Accepted Manuscript Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – a mechanistic study Lauren A. Austin, Samera Ahmad, Bin Kang, Kathryn R. Rommel, Mahmoud Mahmoud, Mary E. Peek, Mostafa A. El-Sayed PII: DOI: Reference:
S0887-2333(14)00218-5 http://dx.doi.org/10.1016/j.tiv.2014.11.003 TIV 3413
To appear in:
Toxicology in Vitro
Received Date: Accepted Date:
11 September 2014 14 November 2014
Please cite this article as: Austin, L.A., Ahmad, S., Kang, B., Rommel, K.R., Mahmoud, M., Peek, M.E., El-Sayed, M.A., Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – a mechanistic study, Toxicology in Vitro (2014), doi: http://dx.doi.org/10.1016/j.tiv.2014.11.003
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Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – a mechanistic study Lauren A. Austin†, Samera Ahmad†, Bin Kang†,‡, Kathryn R. Rommel§, Mahmoud Mahmoud†, Mary E. Peek, and Mostafa A. El-Sayed†,*
†
Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, GA, USA 30332-0400 §
Parker H. Petit Institute for Bioengineering and Biosciences, School of Chemistry and
Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400
*
Corresponding Author Information: email: [email protected] phone: 404.894.0292 fax: 404.894.0294
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ABSTRACT Nanoparticles (NPs), in particular noble metal nanoparticles, have been incorporated into many therapeutic and biodiagnostic applications. While these particles have many advantageous physical and optical properties, little is known about their intrinsic intracellular effects in biological environments. Here, we report the possible cell death mechanisms triggered in human oral squamous cell carcinoma (HSC-3) cells after exposure to extracellular, cytoplasm, and nuclear localized AuNPs and AgNPs. NP uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation, mitochondrial depolarization, and the levels and/or translocation of caspase-dependent and caspase-independent proteins were assessed under control and localized metal nanoparticle exposure. Exposure to AuNPs resulted the adoption of a quiescent cellular state, as AuNPs caused a decrease in intracellular ATP, but no change in viability or cell death populations. However, AgNP exposure significantly reduced HSC-3 cell viability and increased apoptotic populations, especially when localized at the cytoplasm and nucleus.
Increased cell death populations were linked to an increase in intracellular ROS
generation. Western blot analysis indicated cytoplasm localized AgNPs and nuclear localized AgNPs utilized a caspase-independent apoptotic pathway that involved the nuclear translocation of AIF and p38 MAPK proteins. These results demonstrate that the degree of cytotoxicity increases as AgNPs move from extracellular localization to nuclear localization, whereas changing AuNP localization does not trigger any significant cytotoxicity.
KEYWORDS Gold nanoparticles; silver nanoparticles; cytotoxicity; apoptosis; organelle-targeted nanoparticles
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1. Introduction Nanoparticles (NPs), those with dimensions between 1-100 nm, have been heavily implemented in many biomedical and consumer products.(Murthy, 2007) Their small size results in unique optical and physical properties that differ greatly from their bulk counterparts, namely high
surface
area
to
volume
ratios,
increased
reactivity,
and
increased
light
scattering/absorption.(Jain et al., 2007; Jain et al., 2008) While these properties are advantageous and drive the use of nanoparticles, a complete understanding of how these nanoparticles influence biological environments has not been achieved. Noble metal nanoparticles, i.e. gold (AuNPs) and silver (AgNPs), have been extensively used to improve drug solubility, therapeutic delivery and disease diagnostic assays as well as reduce microbial activity.(Austin et al., 2014) However, in order for their biomedical utility to be fully realized, it is imperative that the factors governing their cytotoxicity be completely understood. As such, there have been many investigations into AuNP and AgNP cytotoxicity.(Alkilany and Murphy, 2010; Kim and Ryu, 2013) These studies have mainly focused on the effect that size, shape, and stabilizing agent have on cellular toxicity and the results vary depending on the parameters tested as well as the cell line used. For instance, Pernodet et. al. reported that 14 nm citrate-AuNPs caused a reduction in cellular proliferation, adhesion, and motility in human dermal fibroblast (CF-31) after 24-144 h of exposure.(Pernodet et al., 2006) In 2014, however, citrate stabilized AuNPs, less than 100 nm in diameter, were shown to induce no significant alteration in cellular proliferation and cytotoxicity in Caco-2 cells after 24 h of NP incubation.(Aueviriyavit et al., 2014) When AuNPs, with diameters less than 1.4 nm, were modified with triphenylphosphine monosulfonate, cytotoxicity was observed in metastatic melanoma cells (MV3 and BLM) as well as epithelial (HeLa), endothelial (SK-Mel-28),
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fibroblasts (L929) and phagocytes (j774A1) cells.(Pan et al., 2007; Tsoli et al., 2005) Interestingly, 15 nm AuNPs coated with the same surface ligand was shown to be non-toxic. While there are varying reports of AuNP cytotoxicity, there has been a general consensus that AgNP exposure induces cytotoxicity across a variety of cell lines.
Therefore, these
investigations mainly focus on the mechanisms in which cytotoxicity occurs. In 2008, Asharani et. al. reported that starch-coated AgNPs induced cell death via ROS generation , DNA damage and G2/M cell cycle arrest in human lung fibroblast (IMR-90) and human glioblastoma (U251) cells.(AshaRani et al., 2008) Jiang and co-workers also reported cell death, specifically apoptosis, after human hepatoma (HepG2) cells were exposed to PVP coated AgNPs for 24 h. PVP-AgNP exposure was shown to increased ROS, reduce SOD activity and cause S phase arrest.(Liu et al., 2010) Other groups have further shown AgNP toxicity is linked to cell cycle disruption, DNA damage, reactive oxygen species (ROS) generation, and increased lipid peroxidation.(Arora et al., 2008; Austin et al., 2011; Hussain et al., 2005) Although there have been many studies aimed at assessing the cytotoxic effects of AuNPs and AgNPs, a fundamental, comparative investigation exploring the cytotoxicity of these nanoparticles when they are targeted to specific organelles within a cell has not been reported. In the present study, the cytotoxic effects of extracellular, cytoplasm and nuclear localized AuNPs and AgNPs on an epithelial cell line, human oral squamous cell carcinoma (HSC-3) cells, were investigated. To determine the possible cytotoxic mechanisms triggered, nanoparticle uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation and mitochondrial depolarization were evaluated and compared between control and localized metal nanoparticle treated groups. Moreover, the levels and/or translocation of caspase-dependent and caspase-independent proteins were studied.
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2. Materials and Methods 2.1. Chemicals Dublecco’s
modified
eagle’s
medium
(DMEM), antibiotic-antimycotic (penicillin,
streptomycin, and amphotericin) solution, fetal bovine serum (FBS), trypsin, Dulbecco’s phosphate-buffered saline (PBS), XTT cell viability assay kit, 4’, 6’-diamidino-2-phenylindole (DAPI), Tween 20, TRIS (base), methanol, tetramethylethylenediamine (TEMED), and ammonium persulfate (APS, pro-pure proteomics grade) were purchased from VWR (Randor, PA, USA). Pierce ECL western blotting substrate was purchase from Thermo Scientific Pierce Protein Biology Products (Rockford, IL, USA). Gold (III) chloride trihydrate (≥99.9% trace metal basis), silver nitrate (ACS reagent, ≥99.0%), and trisodium citrate (≥99%) were purchased from Sigma (St. Louis, MO, USA). FITC-Annexin V was purchased from Biolegend (San Diego, CA, USA). The CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega
(Madison,
WI,
USA).
6-carboxy-2’,7’-dichlorodihydrofluorescein
diacetate
(H2DCFDA), and 5x annexin binding buffer were purchased from Invitrogen (Carlsbad, CA, USA).
Laemmli sample buffer, precision plus protein kaleidoscope standards, Quick Start
protein assay, and chelex-100 were purchased from BioRad Laboratories (Hercules, CA, USA). 10x cell lysis buffer, caspase-3 mouse antibody, p38 MAPK rabbit antibody, -actin mouse antibody, AIF rabbit antibody, HtrA2/Omi rabbit antibody, anti-mouse IgG HRP-linked antibody, and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technologies (Danvers, MA, USA). Human oral squamous cell carcinoma (HSC-3) cells were a generous donation from Dr. Ivan H. El-Sayed (UCSF Medical Center). 2.2. Cell culture
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Human oral squamous cell carcinoma (HSC-3) cells were maintained in DMEM supplemented with 4.5g/L glucose and sodium pyruvate, 10 % (v/v) FBS and 1% (v/v) antibiotic-antimycotic solution. Cultures were sustained at 37 C in a 5% CO2 humidified incubator. 2.3. Nanoparticle synthesis and characterization Gold nanoparticles (AuNPs) with an average diameter of 22 nm were synthesized by the citrate reduction method (Kimling et al., 2006). Briefly, 5 mL of 60 mM HAuCl4 was added to 470 mL of deionized (DI) water and heated to boiling while being stirred. Upon boiling, 25 mL of 18 mM trisodium citrate was added to the solution and the reaction was terminated once a deep red solution color was observed.
The AuNP solution was allowed to cool at room
temperature and was then purified by centrifugation (6,000 rpm, 15 min).
AuNPs were
resuspended in DI water and the stock concentration was determined from a previously determined extinction coefficient, = 3.0x109 M-1cm-1 (Darbha et al., 2008). Size and shape distributions were confirmed by UV-vis spectroscopy, transmission electron microscopy (TEM; JOEL 100CX-2, USA), and dynamic light scattering (DLS; Malvern Zetasizer Nano, UK). The hydrodynamic diameter and zeta potential of the synthesized AuNPs as well as functionalized AuNPs were measured by DLS. Silver nanoparticles (AgNPs) with an average diameter of 42 nm were prepared by the reduction of silver ions with sodium citrate (Freund and Spiro, 1985). Briefly, a 500 mL solution of 0.3 mM AgNO3 and 0.1 mM trisodium citrate was brought to a boil under continuous stirring. After 30 min, heating and stirring were discontinued, and the solution was allowed to cool at room temperature. AgNPs were purified by centrifugation (14,000 rpm, 7 min) and resuspended in DI water. The AgNP stock concentration was calculated using = 1.1x1010 M-1cm-1. The size
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and shape of synthesized AgNPs were confirmed by UV-vis spectroscopy, transmission electron microscopy (TEM; JOEL 100CX-2, USA), and dynamic light scattering (DLS; Malvern Zetasizer Nano, UK). DLS measurements were utilized to assess hydrodynamic diameter and zeta potential of the synthesized and functionalized AgNPs. 2.4. PEGylation and peptide conjugation Citrate stabilized AuNPs and AgNPs were modified with thiol-terminated polyethylene glycol (mPEG-SH) through metal-S bonds. A 5 mM mPEG-SH aqueous stock solution was added to nanoparticle solutions to achieve 103 molar excess. Solutions were allowed to shake overnight. Nanoparticles were purified by centrifugation (AuNPs- 6,000 rpm/15 min; AgNPs14,000 rpm/7 min) and redispersed in DI water. Concentrations of the PEGylated nanoparticle (PEG-AuNP and PEG-AgNP) solutions were determined using the extinction coefficients mentioned above. PEGylated nanoparticles were further conjugated with customized arginine-glycineaspartate (RGD) and nuclear localization signal (NLS) peptides purchased from GenScript USA, Inc.
RGD (RGDRGDRGDRGDPGC) and NLS (CGGGPKKKRKVGG) peptides, both
containing C-terminal amidation, were dissolved in DI water to achieve 5 mM stock solutions. RGD and NLS peptides were added to PEGylated nanoparticle solutions to achieve 104 and 105 molar excess, respectively. Solutions were allowed to shake overnight and peptide conjugated nanoparticles were purified by centrifugation (AuNPs- 6,000 rpm/15 min; AgNPs- 14,000 rpm/7 min).
Purified RGD-AuNPs, RGD-AgNPs, NLS/RGD-AuNPs and NLS/RGD-AgNPs were
redispersed in DI water and stock concentrations were determined from molar extinction coefficients described above. The hydrodynamic diameter and zeta potential of the PEGylated
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nanoparticles were measured by DLS. All nanoparticle solutions used for cell culture exposure studies were diluted in supplemented DMEM to achieve treatment concentrations. 2.5. Dark field nanoparticle localization Nanoparticle localization was assessed using dark field microscopy.
HSC-3 cells were
seeded in 12-well plates (VWR, Radnor, PA, USA) containing 18 mm diameter coverslips (Fischer Scientific, Waltham, MA, USA) to achieve 75% final confluence. After 24 h, the medium was discarded and replaced with 0.1 nM nanoparticle solutions. Nanoparticle exposure was for 24 h, after which nanoparticle solutions were removed and coverslips were washed 1x with DPBS. Cells were fixed with 4% paraformaldehyde for 5 min. Images were obtained with an inverted Olympus IX70 microscope equipped with a dark field condenser (U-DCW). A 100x/1.35 oil Iris objective (UPLANAPO) was used to collect the scattered light from the samples. 2.6. In vitro cytotoxicity assays Cytotoxicity was assessed using XTT cell viability and ATP production assays. For the XTT based assay, HSC-3 cells were seeded in 96-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and various concentrations (0.0, 0.1, and 0.4 nM) of conjugated nanoparticles were added. Cells were exposed to nanoparticles for 48 h. After nanoparticle exposure was complete solutions were removed and active XTT reagent was added. After 4-6 h of incubation the absorbance at 450 and 690 nm was obtained using a microplate reader system (Bio-Tek Synergy H4 Multi-Mode Plate Reader).
Cell
viabilities were normalized to control cells (no nanoparticle exposure). For the ATP production assay, HSC-3 cells were seeded in white opaque-walled 96-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was
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removed and 0.0 or 0.1 nM of conjugated nanoparticles were added. Nanoparticle exposure was for 48 h, after which nanoparticle solutions were removed and cells were washed 1x with DPBS. Following washing, 100 L of fresh supplemented DMEM was added to each well and the plate was allowed to equilibrate at room temperature. After equilibration, 100 L of CellTiter-Glo reagent was added to each well and the plate was allowed to shake on an orbital shaker for 2 min. Luminescence readings were acquired using a microplate reader system (Bio-Tek Synergy H4 Multi-Mode Plate Reader). Cell viabilities were normalized to control cells (no nanoparticle exposure). 2.7. Apoptosis vs. necrosis assay To assess whether nanoparticle exposure was triggering apoptotic or necrotic mechanisms, an annexin v-PI assay was used. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve a final confluence of 75%. After 24 h, the medium was discarded and various concentrations (0.0, 0.1, or 0.4 nM) of conjugated nanoparticles were added. Cells were exposed to nanoparticles for 24 or 48 h. At the end of the exposure period, particle solutions were removed and cells were collected by centrifugation at 2,000 rpm for 12 min. Cells were then washed once in cold DPBS and centrifuged at 2,000 rpm for 12 min. The cell pellets were resuspended in 1x annexin binding buffer, and propidium iodide and FITC-annexin v were added. The solutions were allowed to incubate at room temperature for 15 min, after which the solutions were filtered. Samples were run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with a 488 nm-excitation laser and fluorescence detection in the RPhycoerythrin (PE) and Fluorescein (FITC) channels. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). The percentage of cell populations was calculated from the total 10,000 cells in comparison to control cells (no nanoparticle exposure).
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2.8. Intracellular ROS detection Intracellular ROS levels were assessed by confocal microscopy using a H2DCFA assay. Cells were seeded in 12-well plates (VWR, Radnor, PA, USA) containing 18 mm diameter coverslips (Fischer Scientific, Waltham, MA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and immediately replaced with 10 M 6-carboxy-2’, 7’dichlorofluorescein diacetate (H2DCFA) diluted in supplemented DMEM. Cells were allowed to incubate for 45 min at 37 C in a 5% CO2 humidified incubator. After incubation, the ROS indicator solution was removed and 0.4 nM particle solutions were added. Cells were exposed to nanoparticle solutions for 24 h. At the end of exposure, nanoparticle solutions were removed and cells were fixed with 4% paraformaldehyde for 5 min. After fixation, cells were washed three times with DPBS and stained with 4’, 6’-diamidino-2-phenylindole (DAPI) for nuclear staining. Following nuclear staining, cells were washed three times with DI water. Fluorescence images were acquired using a confocal scanning microscope (Zeiss LSM 700, Thronwood, NY, USA) with 405 (DAPI) and 488 nm (FITC) excitation sources. 2.9. Mitochondrial membrane depolarization Depolarization of mitochondria after nanoparticle exposure was assessed using the MitoProbe DiOC2(3) assay kit (Invitrogen, Carlsbad, CA, USA).
In mitochondria with active
membrane potentials, DiOC2(3) is able to accumulate in the mitochondria leading to bright, red fluorescence. Therefore, a decrease in DiOC2(3) staining indicates a decrease in mitochondrial membrane potential. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was discarded and replaced with 0.4 nM nanoparticle solutions.
Cells were exposed to nanoparticles for 48 h, after which particle
solutions were removed and cells were collected via centrifugation at 2,000 rpm for 10 min. Cell
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pellets were resuspended in warm supplemented DMEM containing DiOC2(3) and allowed to incubate for 30 min at 37 C in a 5% CO2 humidified incubator. Following dye incubation, cells washed two times via centrifugation at 2,000 rpm for 10 min. Cell pellets were resuspended in DPBS, filtered, and immediately run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with 488 and 575 nm-excitation lasers and fluorescence detection in the Fluorescein (FITC) and R-Phycoerythrin –Texas Red (PE-Texas Red) channels, respectively. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). Fluorescence intensities were calculated from the total 10,000 cells and compared to negative control (no nanoparticle exposure) and positive control (carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment) cells. 2.10. Western blot analysis Cells were seeded in 10 cm culture dishes (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and 0.4 nM particle solutions were added. Cells were exposed to nanoparticle solutions for 48 h, after which they were washed two times with cold DPBS and treated with 100 L of 1x cell lysis buffer supplemented with 1 mM phenylmthylsulfonyl fluoride (PMSF), a protease inhibitor. Cells were incubated for 5 min at 4 C and then scraped to induce cell membrane breakage. Cell solutions were centrifuged at 14,000 rpm for 5 min and the supernatant was collected. Protein content was detected using the Quick Start Bio-Rad protein assay with bovine serum albumin (BSA) as the standard. Protein lysates were then added in a 1:1 ratio to Laemmli sample buffer supplemented with – mercaptoethanol and boiled for 5 min. Denatured protein samples of equal concentration and a kaleidoscope MW standard (Bio-Rad Laboratories, Hercules, CA, USA) were run a 12.5% SDSPAGE gel (200 V, 40 min) and then transferred (100 V, 75 min) to a nitrocellulose membrane at
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4 C. Membranes were washed in tris-buffered saline (TBS) for 5 min and blocked with 5% non-fat dried milk prepared in tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Blocked membranes were then incubated with primary antibodies at 4 C overnight and either HRP-conjugated anti-rabbit or anti-mouse antibodies for 2 h at room temperature. Protein bands were detected using ECL western blotting substrate (Thermo Scientific, Rockford, IL, USA) and a biomolecular imager (GE Typhoon Imager).
Western blot analyses were
performed using antibodies against –actin, p38 MAP kinase (p38 MAPK), apoptosis inducing factor (AIF), HtrA2/Omi, and caspase-3. Band intensity was analyzed using Image J (NIH, Bethesda, MD, USA) with loading controls based on –actin and control samples (no nanoparticle exposure). 2.11. Caspase inhibition In order to assess the involvement of caspase proteins in apoptotic cell death, a apoptosis and necrosis flow cytometry assay was conducted with or without Q-VD-OPh, a caspase inhibitor. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve a final confluence of 75%.
After 24 h, the medium was discarded and 0.4 nM of conjugated
nanoparticles were added either with or without 50 M of Q-VD-OPh. Cells were exposed to nanoparticles for 48 h. At the end of the exposure period, particle solutions were removed and cells were collected by centrifugation at 2,000 rpm for 12 min. Cells were then washed once in cold DPBS and centrifuged at 2,000 rpm for 12 min. The cell pellets were resuspended in 1x annexin binding buffer, and propidium iodide and FITC-annexin v were added. The solutions were allowed to incubate at room temperature for 15 min, after which the solutions were filtered. Samples were run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with a 488 nm-excitation laser and fluorescence detection in the R-Phycoerythrin (PE) and Fluorescein
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(FITC) channels. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). The percentage of cell populations was calculated from the total 10,000 cells in comparison to control cells (no nanoparticle exposure). 2.12. Statistics The data were presented as mean standard error (SE) of at least three independent culture experiments.
Three technical replicates were performed for each independent cell culture
experiment. Significance differences among samples were determined using the t-test calculator (GraphPad Software, La Jolla, CA, USA). The level of statistical significance was p < 0.05 and was indicated by *.
3. Results 3.1 Characterization of nanoparticles and bioconjugation AuNPs and AgNPs were prepared through the reduction of their respective metal ions via sodium citrate.(Freund and Spiro, 1985; Kimling et al., 2006) UV-Vis spectroscopy indicated characteristic surface plasmon resonance (SPR) peaks at 525 nm for AuNPs and 420 nm for AgNPs.
TEM micrographs further showed successful synthesis of spherical particles with
average diameters of 22 ± 6 nm and 42 ± 12 nm for AuNPs and AgNPs, respectively (Fig. 1). The citrate stabilized AuNPs and AgNPs were also assessed using DLS which revealed hydrodynamic diameters of 25 and 58 nm, and zeta potentials of -20 and -32 mV, respectively. Citrate stabilized particles were then conjugated to thiol terminated polyethylene glycol (PEG, MW = 5,000) to increase their biostability and reduce non-specific binding of serum proteins contained in the cell culture medium. These particles, which are denoted as Ex-AuNPs and Ex-AgNPs, showed ~20 nm increase in their hydrodynamic diameters and a slight increase
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in their zeta potential measurements (Au: -18 mV, Ag: -20 mV). The SPR band of PEGylated AuNPs and AgNPs also showed a 1-2 nm red shift signifying a change in the dielectric constant of the NP’s surrounding environment.
Taken together, these changes indicate PEG was
successfully conjugated to the surface of the nanoparticles. PEGylated AuNPs and AgNPs were then conjugated with arginine-glycine-aspartic acid (RGD) peptides, or RGD and nuclear localization signal (NLS) peptides to promote cytoplasmic (Cy) and nuclear (Nu) localization, respectively. Conjugation of PEGylated nanoparticles with RGD (cytoplasmic targeting) peptides only produced two distinct particles: Cy-AuNPs and CyAgNPs. Conjugation with RGD and NLS (nuclear targeting) peptides resulted in two particles: Nu-AuNPs and Nu-AgNPs. Peptide conjugation was assessed via UV-Vis spectroscopy, as well as hydrodynamic diameter and zeta potential measurements. As seen with PEGylation, the characteristic SPR bands showed a red shift (1-2 nm) after surface modification with RGD and NLS peptides. The hydrodynamic diameter of peptides conjugated particles did not vary from PEG conjugated nanoparticles, however, the zeta potential of all particle formulations increased: Cy-AuNP: -3 mV, Nu-AuNP: +11 mV, Cy-AgNP: -5 mV, Nu-AgNP: +18 mV.
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Fig. 1. Au and Ag nanoparticle design. UV-Vis spectra and TEM micrographs showed successful synthesis of (A) AuNPs and (B) AgNPs with average diameters of 22 6 nm and 42 12 nm, respectively. (C) Synthesized NPs were then separated into three localization groups: extracellular (Ex-NPs), cytoplasmic (Cy-NPs) and nuclear (Nu-NPs), through conjugation with PEG and peptides.
3.2 Nanoparticle localization HSC-3 cells were incubated with 0.1 nM conjugated AuNPs and AgNPs for 24 h after which nanoparticle localization was assessed using dark field microscopy (Fig. 2). PEG conjugated nanoparticles did not show cellular uptake, as the only scattering that was detected was from the cells alone (Fig. 2A). The addition of RGD peptides to PEGylated particles caused nanoparticle internalization and cytoplasmic localization (Fig. 2B). Nu-AuNPs and Nu-AgNPs showed the greatest cellular uptake and were localized at the cell nucleus (Fig. 2C). This marked uptake, indicated by the increased light scattering, was also seen and quantitatively confirmed in
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previous works that utilized NLS in combination with RGD. (Austin et al., 2011; Mackey et al., 2013)
Fig. 2. Dark field images showing cellular uptake and localization of (A) extracellular, (B) cytoplasm, and (C) nuclear localized AuNPs (left) and AgNPs (right). Scale bar = 20 m.
3.3 Effect of nanoparticles on energy production and cell viability ATP production and XTT cell viability assays were conducted to investigate the in vitro cytotoxic effects of conjugated AuNPs and AgNPs on HSC-3 cells after 48 h of particle exposure (Fig. 3). ATP production was significantly reduced after exposure to 0.1 nM AuNP and AgNPs targeted to all cellular locations. Nu-AgNPs showed the greatest reduction in ATP (~92%) when compared to its gold counterpart (Nu-AuNPs, ~50%) and all other NPs tested. Unlike the ATP production assay, XTT assays reported no significant decrease in HSC-3 cellular viability when exposed to AuNPs targeted to the extracellular matrix, cytoplasm, or nucleus. Cy-AgNP and Nu-AgNP exposure at 0.4 nM resulted in ~22% and ~36% reduction in cell viability, respectively.
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Fig. 3. Assessment of cell viability in HSC-3 cells after 48 h of nanoparticle exposure. (A) The relative ATP content significantly decreased after exposure with AuNPs and AgNPs localized at the extracellular matrix (red), cytoplasm (blue) and nucleus (green). Control samples (i.e. “no NPs) are the same sample for each NP. (B) XTT cell viability assays did not show significant cell death after 0.1 nM and 0.4 nM AuNP exposure. (C) AgNPs localized at the cytoplasm (blue) and nucleus (green) showed a significant decrease in cell viability at 0.1 and 0.4 nM exposure concentrations. Ex-AgNPs (red) did not show a significant decrease in cell viability when assessed via mitochondrial enzyme activity. Data are expressed as mean ± SE of the three independent culture experiments. Three technical replicates were performed within each independent culture experiment. Statistical significance is indicated by * (p
S0887-2333(14)00218-5 http://dx.doi.org/10.1016/j.tiv.2014.11.003 TIV 3413
To appear in:
Toxicology in Vitro
Received Date: Accepted Date:
11 September 2014 14 November 2014
Please cite this article as: Austin, L.A., Ahmad, S., Kang, B., Rommel, K.R., Mahmoud, M., Peek, M.E., El-Sayed, M.A., Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – a mechanistic study, Toxicology in Vitro (2014), doi: http://dx.doi.org/10.1016/j.tiv.2014.11.003
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Cytotoxic effects of cytoplasmic-targeted and nuclear-targeted gold and silver nanoparticles in HSC-3 cells – a mechanistic study Lauren A. Austin†, Samera Ahmad†, Bin Kang†,‡, Kathryn R. Rommel§, Mahmoud Mahmoud†, Mary E. Peek, and Mostafa A. El-Sayed†,*
†
Laser Dynamics Laboratory, School of Chemistry and Biochemistry, Georgia Institute of
Technology, Atlanta, GA, USA 30332-0400 §
Parker H. Petit Institute for Bioengineering and Biosciences, School of Chemistry and
Biochemistry, Georgia Institute of Technology, Atlanta, GA 30332-0400
*
Corresponding Author Information: email: [email protected] phone: 404.894.0292 fax: 404.894.0294
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ABSTRACT Nanoparticles (NPs), in particular noble metal nanoparticles, have been incorporated into many therapeutic and biodiagnostic applications. While these particles have many advantageous physical and optical properties, little is known about their intrinsic intracellular effects in biological environments. Here, we report the possible cell death mechanisms triggered in human oral squamous cell carcinoma (HSC-3) cells after exposure to extracellular, cytoplasm, and nuclear localized AuNPs and AgNPs. NP uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation, mitochondrial depolarization, and the levels and/or translocation of caspase-dependent and caspase-independent proteins were assessed under control and localized metal nanoparticle exposure. Exposure to AuNPs resulted the adoption of a quiescent cellular state, as AuNPs caused a decrease in intracellular ATP, but no change in viability or cell death populations. However, AgNP exposure significantly reduced HSC-3 cell viability and increased apoptotic populations, especially when localized at the cytoplasm and nucleus.
Increased cell death populations were linked to an increase in intracellular ROS
generation. Western blot analysis indicated cytoplasm localized AgNPs and nuclear localized AgNPs utilized a caspase-independent apoptotic pathway that involved the nuclear translocation of AIF and p38 MAPK proteins. These results demonstrate that the degree of cytotoxicity increases as AgNPs move from extracellular localization to nuclear localization, whereas changing AuNP localization does not trigger any significant cytotoxicity.
KEYWORDS Gold nanoparticles; silver nanoparticles; cytotoxicity; apoptosis; organelle-targeted nanoparticles
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1. Introduction Nanoparticles (NPs), those with dimensions between 1-100 nm, have been heavily implemented in many biomedical and consumer products.(Murthy, 2007) Their small size results in unique optical and physical properties that differ greatly from their bulk counterparts, namely high
surface
area
to
volume
ratios,
increased
reactivity,
and
increased
light
scattering/absorption.(Jain et al., 2007; Jain et al., 2008) While these properties are advantageous and drive the use of nanoparticles, a complete understanding of how these nanoparticles influence biological environments has not been achieved. Noble metal nanoparticles, i.e. gold (AuNPs) and silver (AgNPs), have been extensively used to improve drug solubility, therapeutic delivery and disease diagnostic assays as well as reduce microbial activity.(Austin et al., 2014) However, in order for their biomedical utility to be fully realized, it is imperative that the factors governing their cytotoxicity be completely understood. As such, there have been many investigations into AuNP and AgNP cytotoxicity.(Alkilany and Murphy, 2010; Kim and Ryu, 2013) These studies have mainly focused on the effect that size, shape, and stabilizing agent have on cellular toxicity and the results vary depending on the parameters tested as well as the cell line used. For instance, Pernodet et. al. reported that 14 nm citrate-AuNPs caused a reduction in cellular proliferation, adhesion, and motility in human dermal fibroblast (CF-31) after 24-144 h of exposure.(Pernodet et al., 2006) In 2014, however, citrate stabilized AuNPs, less than 100 nm in diameter, were shown to induce no significant alteration in cellular proliferation and cytotoxicity in Caco-2 cells after 24 h of NP incubation.(Aueviriyavit et al., 2014) When AuNPs, with diameters less than 1.4 nm, were modified with triphenylphosphine monosulfonate, cytotoxicity was observed in metastatic melanoma cells (MV3 and BLM) as well as epithelial (HeLa), endothelial (SK-Mel-28),
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fibroblasts (L929) and phagocytes (j774A1) cells.(Pan et al., 2007; Tsoli et al., 2005) Interestingly, 15 nm AuNPs coated with the same surface ligand was shown to be non-toxic. While there are varying reports of AuNP cytotoxicity, there has been a general consensus that AgNP exposure induces cytotoxicity across a variety of cell lines.
Therefore, these
investigations mainly focus on the mechanisms in which cytotoxicity occurs. In 2008, Asharani et. al. reported that starch-coated AgNPs induced cell death via ROS generation , DNA damage and G2/M cell cycle arrest in human lung fibroblast (IMR-90) and human glioblastoma (U251) cells.(AshaRani et al., 2008) Jiang and co-workers also reported cell death, specifically apoptosis, after human hepatoma (HepG2) cells were exposed to PVP coated AgNPs for 24 h. PVP-AgNP exposure was shown to increased ROS, reduce SOD activity and cause S phase arrest.(Liu et al., 2010) Other groups have further shown AgNP toxicity is linked to cell cycle disruption, DNA damage, reactive oxygen species (ROS) generation, and increased lipid peroxidation.(Arora et al., 2008; Austin et al., 2011; Hussain et al., 2005) Although there have been many studies aimed at assessing the cytotoxic effects of AuNPs and AgNPs, a fundamental, comparative investigation exploring the cytotoxicity of these nanoparticles when they are targeted to specific organelles within a cell has not been reported. In the present study, the cytotoxic effects of extracellular, cytoplasm and nuclear localized AuNPs and AgNPs on an epithelial cell line, human oral squamous cell carcinoma (HSC-3) cells, were investigated. To determine the possible cytotoxic mechanisms triggered, nanoparticle uptake and localization, cell viability, ATP levels, modes of cell death, ROS generation and mitochondrial depolarization were evaluated and compared between control and localized metal nanoparticle treated groups. Moreover, the levels and/or translocation of caspase-dependent and caspase-independent proteins were studied.
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2. Materials and Methods 2.1. Chemicals Dublecco’s
modified
eagle’s
medium
(DMEM), antibiotic-antimycotic (penicillin,
streptomycin, and amphotericin) solution, fetal bovine serum (FBS), trypsin, Dulbecco’s phosphate-buffered saline (PBS), XTT cell viability assay kit, 4’, 6’-diamidino-2-phenylindole (DAPI), Tween 20, TRIS (base), methanol, tetramethylethylenediamine (TEMED), and ammonium persulfate (APS, pro-pure proteomics grade) were purchased from VWR (Randor, PA, USA). Pierce ECL western blotting substrate was purchase from Thermo Scientific Pierce Protein Biology Products (Rockford, IL, USA). Gold (III) chloride trihydrate (≥99.9% trace metal basis), silver nitrate (ACS reagent, ≥99.0%), and trisodium citrate (≥99%) were purchased from Sigma (St. Louis, MO, USA). FITC-Annexin V was purchased from Biolegend (San Diego, CA, USA). The CellTiter-Glo Luminescent Cell Viability Assay kit was purchased from Promega
(Madison,
WI,
USA).
6-carboxy-2’,7’-dichlorodihydrofluorescein
diacetate
(H2DCFDA), and 5x annexin binding buffer were purchased from Invitrogen (Carlsbad, CA, USA).
Laemmli sample buffer, precision plus protein kaleidoscope standards, Quick Start
protein assay, and chelex-100 were purchased from BioRad Laboratories (Hercules, CA, USA). 10x cell lysis buffer, caspase-3 mouse antibody, p38 MAPK rabbit antibody, -actin mouse antibody, AIF rabbit antibody, HtrA2/Omi rabbit antibody, anti-mouse IgG HRP-linked antibody, and anti-rabbit IgG HRP-linked antibody were purchased from Cell Signaling Technologies (Danvers, MA, USA). Human oral squamous cell carcinoma (HSC-3) cells were a generous donation from Dr. Ivan H. El-Sayed (UCSF Medical Center). 2.2. Cell culture
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Human oral squamous cell carcinoma (HSC-3) cells were maintained in DMEM supplemented with 4.5g/L glucose and sodium pyruvate, 10 % (v/v) FBS and 1% (v/v) antibiotic-antimycotic solution. Cultures were sustained at 37 C in a 5% CO2 humidified incubator. 2.3. Nanoparticle synthesis and characterization Gold nanoparticles (AuNPs) with an average diameter of 22 nm were synthesized by the citrate reduction method (Kimling et al., 2006). Briefly, 5 mL of 60 mM HAuCl4 was added to 470 mL of deionized (DI) water and heated to boiling while being stirred. Upon boiling, 25 mL of 18 mM trisodium citrate was added to the solution and the reaction was terminated once a deep red solution color was observed.
The AuNP solution was allowed to cool at room
temperature and was then purified by centrifugation (6,000 rpm, 15 min).
AuNPs were
resuspended in DI water and the stock concentration was determined from a previously determined extinction coefficient, = 3.0x109 M-1cm-1 (Darbha et al., 2008). Size and shape distributions were confirmed by UV-vis spectroscopy, transmission electron microscopy (TEM; JOEL 100CX-2, USA), and dynamic light scattering (DLS; Malvern Zetasizer Nano, UK). The hydrodynamic diameter and zeta potential of the synthesized AuNPs as well as functionalized AuNPs were measured by DLS. Silver nanoparticles (AgNPs) with an average diameter of 42 nm were prepared by the reduction of silver ions with sodium citrate (Freund and Spiro, 1985). Briefly, a 500 mL solution of 0.3 mM AgNO3 and 0.1 mM trisodium citrate was brought to a boil under continuous stirring. After 30 min, heating and stirring were discontinued, and the solution was allowed to cool at room temperature. AgNPs were purified by centrifugation (14,000 rpm, 7 min) and resuspended in DI water. The AgNP stock concentration was calculated using = 1.1x1010 M-1cm-1. The size
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and shape of synthesized AgNPs were confirmed by UV-vis spectroscopy, transmission electron microscopy (TEM; JOEL 100CX-2, USA), and dynamic light scattering (DLS; Malvern Zetasizer Nano, UK). DLS measurements were utilized to assess hydrodynamic diameter and zeta potential of the synthesized and functionalized AgNPs. 2.4. PEGylation and peptide conjugation Citrate stabilized AuNPs and AgNPs were modified with thiol-terminated polyethylene glycol (mPEG-SH) through metal-S bonds. A 5 mM mPEG-SH aqueous stock solution was added to nanoparticle solutions to achieve 103 molar excess. Solutions were allowed to shake overnight. Nanoparticles were purified by centrifugation (AuNPs- 6,000 rpm/15 min; AgNPs14,000 rpm/7 min) and redispersed in DI water. Concentrations of the PEGylated nanoparticle (PEG-AuNP and PEG-AgNP) solutions were determined using the extinction coefficients mentioned above. PEGylated nanoparticles were further conjugated with customized arginine-glycineaspartate (RGD) and nuclear localization signal (NLS) peptides purchased from GenScript USA, Inc.
RGD (RGDRGDRGDRGDPGC) and NLS (CGGGPKKKRKVGG) peptides, both
containing C-terminal amidation, were dissolved in DI water to achieve 5 mM stock solutions. RGD and NLS peptides were added to PEGylated nanoparticle solutions to achieve 104 and 105 molar excess, respectively. Solutions were allowed to shake overnight and peptide conjugated nanoparticles were purified by centrifugation (AuNPs- 6,000 rpm/15 min; AgNPs- 14,000 rpm/7 min).
Purified RGD-AuNPs, RGD-AgNPs, NLS/RGD-AuNPs and NLS/RGD-AgNPs were
redispersed in DI water and stock concentrations were determined from molar extinction coefficients described above. The hydrodynamic diameter and zeta potential of the PEGylated
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nanoparticles were measured by DLS. All nanoparticle solutions used for cell culture exposure studies were diluted in supplemented DMEM to achieve treatment concentrations. 2.5. Dark field nanoparticle localization Nanoparticle localization was assessed using dark field microscopy.
HSC-3 cells were
seeded in 12-well plates (VWR, Radnor, PA, USA) containing 18 mm diameter coverslips (Fischer Scientific, Waltham, MA, USA) to achieve 75% final confluence. After 24 h, the medium was discarded and replaced with 0.1 nM nanoparticle solutions. Nanoparticle exposure was for 24 h, after which nanoparticle solutions were removed and coverslips were washed 1x with DPBS. Cells were fixed with 4% paraformaldehyde for 5 min. Images were obtained with an inverted Olympus IX70 microscope equipped with a dark field condenser (U-DCW). A 100x/1.35 oil Iris objective (UPLANAPO) was used to collect the scattered light from the samples. 2.6. In vitro cytotoxicity assays Cytotoxicity was assessed using XTT cell viability and ATP production assays. For the XTT based assay, HSC-3 cells were seeded in 96-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and various concentrations (0.0, 0.1, and 0.4 nM) of conjugated nanoparticles were added. Cells were exposed to nanoparticles for 48 h. After nanoparticle exposure was complete solutions were removed and active XTT reagent was added. After 4-6 h of incubation the absorbance at 450 and 690 nm was obtained using a microplate reader system (Bio-Tek Synergy H4 Multi-Mode Plate Reader).
Cell
viabilities were normalized to control cells (no nanoparticle exposure). For the ATP production assay, HSC-3 cells were seeded in white opaque-walled 96-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was
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removed and 0.0 or 0.1 nM of conjugated nanoparticles were added. Nanoparticle exposure was for 48 h, after which nanoparticle solutions were removed and cells were washed 1x with DPBS. Following washing, 100 L of fresh supplemented DMEM was added to each well and the plate was allowed to equilibrate at room temperature. After equilibration, 100 L of CellTiter-Glo reagent was added to each well and the plate was allowed to shake on an orbital shaker for 2 min. Luminescence readings were acquired using a microplate reader system (Bio-Tek Synergy H4 Multi-Mode Plate Reader). Cell viabilities were normalized to control cells (no nanoparticle exposure). 2.7. Apoptosis vs. necrosis assay To assess whether nanoparticle exposure was triggering apoptotic or necrotic mechanisms, an annexin v-PI assay was used. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve a final confluence of 75%. After 24 h, the medium was discarded and various concentrations (0.0, 0.1, or 0.4 nM) of conjugated nanoparticles were added. Cells were exposed to nanoparticles for 24 or 48 h. At the end of the exposure period, particle solutions were removed and cells were collected by centrifugation at 2,000 rpm for 12 min. Cells were then washed once in cold DPBS and centrifuged at 2,000 rpm for 12 min. The cell pellets were resuspended in 1x annexin binding buffer, and propidium iodide and FITC-annexin v were added. The solutions were allowed to incubate at room temperature for 15 min, after which the solutions were filtered. Samples were run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with a 488 nm-excitation laser and fluorescence detection in the RPhycoerythrin (PE) and Fluorescein (FITC) channels. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). The percentage of cell populations was calculated from the total 10,000 cells in comparison to control cells (no nanoparticle exposure).
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2.8. Intracellular ROS detection Intracellular ROS levels were assessed by confocal microscopy using a H2DCFA assay. Cells were seeded in 12-well plates (VWR, Radnor, PA, USA) containing 18 mm diameter coverslips (Fischer Scientific, Waltham, MA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and immediately replaced with 10 M 6-carboxy-2’, 7’dichlorofluorescein diacetate (H2DCFA) diluted in supplemented DMEM. Cells were allowed to incubate for 45 min at 37 C in a 5% CO2 humidified incubator. After incubation, the ROS indicator solution was removed and 0.4 nM particle solutions were added. Cells were exposed to nanoparticle solutions for 24 h. At the end of exposure, nanoparticle solutions were removed and cells were fixed with 4% paraformaldehyde for 5 min. After fixation, cells were washed three times with DPBS and stained with 4’, 6’-diamidino-2-phenylindole (DAPI) for nuclear staining. Following nuclear staining, cells were washed three times with DI water. Fluorescence images were acquired using a confocal scanning microscope (Zeiss LSM 700, Thronwood, NY, USA) with 405 (DAPI) and 488 nm (FITC) excitation sources. 2.9. Mitochondrial membrane depolarization Depolarization of mitochondria after nanoparticle exposure was assessed using the MitoProbe DiOC2(3) assay kit (Invitrogen, Carlsbad, CA, USA).
In mitochondria with active
membrane potentials, DiOC2(3) is able to accumulate in the mitochondria leading to bright, red fluorescence. Therefore, a decrease in DiOC2(3) staining indicates a decrease in mitochondrial membrane potential. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was discarded and replaced with 0.4 nM nanoparticle solutions.
Cells were exposed to nanoparticles for 48 h, after which particle
solutions were removed and cells were collected via centrifugation at 2,000 rpm for 10 min. Cell
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pellets were resuspended in warm supplemented DMEM containing DiOC2(3) and allowed to incubate for 30 min at 37 C in a 5% CO2 humidified incubator. Following dye incubation, cells washed two times via centrifugation at 2,000 rpm for 10 min. Cell pellets were resuspended in DPBS, filtered, and immediately run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with 488 and 575 nm-excitation lasers and fluorescence detection in the Fluorescein (FITC) and R-Phycoerythrin –Texas Red (PE-Texas Red) channels, respectively. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). Fluorescence intensities were calculated from the total 10,000 cells and compared to negative control (no nanoparticle exposure) and positive control (carbonyl cyanide 3-chlorophenylhydrazone (CCCP) treatment) cells. 2.10. Western blot analysis Cells were seeded in 10 cm culture dishes (VWR, Radnor, PA, USA) to achieve 75% final confluence. After 24 h, the medium was removed and 0.4 nM particle solutions were added. Cells were exposed to nanoparticle solutions for 48 h, after which they were washed two times with cold DPBS and treated with 100 L of 1x cell lysis buffer supplemented with 1 mM phenylmthylsulfonyl fluoride (PMSF), a protease inhibitor. Cells were incubated for 5 min at 4 C and then scraped to induce cell membrane breakage. Cell solutions were centrifuged at 14,000 rpm for 5 min and the supernatant was collected. Protein content was detected using the Quick Start Bio-Rad protein assay with bovine serum albumin (BSA) as the standard. Protein lysates were then added in a 1:1 ratio to Laemmli sample buffer supplemented with – mercaptoethanol and boiled for 5 min. Denatured protein samples of equal concentration and a kaleidoscope MW standard (Bio-Rad Laboratories, Hercules, CA, USA) were run a 12.5% SDSPAGE gel (200 V, 40 min) and then transferred (100 V, 75 min) to a nitrocellulose membrane at
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4 C. Membranes were washed in tris-buffered saline (TBS) for 5 min and blocked with 5% non-fat dried milk prepared in tris-buffered saline with Tween 20 (TBST) for 1 h at room temperature. Blocked membranes were then incubated with primary antibodies at 4 C overnight and either HRP-conjugated anti-rabbit or anti-mouse antibodies for 2 h at room temperature. Protein bands were detected using ECL western blotting substrate (Thermo Scientific, Rockford, IL, USA) and a biomolecular imager (GE Typhoon Imager).
Western blot analyses were
performed using antibodies against –actin, p38 MAP kinase (p38 MAPK), apoptosis inducing factor (AIF), HtrA2/Omi, and caspase-3. Band intensity was analyzed using Image J (NIH, Bethesda, MD, USA) with loading controls based on –actin and control samples (no nanoparticle exposure). 2.11. Caspase inhibition In order to assess the involvement of caspase proteins in apoptotic cell death, a apoptosis and necrosis flow cytometry assay was conducted with or without Q-VD-OPh, a caspase inhibitor. HSC-3 cells were seeded in 12-well plates (VWR, Radnor, PA, USA) to achieve a final confluence of 75%.
After 24 h, the medium was discarded and 0.4 nM of conjugated
nanoparticles were added either with or without 50 M of Q-VD-OPh. Cells were exposed to nanoparticles for 48 h. At the end of the exposure period, particle solutions were removed and cells were collected by centrifugation at 2,000 rpm for 12 min. Cells were then washed once in cold DPBS and centrifuged at 2,000 rpm for 12 min. The cell pellets were resuspended in 1x annexin binding buffer, and propidium iodide and FITC-annexin v were added. The solutions were allowed to incubate at room temperature for 15 min, after which the solutions were filtered. Samples were run on a BD LSR II (BD Biosciences, San Jose, CA, USA) flow cytometer with a 488 nm-excitation laser and fluorescence detection in the R-Phycoerythrin (PE) and Fluorescein
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(FITC) channels. Data were analyzed using FlowJo (Tree Star Inc, Ashland, OR, USA). The percentage of cell populations was calculated from the total 10,000 cells in comparison to control cells (no nanoparticle exposure). 2.12. Statistics The data were presented as mean standard error (SE) of at least three independent culture experiments.
Three technical replicates were performed for each independent cell culture
experiment. Significance differences among samples were determined using the t-test calculator (GraphPad Software, La Jolla, CA, USA). The level of statistical significance was p < 0.05 and was indicated by *.
3. Results 3.1 Characterization of nanoparticles and bioconjugation AuNPs and AgNPs were prepared through the reduction of their respective metal ions via sodium citrate.(Freund and Spiro, 1985; Kimling et al., 2006) UV-Vis spectroscopy indicated characteristic surface plasmon resonance (SPR) peaks at 525 nm for AuNPs and 420 nm for AgNPs.
TEM micrographs further showed successful synthesis of spherical particles with
average diameters of 22 ± 6 nm and 42 ± 12 nm for AuNPs and AgNPs, respectively (Fig. 1). The citrate stabilized AuNPs and AgNPs were also assessed using DLS which revealed hydrodynamic diameters of 25 and 58 nm, and zeta potentials of -20 and -32 mV, respectively. Citrate stabilized particles were then conjugated to thiol terminated polyethylene glycol (PEG, MW = 5,000) to increase their biostability and reduce non-specific binding of serum proteins contained in the cell culture medium. These particles, which are denoted as Ex-AuNPs and Ex-AgNPs, showed ~20 nm increase in their hydrodynamic diameters and a slight increase
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in their zeta potential measurements (Au: -18 mV, Ag: -20 mV). The SPR band of PEGylated AuNPs and AgNPs also showed a 1-2 nm red shift signifying a change in the dielectric constant of the NP’s surrounding environment.
Taken together, these changes indicate PEG was
successfully conjugated to the surface of the nanoparticles. PEGylated AuNPs and AgNPs were then conjugated with arginine-glycine-aspartic acid (RGD) peptides, or RGD and nuclear localization signal (NLS) peptides to promote cytoplasmic (Cy) and nuclear (Nu) localization, respectively. Conjugation of PEGylated nanoparticles with RGD (cytoplasmic targeting) peptides only produced two distinct particles: Cy-AuNPs and CyAgNPs. Conjugation with RGD and NLS (nuclear targeting) peptides resulted in two particles: Nu-AuNPs and Nu-AgNPs. Peptide conjugation was assessed via UV-Vis spectroscopy, as well as hydrodynamic diameter and zeta potential measurements. As seen with PEGylation, the characteristic SPR bands showed a red shift (1-2 nm) after surface modification with RGD and NLS peptides. The hydrodynamic diameter of peptides conjugated particles did not vary from PEG conjugated nanoparticles, however, the zeta potential of all particle formulations increased: Cy-AuNP: -3 mV, Nu-AuNP: +11 mV, Cy-AgNP: -5 mV, Nu-AgNP: +18 mV.
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Fig. 1. Au and Ag nanoparticle design. UV-Vis spectra and TEM micrographs showed successful synthesis of (A) AuNPs and (B) AgNPs with average diameters of 22 6 nm and 42 12 nm, respectively. (C) Synthesized NPs were then separated into three localization groups: extracellular (Ex-NPs), cytoplasmic (Cy-NPs) and nuclear (Nu-NPs), through conjugation with PEG and peptides.
3.2 Nanoparticle localization HSC-3 cells were incubated with 0.1 nM conjugated AuNPs and AgNPs for 24 h after which nanoparticle localization was assessed using dark field microscopy (Fig. 2). PEG conjugated nanoparticles did not show cellular uptake, as the only scattering that was detected was from the cells alone (Fig. 2A). The addition of RGD peptides to PEGylated particles caused nanoparticle internalization and cytoplasmic localization (Fig. 2B). Nu-AuNPs and Nu-AgNPs showed the greatest cellular uptake and were localized at the cell nucleus (Fig. 2C). This marked uptake, indicated by the increased light scattering, was also seen and quantitatively confirmed in
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previous works that utilized NLS in combination with RGD. (Austin et al., 2011; Mackey et al., 2013)
Fig. 2. Dark field images showing cellular uptake and localization of (A) extracellular, (B) cytoplasm, and (C) nuclear localized AuNPs (left) and AgNPs (right). Scale bar = 20 m.
3.3 Effect of nanoparticles on energy production and cell viability ATP production and XTT cell viability assays were conducted to investigate the in vitro cytotoxic effects of conjugated AuNPs and AgNPs on HSC-3 cells after 48 h of particle exposure (Fig. 3). ATP production was significantly reduced after exposure to 0.1 nM AuNP and AgNPs targeted to all cellular locations. Nu-AgNPs showed the greatest reduction in ATP (~92%) when compared to its gold counterpart (Nu-AuNPs, ~50%) and all other NPs tested. Unlike the ATP production assay, XTT assays reported no significant decrease in HSC-3 cellular viability when exposed to AuNPs targeted to the extracellular matrix, cytoplasm, or nucleus. Cy-AgNP and Nu-AgNP exposure at 0.4 nM resulted in ~22% and ~36% reduction in cell viability, respectively.
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Fig. 3. Assessment of cell viability in HSC-3 cells after 48 h of nanoparticle exposure. (A) The relative ATP content significantly decreased after exposure with AuNPs and AgNPs localized at the extracellular matrix (red), cytoplasm (blue) and nucleus (green). Control samples (i.e. “no NPs) are the same sample for each NP. (B) XTT cell viability assays did not show significant cell death after 0.1 nM and 0.4 nM AuNP exposure. (C) AgNPs localized at the cytoplasm (blue) and nucleus (green) showed a significant decrease in cell viability at 0.1 and 0.4 nM exposure concentrations. Ex-AgNPs (red) did not show a significant decrease in cell viability when assessed via mitochondrial enzyme activity. Data are expressed as mean ± SE of the three independent culture experiments. Three technical replicates were performed within each independent culture experiment. Statistical significance is indicated by * (p
