Accepted Manuscript
Glutathione reduces cytotoxicity of polyethyleneimine coated
magnetic nanoparticles in CHO cells
Klemen Strojan, Jasna Lojk, Vladimir B. Bregar, Peter Veranič,
Mojca Pavlin
PII:
DOI:
Reference:
S0887-2333(17)30029-2
doi: 10.1016/j.tiv.2017.02.007
TIV 3932
To appear in:
Toxicology in Vitro
Received date:
Revised date:
Accepted date:
15 December 2016
13 February 2017
14 February 2017
Please cite this article as: Klemen Strojan, Jasna Lojk, Vladimir B. Bregar, Peter Veranič,
Mojca Pavlin , Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic
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as affiliation for all authors. Please check if appropriate. Tiv(2017), doi: 10.1016/
j.tiv.2017.02.007
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Glutathione reduces cytotoxicity of polyethyleneimine coated magnetic nanoparticles in
CHO cells
Klemen Strojan a,1, Jasna Lojk a,b,1,
Vladimir B. Bregar a, Peter Veranič b and Mojca Pavlin a,c,*
a
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Faculty of electrical Engineering, University of Ljubljana, Trzaska cesta 25, SI-1000 Ljubljana,
Slovenia (klemen.strojan@fe.uni-lj.si, jasna.lojk@fe.uni-lj.si, vladimir.bregar@kolektor.com)
b
Institute of Cell Biology, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000
Ljubljana, Slovenia (peter.veranic@mf.uni-lj.si)
c
Institute of Biophysics, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, SI-1000
Ljubljana, Slovenia (mojca.pavlin@fe.uni-lj.si)
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Authors contributed equally to this work
Abstract
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Polyethyleneimine (PEI) is a polycationic compound frequently used as a transfection agent.
However, cytotoxicity of PEI and PEI-coated nanoparticles (PEI NPs) is still a major obstacle in
its use. In this study we report a method for reducing cytotoxicity of PEI NPs by addition of
glutathione in NPs synthesis. Glutathione reduced cytotoxic effects for at least 30% and
decreased observed oxidative stress response compared to standard formulation. Results showed
that the effect was partially due to reduced zeta potential and partially due to protective
antioxidant properties of glutathione. Addition of glutathione to cell culture media with
concurrent exposure to PEI NPs proved to be insufficient for cytotoxicity reduction. Additionally,
we compared internalization pathways of both PEI NPs and GSH NPs. NPs were only found in
endosomes and no NPs were found free in the cytosol, as would be expected according to so
called proton sponge hypothesis.
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Keywords: Magnetic nanoparticle; Glutathione; Polyethyleneimine; Cytotoxicity
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*Correspondence to:
Assist. Prof., Ph.D. Mojca Pavlin
Head of the Group for nano and biotechnological applications
Faculty of electrical Engineering, University of Ljubljana
Trzaska cesta 25, SI-1000 Ljubljana
Slovenia
Phone: +386 1 4768 949
Fax:
+386 1 4264 850
E-mail: mojca.pavlin@fe.uni-lj.si
NPs, nanoparticles; PEI, polyethyleneimine; CHO, Chinese hamster ovary cells; ROS, reactive
oxygen species; PEI NPs, polyethyleneimine coated magnetic nanoparticles; GSH NPs,
glutathione modified PEI NPs; PAA, polyacrylic acid sodium salt; TEM, transmission electron
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microscopy; SEM, scanning electron microscopy; MWCO, molecular weight cut-off; DLS,
dynamic light scattering, FBS, fetal bovine serum; PI, propidium iodide; pGFP, plasmid DNA
encoding green fluorescent protein
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1. Introduction
In the last two decades, nanotechnology became an important research field and nanomaterials
quickly found their way also in biomedicine and biotechnology. In biomedicine, nanoparticles
(NPs) are commonly used as contrasting agents (Arami et al., 2015; Gupta and Gupta, 2005;
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Pankhurst et al., 2003; Wang et al., 2001) and for drug delivery (Gabizon et al., 1994; Gad et al.,
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2012; Gradishar, 2006; Kalia et al., 2014; Werner et al., 2011; Zhu and Liao, 2015). Magnetic
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NPs are a special type of NPs based on magnetic core that is combined with additional coatings
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(Bee et al., 1995; Schroder et al., 1986). Their magnetic properties in combination with specific
coatings (e.g. polymers, small-molecule ligands, surfactants) make them suitable for specialized
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applications such as molecular imaging (Laurent et al., 2008; Yu et al., 2000), hyperthermia
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(Thiesen and Jordan, 2008) and magnetofection (Chen et al., 2012; Scherer et al., 2002).
Moreover, the electron dense core that enables visualization with NMR and electron microscopy
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in combination with other properties enables theranostic approach (Rizzo et al., 2013). Still, the
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cytotoxicity of certain types of NPs limits their efficiency and applicability (Buyukhatipoglu and
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Clyne, 2011; De Jong and Borm, 2008).
Polyethyleneimine (PEI) is a cationic polymer with one of the highest cationic-charge-density
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potentials; namely every third atom is amino nitrogen that can be protonated. This allows PEI to
form stable complexes with DNA and transfer them into the cytosol once internalized (Boussif et
al., 1995). Moreover, PEI is used also as a coating that stabilizes the NPs and enables
electrostatically based delivery of nucleic acids into mammalian cells, i.e. transfection (Prijic et
al., 2012; Scherer et al., 2002; Tai et al., 2003), also referred to as magnetofection in case when
magnetic field is used to stimulate NPs uptake (Mykhaylyk et al., 2007).
However, PEI and similar polycations can induce cytotoxic responses and trigger cell death
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(Fischer et al., 2003; Fröhlich, 2012; Hoskins et al., 2012; Hwang et al., 2015; Larsen et al.,
2012; Moghimi et al., 2005). Cytotoxicity is the major obstacle of its use especially in
applications that require delivery of higher concentrations of NPs (Moghimi et al., 2005). Several
approaches have been proposed to reduce the cytotoxicity (Beyerle et al., 2010; Hoskins et al.,
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2012; Huang et al., 2014; Ratanajanchai et al., 2014), such as combining PEI and
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tripolyphosphate (Huang et al., 2014) or incorporation of PEI into core-shell nanostructures
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(Ratanajanchai et al., 2014). In the present paper we propose addition of reduced form of
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glutathione as the final step of NPs preparation.
Glutathione is the most prevalent intracellular thiol with many different functions; the most
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obvious one being the protection of cells against reactive oxygen species (ROS) (Meister and
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Anderson, 1983; Sies, 1999). Reduced glutathione prevents oxidation of cellular thiols by acting
as a sacrificial antioxidant and it also assist in protein folding by participating in thiol transfer
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reactions. Moreover, significant amounts of glutathione have been found covalently bound to
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proteins, which might have a protective and reparative role (Hill and Bhatnagar, 2007).
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The aim of our study was to test the hypothesis that addition of glutathione in the NPs preparation
protocol reduces cytotoxicity of PEI-coated NPs (PEI NPs). The obtained results show reduced
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cytotoxicity for glutathione modified NPs (GSH NPs) that correlates with decreased ROS levels
and increased levels of glutathione content in cell lysates. We also showed that GSH NPs had
similar transfection efficiency to PEI NPs. Furthermore, we visualized NP-membrane interactions
with scanning electron microscopy (SEM) and internalization pathways of PEI NPs and GSH
NPs with transmission electron microscopy (TEM), showing the uptake of PEI NPs and GSH
NPs. Interestingly, no sign of lysosomal escape of NPs was present since no NPs were observed
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in the cytosol. Although, this was not the question of our research, it may shed some light to the
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“proton sponge” hypothesis (Behr, 1997; Benjaminsen et al., 2012).
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2. Materials and methods
2.1. Synthesis
Cobalt ferrite (CoFe2O4) polyacrylic acid NPs (CoFe2O4-PAA NPs) and PEI coated CoFe2O4PAA NPs (PEI NPs) were prepared as described previously (Bregar et al., 2013; Prijic et al.,
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2012). Briefly, cobalt ferrite NPs were prepared by co-precipitation and stabilized in alkaline
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medium (Tourinho et al., 1990). Alkaline medium was removed and NPs were re-suspended in
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distilled water. The process was repeated three times and after the last washing step nitric acid
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was added to stabilize NPs in acidic media. NPs were coated in situ with water solution of
polyacrylic acid sodium salt (PAA) with molecular mass of 8 kDa (Sigma-Aldrich, St. Luis,
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Missouri, USA) by mixing equal volumes of CoFe2O4 NPs (pH 2.0; 3 wt. %) and PAA (pH 7.3; 3
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wt. %) for 10 min at room temperature. Unbound PAA was removed with 24 hours of dialysis
against 2 l of distilled water using dialysis membrane with 20 kD MWCO (Spectrum
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Laboratories, Rancho Dominguez, CA, USA). Water was replaced three times to ensure complete
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removal of unbound PAA. At this step we obtained CoFe2O4-PAA NPs. PEI functionalization
was performed using branched cationic polymer PEI with molecular weight of 25 kDa (Sigma-
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Aldrich) by mixing equal volumes of CoFe2O4-PAA NPs (pH 7.4; 2 wt. %) with PEI water
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solution (pH 7.8; 3 wt. %) for 10 min at room temperature. Unbound PEI was removed with 24
hours of dialysis against 2 l of distilled water using dialysis membrane with 20 kD MWCO.
Water was replaced three times to ensure complete removal of unbound PEI. At this step we
obtained PEI NPs. To obtain GSH NPs, PEI NPs (pH 7.4; 2 wt. %) were mixed with Lglutathione reduced (Sigma-Aldrich, St. Luis, Missouri, USA; pH 5.5; 0.5 wt. %) in 1:1 ratio.
Again, NPs were dialyzed against 2 l of distilled water for 24 hours using dialysis membrane with
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20 kD MWCO. Water was replaced three times to ensure complete removal of unbound
glutathione. Before application to cells, the pH of all NP-formulations was adjusted to 7.2 ± 2.
2.2. Characterization
ATR-FTIR spectra of dry samples were recorded on a Bruker FTIR (Fourier Transform Infrared
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Spectroscopy) Alpha Platinum ATR spectrophotometer (Bruker, Billerica, Massachusetts, USA).
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Samples were dried on to the surface of the ATR diamond crystal prior to measurements. For size
characterization of the NPs we used dynamic light scattering (DLS) (Malvern Zetasizer Nano ZS;
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Malvern Industries, Malvern, UK) with the NIBS (noninvasive backscatter) 173° backscatter
algorithm. Zeta potential measurements were done with disposable folded capillary cells and the
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M3-PALS measurements technology, built in the Malvern Nanosizer NanoZS system. Before any
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2.3. Cell lines and cell culturing
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measurements the pH of all NP-formulations was adjusted to 7.2 ± 2.
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All experiments were performed on adherent Chinese Hamster Ovary (CHO) cells. Cells were
grown in HAM culture medium for mammalian cells (Sigma-Aldrich), supplemented with 10%
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fetal bovine serum (FBS) (Sigma-Aldrich), 0.5% L-glutamine (Sigma-Aldrich), 0.1% gentamicin
(PAA Laboratories, Pasching, Austria), and 0.01% penicillin (PAA Laboratories), at 37°C in a
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humidified 5% CO2 atmosphere. All experiments were performed on a 24-hour old cell culture.
NPs were incubated in cell culture medium with 10% FBS, NPs concentration and NPs mass per
well surface ratio was maintained constant through all experiments.
2.4. Propidium iodide viability assay
Propidium iodide (PI) viability assay (PI assay) was performed as described previously (Bregar et
al., 2013; Jasna Lojk et al., 2015). Cells were grown in a 24-well plate (Corning Inc., Corning,
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NY, USA), seeded at 2 × 104 cells/well and incubated with different concentration of NPs (0, 2,
4, 6, 8 and 10 µg/ml) for 24 hours. After incubation, cell culture medium containing NPs was
removed and cell nuclei were stained with 0.2 mM Hoechst 33342 (Thermo Fisher Scientific,
Waltham, MA, USA). Hoechst stain enabled us to obtain the total cell number (N). To label dead
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cells, cells were incubated with 0.15 mM propidium iodide (Sigma-Aldrich) for 5 minutes,
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allowing the fluorescent dye to enter only dead cells. For each sample, 20 visual fields at 20×
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objective magnification were taken using MetaMorph imaging system software (Visitron,
Puchheim, Germany) connected to fluorescence microscope (Zeiss 200, Axiovert, Jena,
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Germany). Stained nuclei were counted using CellCounter software (Lojk et al., 2015b).
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Approximately 2000 cells were counted for each parameter. The number of dead cells (Nd) was
subtracted from the total number of cells (N). The percentage of viable cells (Viability) in a given
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sample was determined as the ratio between the number of viable cells in the sample (Ns) and the
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number of all cells in the control sample (N0):
(1)
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(2)
The
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2.5. MTS viability assay
assay
was
performed
using
MTS
reagent
3-(4,5-dimethylthiazol-2-yl)-5-(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (Cell Titer®96 AQueous
One Solution Cell Proliferation Assay; Promega Corp, Fitchburg, WI, USA) according to
manufacturer’s instructions. Briefly, CHO cells were grown in 24-well plate (Corning Inc.) and
incubated with different concentration of NPs (0, 2, 4, 6, 8, 10 µg/ml) for 24 hours. After
incubation, cell culture medium containing NPs was removed and replaced with 200 µl of fresh
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cell culture medium per well. Then, 40 µl of MTS reagent 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt was added to each well and
left to incubate for 90 minutes. Absorbance was measured at 490 nm using Tecan Infinite 200
(Tecan Group Ltd, Männedorf, Switzerland). None of the tested NPs interfered with the assay at
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concentrations used for experiments. The results are presented as percentage of relative
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absorbance units compared to the untreated control.
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2.6. Transmission electron microscopy
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CHO cells were grown in 6-well plates (Corning Inc.) and incubated with 4 µg/ml NPs
concentration for 2 or 24 hours. After incubation, cells were washed and fixed with a mixture of
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4% (w/v) paraformaldehyde and 2% (v/v) glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, for
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2 hours at room temperature. Post-fixation was carried out in 1% osmium tetroxide in 0.1 M
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cacodylate buffer for 2 hours, followed by dehydration in graded ethanol and embedding in Epon
812 resin (Electron microscopy sciences, Hatfield, PA, USA) as described previously (Bregar et
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al., 2013). Ultrathin sections were counterstained with uranyl acetate and lead citrate and
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examined with TEM (CM100; Royal Philips Electronics, Amsterdam, The Netherlands).
2.7. Scanning electron microscopy
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CHO cells were grown on cover glass in 6-well plates (Corning) to 40% confluency and
incubated with 8 μg/ml NPs for 30 minutes at 37°C. After incubation, cells were fixed with a
mixture of 4% (w/v) formaldehyde and 2% (w/v) glutaraldehyde in 0.1 M cacodylate buffer for 2
hours. Samples were washed with cacodylate buffer and post-fixated in 4% osmium tetroxide for
1 hour; washed again and incubated with 1% tannic acid in 0.05 M cacodylate buffer for 30
minutes; followed by washing in cacodylate buffer and dehydration in progressive series of
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ethanol, which was exchanged in a progressive series with hexamethyldisilazane (HMDS)
(Structure Probe Inc., West Chester, PA, USA); and incubated overnight. The samples in HMDS
were air dried and carbon sputtered. The whole procedure was carried out at room temperature.
SEM analysis was performed with VEGA3 scanning electron microscope (TESCAN Brno, s.r.o.
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Brno, Czech Republik).
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2.8. Oxidative stress
Oxidative stress was evaluated measuring ROS production, using 5-(and-6)-chloromethyl-2',7'-
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dichlorodihydrofluorescein diacetate fluorescent probe (CM-H2DCFH-DA; Molecular Probes,
Invitrogen) according to manufacturer’s instructions. Briefly, CHO cells were grown in 24-well
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plate (Corning Inc.) and incubated with different concentration of NPs (0, 2, 4, 6, 8, 10 µg/ml) for
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24 hours. After incubation, cell culture medium containing NPs was removed and cells were
washed two times with PBS. Cells were incubated with 10 µM CM-H2DCFH-DA in PBS
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(containing Ca2+ and Mg2+ ions). After 45 minutes incubation at 37°C, fluorescence intensity was
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measured with excitation at 492 nm and emission at 527 nm using Tecan Infinite 200. None of
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the tested NPs interfered with the assay at concentrations used for experiments. The results are
presented as fold increase of fluorescence compared to the untreated control.
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2.9. Total glutathione content
Total glutathione concentration (GSH+GSSG) was evaluated using ApoGSHTM Glutathione
Colorimetric Detection Kit (Biovision Inc, Milpitas, CA, USA) according to manufacturer’s
instructions. Briefly, CHO cells were grown in 60 mm petri dishes (Corning Inc.) (seeded 3 × 105
cells/dish) and incubated with different concentration of NPs (4, 6, 8 µg/ml) for 24 hours. After
incubation, cells were collected by trypsinisation and centrifuged at 700 × g for 5 minutes. Pellets
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were suspended in 500 µl of ice-cold PBS and centrifuged at 700 × g for 5 minutes. Lysis buffer
was added to the pellet. 15 µl of 5% 5-Sulfosalicylic acid was added to the sample, mixed and
centrifuged at 8000 × g for 10 minutes (all centrifugation steps were performed at 4°C). 20 µl of
supernatant was added to 160 µl of reaction mix, containing NADPH generating mix, glutathione
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reductase (GR) and lysis buffer. After 10 minutes of incubation at room temperature, 20 µl of
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5,5´-dithiobis(2-nitrobenzoic acid) (DTNB) was added. Glutathione reduces DTNB to 5-thio(2-
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nitrobenzoic acid), a yellow by-product, absorbing at 405 nm. Absorbance at 405 nm was
measured after 5 minutes of incubation using Tecan Infinite 200. Total glutathione concentration
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in cell lysates was determined by inverse linear regression from calibration curve and normalized
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to total protein content obtained with Pierce® BCA Protein Assay Kit (Thermo Fisher Scientific
Inc., Waltham, MA, USA). None of the tested NPs interfered with the assay at concentrations
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used for experiments. The results are presented as micrograms of glutathione per milligram of
Transfection with PEI NPs and GSH NPs
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2.10.
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total protein content.
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Preparation of NP-DNA complexes is described elsewhere (Mykhaylyk et al., 2007). Briefly, NPDNA complexes were obtained by mixing equal volumes of 180 µg/ml PEI NPs or GSH NPs and
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90 µg/ml of plasmid DNA encoding for green fluorescent protein (pGFP). The suspension was
thoroughly mixed and incubated for 15 minutes at room temperature. Volumes corresponding to
0, 4, 6, and 8 µg/ml of NPs were added to CHO cells grown in 24-well plate (Corning Inc.).
Additional control was performed by adding pGFP without NPs to CHO cells. Transfection
efficiency was determined after 24 hours of incubation using fluorescence microscope with
excitation at 475 nm and emission at 510 nm (Zeiss 200, Axiovert, Jena, Germany). Transfection
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efficiency is shown as a percent of green fluorescent protein expressing viable cells compared to
control (Pavlin and Kandušer, 2015).
2.11.
Statistical analysis
Each experiment was performed in at least three independent repeats. If not stated otherwise, data
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are presented as a mean percent difference from control with corresponding standard error of the
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mean. Statistical tests were run by GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA,
USA) using ANOVA followed by Tukey's multiple comparisons test. Statistical significance is
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displayed as follows: ns – not significant (p>0.05); *p ≤ 0.05; **p ≤ 0.001; ***p ≤ 0.0001; ****
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p ≤ 0.00001.
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3. Results
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3.1. Nanoparticle synthesis and characterization
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To test our hypothesis, we prepared L-glutathione reduced modified PEI NPs (GSH NPs). To
determine the changes in the properties of NPs due to additional coatings, we characterized the
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NPs using TEM, DLS, ATR-FTIR, and zeta potential measurements. TEM micrographs of NPs
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dispersed in distilled water are shown in Fig. 1, where we can see 10-20 nm large cores.
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Fig. 1. TEM micrographs of PEI NPs and GSH NPs dispersed in distilled water. Scale bar
corresponds to 100 nm.
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DLS and zeta potentials were measured in distilled water and HAM cell culture media.
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Dispersion in HAM increased the hydrodynamic diameters of both NP types, which was further
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increased when NPs were incubated in HAM + 10 % FBS (Table 1), leading to formation of
protein corona and agglomeration of NPs. On average, hydrodynamic diameters of NPs
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agglomerates in the medium with serum were 3 µm for PEI NPs and 2.3 µm for GSH NPs (Table
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1). Size distributions of PEI NPs and GSH NPs are shown in Fig. 3. The values of zeta potential
of PEI NPs and GSH NPs in water suspension were 54 mV and 45 mV, respectively. Zeta
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potential decreased in HAM cell culture medium to approximately 22 mV for both NPs (Table 1).
ATR-FTIR measurements confirmed additional coatings of NPs between different steps of the
synthesis (Fig. 2A). Peak shifts were obvious when Co ferrite NPs were coated with PAA and
additionally coated with PEI. Modification with glutathione (GSH NPs) resulted only in minor
changes of the spectra (Fig. 2B).
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Table 1: Physical characterization of NPs
Distilled water
HAM
HAM + 10% FBS
GSH NPs
Distilled water
HAM
HAM + 10% FBS
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59
2300b
Zeta potential (mV)
54 ± 4
21 ± 5
NAb
45 ± 5
22 ± 1
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PEI NPs
Mean hydrodynamic
diametera (nm)
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81
3000b
Size distribution obtained by dynamic light scattering (distribution based on particle number)
Formation of agglomerates
2.
FTIR
measurements.
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Fig.
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Dispersion media
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were dried
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crystal
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solutions/dispersions prior to ATR-FTIR measurements. ATR-FTIR spectra of cobalt ferrite NPs
(Co ferrite NPs), PAA, PEI, Co ferrite PAA NPs, PEI NPs and GSH NPs (A). ATR-FTIR spectra
of PEI NPs and GSH NPs in the 1800-1000 cm-1 region (B).
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Fig. 3. Dynamic light scattering measurements of PEI NPs and GSH NPs. Particle number
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distributions in water (A), cell culture media (B) and cell culture media with 10% FBS (C) are
shown. There were no significant differences between distributions of hydrodynamic diameters
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of PEI NPs and GSH NPs in cell culture media and cell culture media with 10% FBS. *p<0.05
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3.2. Cell viability
Cell viability was determined 24 hours after incubation with increasing concentrations of PEI
NPs and GSH NPs using two independent assays: MTS viability assay and PI viability assay
(Fig. 4). Both assays showed a similar concentration dependent decrease in viability, however,
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the rate of decrease significantly depended on NP-type. PEI NPs were more cytotoxic than GSH
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NPs. For example, at 6 µg/ml NPs concentration, viability of cells determined by PI assay was
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40.2% when exposed to PEI NPs, and 84.7% when exposed to GSH NPs. MTS assay showed
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even larger differences in viability of CHO cells exposed to PEI NPs and GSH NPs at
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concentration 6 µg/ml: 30.8% and 87.4% respectively.
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Fig. 4. Viability of CHO cells exposed to different concentrations of PEI NPs and GSH NPs.
Viability was determined 24 hours after exposure to NPs with PI assay (A) and MTS assay (B).
The results are presented as percentage of viable cells compared with the number of cells in the
control sample (PI assay) or percentage viability determined with spectrofluorimetry (MTS
assay). Means with standard errors are shown (n=3). **p ≤ 0.001; ***p ≤ 0.0001; **** p ≤
0.00001
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In order to analyze separately the potential reduction of NP cytotoxicity by the glutathione
present free (not bound to NPs) in the cell culture media we incubated the cells with increasing
concentration of glutathione in the media (0.05 – 500 µM) in absence or presence of 4 µg/ml PEI
NPs (Fig. 5). Glutathione showed no significant effect on viability of CHO cells exposed to 4
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µg/ml PEI NPs in the concentration range tested.
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Fig. 5. Viability of CHO cells exposed to different concentration of glutathione, PEI NPs or both.
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Viability was determined 24 hours after exposure with PI assay. PEI NPs with concentration 4
μg/ml were used. The results are presented as percentage of viable cells in each sample compared
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to the control sample. Means with standard errors are shown (n=3). There were no significant
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differences between treatments and control samples.
3.3. Oxidative stress and total glutathione content
The effects of PEI NPs and GSH NPs on oxidative stress were determined by measuring ROS
levels and intracellular total glutathione content after 24 hour exposure (Fig. 6). Both PEI and
GSH NPs induced ROS in a concentration dependent manner. Regardless of NP-type, lower
concentrations (2-6 µg/ml) did not induced ROS formation, while at higher concentrations PEI
NPs showed significant increase in ROS compared to the same concentrations of GSH NPs.
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To further understand the effect of PEI NPs and GSH NPs on CHO cells, levels of total
glutathione (GSH+GSSG) were measured. In samples incubated with GSH NPs, total glutathione
levels were higher for all tested concentrations compared to PEI NPs. With GSH NPs, total
glutathione concentration slowly decreased with increasing NPs concentration up to 8 µg/ml
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while for PEI NPs total glutathione concentration dropped already at 4 µg/ml and then slowly
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decreased with increasing NPs concentration until depletion at 8 µg/ml. Redox state of the cells
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was closer to normal, when cells were exposed to GSH NPs compared to PEI NPs.
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Fig. 6. Oxidative stress induced by PEI NPs and GSH NPs. Increase in reactive oxidative species
(ROS) compared to control (A) and total glutathione content per mg of protein (B) were
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measured 24 h after exposure of CHO cells to PEI NPs or GSH NPs. Means with standard errors
are shown (n=4). ns – not significant (p>0.05); *p ≤ 0.05; **p ≤ 0.001; **** p ≤ 0.00001.
3.4. NP-membrane interactions and NP internalization
To analyze NP-membrane interaction and to determine the internalization pathways and
intracellular fate, SEM and TEM were performed. SEM showed several NPs attached to the cell
membrane already after 30 min of incubation (Fig. 7). Using TEM, we showed that both PEI NPs
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and GSH NPs enter the cells with endocytosis, most probably with macropinocytosis (Fig. 8A &
B) and were found only inside the vesicles of the endo-lysosomal system, most of which
resembled late endosomes (Fig. 8C & E) and amphisomes (Fig. 8D & F) already 2 hours after
internalization. Similarly, both types of NPs were mostly found in vesicles which resembled
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amphisomes also after 24 hours of incubation (Fig. 8G & J). No NPs were found free in the
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cytosol or associated with any other cell organelles.
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Fig. 7. SEM of CHO cells exposed to PEI NPs and GSH NPs. CHO cells were exposed to 8
μg/ml of PEI NPs (B, E) or GSH NPs (C, F) for 30 minutes. Black arrows denote NPs on the
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surface of the cells. Scale bars correspond to 10 μm in panels A-C and to 1 μm in panels D-F.
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Fig. 8. TEM micrographs of CHO cells exposed to PEI NPs and GSH NPs. CHO cells were
incubated with 4 μg/ml PEI NPs (A, C, D, G, H) or GSH NPs (B, E, F, I, J) for 2 h (C, D, E, F) or
24 h (A, B, G, H, I, J). Scale bars correspond to 500 nm.
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3.5. Transfection of cells with PEI and GSH NPs as delivery vectors
To confirm that GSH NPs can still be used as transfection vectors, we compared transfection
efficiencies of GSH and PEI NPs as delivery vectors. Phase contrast and fluorescent microscopy
images of transfected cells are shown in Fig. 9. Both types of NPs showed relatively low but
comparable transfection efficiency, ranging from 2.0% and 1.3% at the lowest NP-concentration
to 5.1% and 7.3% at 8 µg/ml concentration for PEI NPs and GSH NPs respectively (Figs. 9 &
10), with no significant differences in transfection efficiency between PEI NPs and GSH NPs.
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Moreover, there were no CHO cells expressing green fluorescent protein detected when the cells
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were exposed solely to PEI NPs, GSH NPs or pGFP (Fig. 10).
Fig. 9. Transfection of CHO cells with plasmid DNA encoding for green fluorescent protein
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(pGFP). 8 μg/ml of PEI NPs or GSH NPs with pGFP were used for transfection. From left to
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right: phase contrast (A), Hoechst 33342 stained nuclei (B), PI stained nuclei (dead cells) (C),
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corresponds to 45 µm.
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green fluorescent protein positive (transfected) cells (D) and merge (E) are shown. Scale bar
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Fig. 10. Transfection efficiency of PEI NPS and GSH NPs. Transfection efficiency on CHO cells
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24 hours after transfection with pGFP using PEI NPs and GSH NPs. Means with standard error
are shown (n=3). ns – not significant (p>0.05)
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4. Discussion
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High cationic charge-density potentials and branched network enables PEI to form complexes
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with DNA that can be used to transfect cells (Boussif et al., 1995). PEI-coated magnetic NPs are
therefore an effective tool for transfection and magnetofection (Mykhaylyk et al., 2007; Scherer
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et al., 2002). However, very high surface charge of PEI is also responsible for cytotoxicity of PEI
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NPs, which affects the cells mainly through two mechanisms: i) plasma membrane destabilization
and perturbation, (Fischer et al., 2003; Larsen et al., 2012; Moghimi et al., 2005) and ii)
lysosomal disruption that is based on the so called proton-sponge hypothesis (Behr, 1997;
Benjaminsen et al., 2012). Reducing cytotoxicity of PEI NPs would thus allow the use of higher
NP-concentrations and possibly increase their applicability. Glutathione has already been used
for NP-functionalization (Pan et al., 2011; Simpson et al., 2013), but not in combination with PEI
NPs or for reduction of their cytotoxicity. Our hypothesis was that modifying PEI NPs with
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glutathione could potentially hide the reactive amino groups on PEI and thus decrease the high
surface charge and strong binding to the membrane. Furthermore, anti-oxidative properties of
glutathione could reduce the indirect damage caused by ROS.
FTIR analysis confirmed changes of NPs surface after consecutive coating with PAA and PEI
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(PEI NPs). Additional glutathione coating (GSH NPs) resulted only in minor changes of the
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spectra, which was expected as glutathione formed an electrostatically bound corona that only
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partially covered the surface. GSH NPs had significantly smaller hydrodynamic diameter in
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distilled water (41 nm) compared to PEI NPs (78 nm) which can be explained with additional
electrostatic stabilization of NPs with glutathione. Moreover, the zeta potential of GSH NPs
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dropped from 54 mV to 45 mV.
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Despite the initial differences between PEI NPs and GSH NPs, zeta potential in HAM cell culture
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medium was similar for both NP formulations. This can be explained by counter ion layer
formation. Counter layer formation is a complex function of NPs surface chemistry and different
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ions and macromolecules (e.g. salts and amino acids) present in the media. Counter layer was
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responsible also for the increase of hydrodynamic diameters of both NPs dispersed in HAM cell
culture medium compared to NPs dispersed in distilled water (Pavlin and Bregar, 2012). When
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NPs were suspended in HAM cell culture medium with 10% FBS, protein coronas formed around
NPs due to relatively high effective surface potential (Treuel et al., 2015). Proteins destabilized
NPs and caused their agglomeration (Pavlin and Bregar, 2012). The differences in agglomeration
in physiological conditions between PEI NPs and GSH NPs were also clearly visible in TEM
micrographs (Fig. 8).
Following internalization, GSH NPs showed significantly increased cell viability compared to
PEI NPs (Fig. 4). The effect can be explained with i) the observed reduction of zeta potential
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(Nimesh et al., 2006) and ii) by antioxidant properties of glutathione. Moreover, to evaluate if the
effect was due to the presence of unbound glutathione in the media alone, we incubated CHO
cells with increasing concentration of glutathione (Fig. 5). Additionally added glutathione in the
media did not increase viability of CHO cells when the cells were exposed to 4 μg/ml PEI NPs,
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proving that glutathione must be bound to NPs already during the synthesis procedure to reduce
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the cytotoxicity of PEI NPs.
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The reduced cytotoxicity of GSH NPs compared to PEI NPs was further confirmed with
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significantly lower ROS formation at the same NP concentrations. Similarly, GSH NPs reduced
the total glutathione (GSH+GSSG) contents at a lower rate compared to PEI NPs, which
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coincided with ROS formation and further confirmed the effect of the bound glutathione on the
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cells redox state and viability. Observed difference in the effects of glutathione present freely in
the cell culture media and the glutathione bound on the GSH NPs can be explained with the
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utilization of different uptake mechanisms. In the case of glutathione in the media, glutathione
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was primarily degraded and the products were internalized by the cells, which was followed by
intracellular re-synthesis (Bachhawat et al., 2013; Meister and Anderson, 1983). On the other
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hand, glutathione on GSH NPs entered the cells in its completely functional, non-degraded form,
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most likely through the process of macropinocytosis. After internalization, glutathione might
have dissociated from GSH NPs due to pH changes in lysosomes and performed its antioxidant
functions. Moreover, glutathione was present at the site of NPs induced damage and was
therefore immediately available for enzymatically driven ROS scavenging processes.
Glutathione controls a variety of key molecular mechanisms linked to cell signaling, immune
response, cell proliferation, apoptosis and cell death (Jacob and Winyard, 2009). Its depletion is
usually an indication of overload of the antioxidant defense systems (Oberdörster, 2004).
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Glutathione depletion can be mainly explained by protein glutathiolation (Hill and Bhatnagar,
2007). When cells are exposed to an oxidant challenge, abundance of glutathiolated proteins
increases. Adduction of glutathione to proteins may have a protective and reparative role;
glutathione could serve as a protective cap to blunt further reactivity or trigger adaptive responses
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(Hill and Bhatnagar, 2007). In the case of GSH NPs, excess of glutathione can non-enzymatically
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dethiolate glutathiolated proteins, resulting in higher glutathione concentration in CHO cells
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compared to cells exposed to PEI NPs.
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Despite the changed surface properties, PEI NPs and GSH NPs showed similar interactions with
the cell membrane. They adhered to the cell membrane already after 30 min of incubation (Fig.
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7), entered the cells through endocytosis (most probably macropinocytosis) (Figs. 8A & B) and
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were found in different vesicles of the endolysosomal system, most of which resembled
amphisomes (Fig. 8C & J), vesicles formed by fusion of endosomes and autophagosomes. This is
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also in agreement with previous studies showing that PEI polymer and PEI NPs can induce
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autophagy (Gao et al., 2011), which is considered as one of the mechanisms for cell survival
under stress. Interestingly, no PEI NPs or GSH NPs were found free in the cytosol or associated
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with any other cell organelles even after 24 h of incubation in our experimental setup, as would
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be expected for NPs with the ability to escape the lysosomes according to the proton sponge
hypothesis (Behr, 1997; Benjaminsen et al., 2012). Since our NPs are still able to transfect cells
(Figs. 9 & 10), at least the delivered plasmid DNA had to escape the lysosomes and enter the
nuclei, confirming that a certain amount of lysosomal damage and leakage was present.
Nevertheless, the amount of escaped NPs might be too low to be detected using TEM. The
lysosomal escape might thus have been restricted to smaller molecules and not bigger NPagglomerates. This is also in agreement with other studies, which showed that DNA detaches
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from NPs before entering the nuclei (Cebrián et al., 2011), although NPs entering the nuclei have
also been observed previously (Siu et al., 2012; Thomas and Klibanov, 2003).
The efficiency of GSH NPs as delivery vectors was confirmed by transfection experiment.
Transfection of cells with plasmid DNA encoding green fluorescent protein using PEI NPs and
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GSH NPs showed similar efficiency (Fig. 10). We would like to stress that the transfection served
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only as a proof of principle; transfection could be improved by intensive optimization, which was
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not the focus of this study.
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5. Conclusions
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In conclusion, our results showed that glutathione modified PEI NPs (GSH NPs) NPs were
considerably less cytotoxic to CHO cells as PEI NPs. The main mechanisms behind reduced
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cytotoxicity were i) reduced membrane damage and ii) reduction of ROS formation. Cytotoxicity
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of GSH NPs was reduced due to the antioxidant effect of glutathione, as indicated by lower
depletion of total glutathione levels and lower zeta potential, which consequently decreased
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membrane damage. Moreover, following internalization, NPs were only found in endosomal
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compartments, indicating that any damage to the lysosomes either via proton sponge effect or
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direct membrane damage did not result in release of NPs into the cytosol.
Acknowledgments
This work was supported by Slovenian Research Agency (grant numbers: J2-6758, J3-6794, J77424, P1-0055), Young Researchers Program and MRIC UL IP-0510 Infrastructure Program.
The authors would like to thank Nanotesla Institute Ljubljana for their technical support in the
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preparation of nanoparticles and Prof. dr. Jurij Svete for his help with FTIR measurements. We
express gratitude to dr. Samo Hudoklin, Maruša Bizjak, Maruša Lokar, Jerneja Kladnik, Sanja
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Čabraja, Linda Štrus, Nada Pavlica Dubarič, and Sabina Železnik for their technical assistance.
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Highlights
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PEI NPs induce toxicity, ROS production and glutathione depletion in Chinese hamster
ovary cells.
Cytotoxicity is reduced by adding glutathione to the nanoparticle-synthesis procedure.
Glutathione added to the cell culture media does not have the same effect.
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