of AuNPs was performed on MRC5 cells, in the presence of endocytosis inhibitors by inductively coupled plasma mass spectrometry (ICPMS). Using the AuNPs of the same size, we then verified the endocytic pathway in Chang liver cells, an epithelial cell line with a different tissue of origin.

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MATERIALS AND METHODS Synthesis and coating of AuNPs with FBS AuNPs were synthesized from citrate reduction of gold salts following the method of Turkevitch (Li et al., 2010). AuNPs of 20 nm diameter were concentrated from 2 nM stock solutions to 10 nM by 20 min centrifugation cycles at 7000 g. 10 nM AuNP solution was incubated for 5 h with an equal volume of FBS in a 37°C water bath. FBS-coated AuNPs were sterile filtered and stored at 4°C in the dark. Cell Culture and Exposure to AuNPs MRC5 lung fibroblasts (ATCC CCL-171) were cultured in FBS and penicillinstreptomycin supplemented RPMI 1640 media and Chang liver cells (ATCC CCL-13) were cultured with DMEM medium and 10% heat-inactivated FBS (Gibco, NY) in a 5% carbon dioxide incubator at 37°C. For AuNP treatment, 1 nM of AuNPs, which is the dose preoptimized from previous studies (Li et al., 2008; Ng et al., 2013), was used to treat the cells for 24 h – 72 h. Automegallography (AMG) 50 µl GoldEnhance™ from the silver enhancement kit (Cytodiagnostics, Ontario, Canada) sufficient to cover the specimens, was applied after 24 h post exposure to AuNPs, and allowed to develop for 10 min. The reaction was then stopped by rinsing with deionized water, before micrographs were taken using a Nikon Eclipse TS100 microscope (Nikon Corporation, Japan). TEM Cells were seeded on coverglass chambers (Nalge Nunc, NY) and AuNPs were added into the complete culture medium for 72 h. After which, cells were fixed for 1 h in 2.5%

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glutaraldehyde, before osmification with 1% osmium tetroxide (Agar Scientific Ltd, Stansted, UK), dehydration in ethanol and embedding in araldite. Semithin sections were cut followed by ultrathin sections, which were stained with uranyl acetate (BDH, Poole, UK) as well as lead citrate (BDH). The ultrathin sections were then viewed under a Philips CM120 BioTWIN transmission electron microscope. SEM Cells were initially fixed in 2.5% glutaraldehyde solution. After rinsing with phosphate buffered saline (PBS) solution, post-fixation was carried out with 1% osmium tetroxide for 30 min, before dehydration with increasing concentrations of ethanol. Samples were then dried in a Critical Point Dryer before examination under a JEOL JSM 6701F scanning electron microscope. Cytotoxicity Assay The Promega CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay Kit (Promega, Madison, USA) was used to quantify cell viability by measuring the amount of formazan formed. A negative control set (complete medium DMSO as vehicle control) and a blank set (wells with only culture medium and no cells) were also prepared. The percentage of cell viability was calculated using the formula: % cell viability = (AbsTest – AbsBlank) / (AbsControl - AbsBlank) x 100% Cytoskeleton Staining Cells were initially fixed with 4% paraformaldehyde in PBS before washing with PBS and staining with F-actin (Sigma Aldrich, MO) at a dilution factor of 1:50 for 30 min. After washing with PBS, the cells were mounted onto a glass slide using the Dako mounting medium (Dako Corporation, CA) and kept moist in the dark. The specimens were observed using an

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Olympus Fluoview™ FV1000 confocal microscope equipped with Fluoview v5.0 software to capture images of the cellular architecture. A negative control was included and processed similarly. Endocytosis Inhibition and Inductively Coupled Plasma Mass Spectrometry (ICPMS) Endocytosis inhibitors, concanavalin A, chlorpromazine and nystatin (Sigma Aldrich), were applied to MRC5 lung fibroblasts and Chang liver cell cultures. Inhibition of clathrinmediated endocytosis was performed with 0.5 µg/ml concanavalin A and 10 µg/ml chlorpromazine; and caveolae-mediated endocytosis inhibition with 25 µg/ml nystatin. All the inhibitor concentrations used were according to reported values in the literature (Singh et al., 2003; Morisco et al., 2008; Vercauteren et al., 2010) and carried out for 4 h at 37°C. Subsequently, the medium was replaced with 1 nM AuNP, incubated at 37°C for 24 h and trypinised, before centrifugation at 1000 rpm for 5 min. Acid digestion was performed using Aqua Regia on cells which were collected overnight. Samples were diluted with ultrapure water and platinum was used as an internal standard for all samples, before analysis with the ICPMS instrument using an Agilent 7500cx system (Perkin Elmer, MA). Statistical Analysis Data analysis was performed using the Graph Pad Prism Version 5.0. The Student’s t-test was used for comparison of two variables and One-way ANOVA with post-hoc Tukey test for comparison of three or more groups. P < 0.05 was considered as statistically significant.

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RESULTS AuNP internalization into MRC5 lung fibroblasts Characterization of the AuNPs by TEM showed that the particles were uniform in size and shape with a mean diameter of 20 nm (Fig. 1A). FBS-coated AuNPs were observed as single particles of similar size and in aggregate forms under SEM (Fig. 1B). Previous measurement of the size of NPs by dynamic light scattering, had revealed the size of AuNPs to be ~22 nm and ~36 nm, before and after coating with FBS respectively (Li et al., 2010). The concentration of 1 nM AuNPs utilized in this study, falls within the mid-range of concentrations that are used in nanotoxicity studies (Khlebtsov and Dykman, 2011). Exposure of MRC5 lung fibroblasts to 1 nM AuNPs resulted in internalization of the AuNPs as observed under LM (Fig. 2B cf Fig. 2A), which was more obviously seen with AMG enhancement (Fig. 2C). AuNPs were mainly observed as small aggregates of ≤ 100 nm diameter appearing as dark blue clusters (Fig. 2B). After enhancement with AMG, the internalized NP aggregates were clearly observed as AMG grains as a result of silver deposition on the AuNPs, inside the cells (Fig. 2C). SEM showed clusters of AuNPs being adsorbed on the surface of an MRC5 lung fibroblast (Fig. 3). TEM was performed to validate that AuNPs were indeed internalized by the fibroblasts. AuNPs were found as agglomerates in lysosomes 72 h post AuNP exposure (Fig. 4B cf Fig. 4A), cytoplasmic vesicles or as free particles in the cytoplasm of the fibroblasts (both not shown). At the ultrastructural level, the well defined spherical shape and electron dense property of AuNPs enabled their unambiguous identification in ultrathin tissue sections.

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AuNP internalization by clathrin-mediated endocytosis in MRC5 lung fibroblasts Inhibition of the clathrin-mediated pathway using concanavalin A significantly inhibited the uptake of AuNPs into MRC5 lung fibroblasts by 18.2% as compared with control cells (Fig. 5). To ensure that the endocytosis inhibitors did not have an effect on cellular toxicity, thereby influencing uptake of the AuNPs, we also investigated the viability of the MRC5 cells after treatment with the inhibitors. They were found to be relatively insensitive to concanavalin A and chlorpromazine treatment since there were no significant differences in cell viability, although nystatin exhibited slight cytotoxicity at 25 µg/ml, but still maintaining an average of 82% cell viability (Fig. 6A). The F-actin structure which can affect uptake of AuNPs (Papakonstanti and Stournaras, 2008; dos Santos et al., 2011), was also not affected in cells treated with the endocytosis inhibitors (Fig. 6B).

Cellular uptake of AuNPs mediated via clathrin-mediated endocytosis in Chang liver epithelial cells AuNPs were taken up by Chang liver cells as observed by LM (Fig. 7B cf Fig. 7A) and TEM (Fig. 8) after AuNP exposure. At the ultrastructiral level, AuNPs were seen in cytoplasmic vesicles that resembled endosomes (Fig. 8B and 8C). The addition of chlorpromazine (but not the other endocytosis inhibitors) during the NP exposure significantly inhibited the uptake of AuNPs into Chang liver cells by 17% as compared with control cells (Fig. 9A, P

Clathrin-mediated endocytosis of gold nanoparticles in vitro.

Gold nanoparticles (AuNPs) have potential biomedical and scientific applications. In this study, we evaluated the uptake and internalization of FBS-co...
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