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Evaluation of gold nanorods toxicity on isolated mitochondria
Accepted Manuscript Title: Evaluation of gold nanorods toxicity on isolated mitochondria ´ Authors: Abner M. Nunes, Kleyton R.M. da Silva, Claudia M.S. Calado, Karina L.A. Saraiva, Regina C.B. Q. Figueiredo, Ana Catarina R. Leite, Mario R. Meneghetti PII: DOI: Reference:
S0300-483X(18)30245-2 https://doi.org/10.1016/j.tox.2018.12.002 TOX 52141
To appear in:
Toxicology
Received date: Revised date: Accepted date:
14 August 2018 22 November 2018 5 December 2018
´ Please cite this article as: Nunes AM, da Silva KRM, Calado CMS, Saraiva KLA, Q. Figueiredo RCB, Leite ACR, Meneghetti MR, Evaluation of gold nanorods toxicity on isolated mitochondria, Toxicology (2018), https://doi.org/10.1016/j.tox.2018.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of gold nanorods toxicity on isolated mitochondria
Ábner M. Nunes1, Kleyton R. M. da Silva2, Claudia M. S. Calado1, Karina L. A.
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Saraiva3, Regina C. B. Q. Figueiredo3, Ana Catarina R. Leite2*, Mario R. Meneghetti1*.
Grupo de Catálise e Reatividade Química, Instituto de Química e Biotecnologia,
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Universidade Federal de Alagoas, Av. Lourival de Melo Mota, CEP 57072-970, Maceió, Alagoas, Brazil 2
Laboratório de Bioenergética, Instituto de Química e Biotecnologia, Universidade
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Federal de Alagoas, Av. Lourival de Melo Mota, CEP 57072-970, Maceió, Alagoas,
Laboratório de Biologia Celular de Patógenos, Departamento de Microbiologia,
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Brazil
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Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Cidade Universitária, CEP
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50670-420, Recife, Pernambuco, Brazil
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*Corresponding Authors details:
E-mail: [email protected] (M.R.M)
Phone: +55 82 32141344 Phone: +55 82 998104445
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E-mail: [email protected] (A.C.R.L.)
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Graphical abstract
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Abstract
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Gold nanorods (AuNRs) have been studied extensively in biomedicine due to their
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biocompatibility and their unique properties. Some studies reported that AuNRs
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selectively accumulate on cancer cell mitochondria causing its death. However, the immediate effects of this accumulation needed further investigations. In this context, we
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evaluated the effect of AuNRs on the mitochondrial integrity of isolated rat liver mitochondria. We verified that AuNRs decreased the mitochondrial respiratory ratio by
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decreasing the phosphorylation and maximal states. Additionally, AuNRs caused a
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decrease in the production of mitochondrial ROS and a delay in mitochondrial swelling. Moreover, even with cyclosporine A treatment, AuNRs disrupted the mitochondrial potential. With the highest concentration of AuNRs studied, disorganized mitochondrial
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crests and intermembrane separation were observed in TEM images. These results indicate that AuNRs can interact with mitochondria, disrupting the electron transport chain. This study provides new evidence of the immediate effects of AuNRs on mitochondrial bioenergetics.
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Abbreviations: AuNRs, gold nanorods; AuNPs, gold nanoparticles; AuNSs, gold nanospheres;
BSA,
bovine
serum
protein;
CCCP,
carbonyl
cyanide
m-
chlorophenylhydrazone; CsA, cyclosporine A; CTAB, hexadecyltrimethylammonium bromide; MS, maximal state; OLIGO, oligomycin; PS, phosphorylation state; RCP, respiratory control rate; RLM, rat liver mitochondria; ROS, reactive oxygen species;
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RS, the resting state; SPR, surface plasmon resonance; TEM, transmission electron
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Keywords: gold nanorods; mitochondria; bioenergetics
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microscope.
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1. Introduction
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Due to their singular physicochemical properties, gold nanorods (AuNRs) are
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promising nanomaterials for a wide range of applications. AuNRs can be applied as
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catalysts for important chemical transformations (Priecel et al., 2016) in the biomedical field (Bobo et. al., 2016; Versiani et al., 2016 ). In this last area in particular, AuNRs
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have been used in the development of biosensors (Beuwer et. al., 2015), optical imaging (Zerda et. al., 2015; Qu et. al., 2016), gene (Nakatsuji et. al., 2017) and drug (Ali et. al.,
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2016; Feng et. al., 2015 ) delivery, photothermal therapy (Ali et. al., 2017), and a wide range of strategies for developing cancer treatments. It is worth mentioning here that
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many studies have reported the low toxicity of gold nanoparticles (AuNPs) (Patra et. al., 2007; Peng et al., 2009), nevertheless, their toxicity can be tuned by their surface modifications (Locatelli et. al., 2015; Wan et. al., 2015 ) and dimensions (size and shape) (Li et. al., 2014; Pietro et. al., 2016; Truong et. al., 2015).
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Due to the singular biological proprieties and features of AuNRs have been directly applied for therapeutic purposes in antitumor therapy through subcellular targeting, (Kodiha et. al., 2015; Zhang, 2015) with a strong focus on the nucleus and mitochondria. Mitochondria provide cancer cells with altered fuel availability, bioenergetics, oxidative stress, and cell death susceptibility, allowing them to survive in
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the face of adverse environmental conditions, such as starvation and cancer treatments. These features show the potential of mitochondria as a target for cancer therapy (Vyas
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et. al., 2016; Weinberg and Chandel, 2015). Thus, improvements based on the concept that AuNPs can target specific organelles will maximize their impact on tumor cells
(Kodiha et. al., 2015; Zhou et al., 2017). AuNRs often impair mitochondrial functions
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and thereby induce cell death (Zhang et. al., 2016). Among other things, CTAB-capped
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AuNRs were able to damage lipid bilayers and facilitate the permeabilization of
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membranes because AuNRs were capable of targeting mitochondria via their negative
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mitochondrial membrane potential (Kodiha et. al., 2015). It has been reported that
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AuNRs treated with fetal bovine serum albumin (BSA) caused cancer cell death with minimal injury to healthy cells (Wang et al., 2011). Both healthy and cancer cells were
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able to endocytose protein-coated AuNRs. Nevertheless, lysosome disruption was observed only in cancer cells and led to high AuNR accumulation in the mitochondria.
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AuNRs are able to induce cancer cell apoptosis and necrosis via lysosome- and mitochondria-mediated routes (Zhang et. al., 2017). Wang and coworkers demonstrated
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that AuNRs selectively accumulate in cancer cell mitochondria (Wang et al., 2011). The authors suggested that AuNR accumulation reduces the mitochondrial membrane potential and that the resulting dysfunction causes cell death. These works show again how important is the role of mitochondria in cancer cells for the development of new cancer therapies. For that reason, we decided to 4
investigate the effects of protein-coated AuNRs on the bioenergetics of isolated rat liver mitochondria (RLM) to obtain more information about how the mitochondriananoparticle interaction occurs and the effects on the organelle. Part of our studies are presented in this work.
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We would like to mention that despite of their interesting properties and applications of nanoparticulated materials, the risk of exposure to these materials for the
environment and/or humans must be subject of further studies (Ye et al. 2011;
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Kunzmann et al. 2011).
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2. Methods
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2.1. Chemicals
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All chemicals were obtained from commercial suppliers and used without
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further purification. Gold(III) chloride trihydrate (HAuCl4.3H2O, ≥ 99.9%), sodium borohydride (NaBH4, > 98%), hexadecyltrimethylammonium bromide (CTAB, ≥ 99%),
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bovine serum albumin (BSA, ≥ 96%), ethylene glycol-bis(2-aminoethylether)N,N,N′,N′-tetraacetic
acid
(EGTA,
≥
97%),
4-(2-hydroxyethyl)piperazine-1-
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ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, 99.5%), magnesium chloride (MgCl2, ≥ 98%), potassium phosphate monobasic
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(KH2PO4, ≥ 98%), dibasic potassium phosphate (K2HPO4, ≥ 98%), sodium pyruvate (≥ 99.0%), D-malic acid (≥ 97%), α-ketoglutaric acid (≥ 98.5%), aspartic acid (≥ 98%), succinic acid (≥ 99.0%), rotenone (≥ 95%), oligomycin, adenosine 5′-diphosphate sodium salt (ADP ≥ 95%), safranin O (≥ 85%), L-ascorbic acid p.a. (≥ 99%), H2O2 solution (30 wt % H2O), potassium chloride (KCl, ≥ 99%), carbonyl cyanide m-
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chlorophenyl hydrazone (CCCP) and horseradish peroxidase (HRP) were purchased from
Sigma-Aldrich
(Saint
Louis,
MO,
USA).
Amplex-Red®
and
2’,7’-
dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Thermo Fisher (Waltham, MA, USA). Sucrose, silver nitrate (AgNO3, > 99%), and ethyl alcohol (99%)
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were purchased Dinâmica (Diadema, SP, Brazil). 2.2. Gold nanorods preparation
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AuNRs were prepared by the seed-mediated methodology (Nikoobakht and ElSayed, 2003; Silva et al., 2013). Briefly, in a 20 mL cylindrical flask, gold seeds were prepared, beginning with the addition of 2.5 mL of an aqueous solution of CTAB (0.2
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M) and 5.0 mL of an aqueous solution of HAuCl4 (0.5 mM). Then, 0.6 mL of ice-cold
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NaBH4 solution (0.01 M) was added to the mixture with rapid stirring. The mixture was
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stirred for 2 minutes, and the final solution was left undisturbed at 27 °C for 2 h. The
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seed particles had a diameter of ca. 4 nm. In another flask, a mixture of aqueous
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solutions of 5.0 mL of HAuCl4 (1 mM), 2.5 mL of CTAB (0.2 M), and 0.15 mL of AgNO3 (4 mM) was prepared. Then, 70 µL of freshly prepared ascorbic acid aqueous
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solution (0.08 M) was added. A change in the color of the solution from light yellowish to colorless was observed. In the final step, 60 µL of seed solution was added to the
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growth solution, rapidly stirred for 10 seconds and allowed to age (4 hours) at 27 °C. After synthesis, the AuNRs were washed at least 3 times by centrifugation (13,500 rpm,
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15 minutes) and resuspended in a 10% bovine serum albumin (BSA) phosphatebuffered saline (PBS). AuNRs thus obtained were characterized by UV-vis spectroscopy and transmission electron microscope. 2.3. Gold nanorods characterization
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The extinction spectra of the colloidal solutions containing AuNRs were recorded in a Shimadzu UV-3600 spectrophotometer with photomultiplier tube (PMT) and InGaAs detectors (slit 2 nm). The transmission electron microscopy (TEM) analyses were performed on a FEI Tecnai 20 model electron microscope (FEI, Hillsboro, OR, USA) at an accelerating voltage of 200 kV, and the samples were
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prepared by the addition of a drop of the gold colloidal solution on a copper grid coated with a porous carbon film. For particle size analysis, the image processing software
extract the mean size of the AuNRs (length and width).
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2.4. Mitochondrial Assay
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developed ImageJ Version 1.48v was used and more than 200 particles were analyzed to
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Rat liver mitochondria (RLM) were isolated from male Wistar rats by
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conventional differential centrifugation at 4 °C (Schneider and Hogeboom, 1950) and
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exposed to AuNRs in different concentrations (6.25 to 100 µM). The mitochondrial
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content was calculated in terms of proteins by the Bradford method, and all experiments were performed in a standard medium (125 mM sucrose, 60 mM KCl, 1 mM MgCl 2, 2
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mM K2HPO4 and 10 mM HEPES) at pH 7.2. All concentrations of AuNRs are expressed in μM of gold atoms and the data represent three separate experiments
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performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05). All mitochondrial assay have been carried out in the presence of BSA-coated AuNRs,
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excepted as indicated. The authors state that they have obtained appropriate institutional review board
approval. Wistar rats (age, 60 days; weight, 250 g; housing, 5 per cage) were obtained from the vivarium of the Universidade Federal de Alagoas. All procedures were carried
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out in strict with the animal care guidelines of the Committee of National Research Council. 2.5. Oxygen consumption The oxygen consumed by the mitochondria was measured by using an OXIGY
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Oxygraph electrode (Hansatech Instrument, USA) in a 1.0 mL glass chamber with magnetic stirring (Chance and Williams, 1955; Robinson and Cooper, 1970). The initial
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concentration was fixed at 225 nmol/mL O2, and the sample was kept at 28 °C during the experiments. For the oxygen consumption evaluation, mitochondria (0.5 mg protein/mL) were incubated in the standard medium supplemented with 5 mM
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pyruvate/malate/glutamate/α-ketoglutarate and 200 µM EGTA. The phosphorylation
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state was induced by the addition of 250 µM ADP. After all of the ADP was consumed,
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the resting state began, and after several minutes, 1 µg/mL oligomycin was added to
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force this state to change. The maximal state was induced by the addition of 1 µM
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CCCP. In the experiments performed with AuNRs, the colloidal solution of AuNRs were added just after the mitochondria were added.
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2.6. Mitochondrial electrochemical membrane potential determination
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The mitochondrial membrane potential was evaluated by monitoring the
fluorescence of safranin O (Palmeira and Moreno, 2012) with the Shimadzu spectrofluorometer (RF, 5301PC, Japan) with an excitation wavelength of 495 nm and
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emission wavelength of 586 nm (slit: 5 nm). The mitochondrial suspension (0.5 mg protein/mL) was incubated with 5 mM pyruvate/malate/glutamate/α-ketoglutarate and 5 µM safranin O. The different amounts of AuNRs were added immediately after safranin O was added.
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2.7. Mitochondrial swelling The mitochondrial swelling was evaluated by monitoring the absorbance of the mitochondrial suspension in a UV-Vis spectrophotometer (Shimadzu UV-3600) at 520 nm. The mitochondria (0.5 mg protein/mL) were added to the standard medium, and the
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absorbance was recorded every 60 seconds for 10 minutes at 27 °C. 2.8. Mitochondrial reactive oxygen species production
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The ROS production was estimated by monitoring the fluorescence of
H2DCFDA (Oliveira et. al., 2004) and the H2O2-specific probe Amplex® Red.
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H2DCFDA (1 µM) is chemically converted to fluorescein (Ex/Em wavelength: 488/525
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nm, slit width: 3 nm) by reactive oxygen species. On the other hand, in the presence of
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horseradish peroxidase (HRP, 1 U/mL) and H2O2, Amplex® Red is converted to the
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2.9. Statistical analysis
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fluorescent product resorufin (Ex/Em wavelengths: 563/587 nm, slit width: 3 nm).
The results of all experiments are expressed as the mean of at least three
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experiments, which were each performed in duplicate. Data were analyzed by one-way
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analysis of variance (ANOVA) followed by the Dunnett post hoc test, and p < 0.05 was
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considered statistically significant.
3. Results 3.1. Colloids and nanoparticles characterization
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The colloidal solution of AuNRs was characterized by UV-Vis spectroscopy, and the morphology of the nanoparticles was analyzed by transmission electron microscopy (TEM). The UV-Vis absorption spectra showed two typical bands; the longitudinal and transverse surface plasmon resonances (SPR), see Figure 1a. The TEM images revealed AuNRs with a mean length of 42 nm and an aspect ratio of 3.7 (Figure
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1b). The CTAB-capped AuNRs were isolated and washed with deionized water (3x, 10
mL each) to remove the residual CTAB and were incubated with a 10% BSA in buffer
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solution (10 mL) at pH 7.4. The presence of BSA induced a 20 nm redshift of the
longitudinal SPR of the AuNRs (Figure 1c) (Wang et. al., 2007). In addition, adsorption of proteins by AuNRs was confirmed by the reduction of the molecular fluorescence of
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the BSA solution (Santos et al., 2018) after incubation and ablation of AuNR (Figure
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S1). The protein can bond to the AuNR via electrostatic attraction, hydrophobic
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interaction between an imperfect residual CTAB coating, and the buried hydrophobic
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residues of the protein (Alam and Mukhopadhyay, 2014), as well as covalent linkages
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through at least 12 Au-S bonds (Wang et. al., 2013).
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3.2. Evaluation of the mitochondria-AuNR interaction Our results indicate that the exposure of the mitochondrial suspension to 100 µM
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Au was capable of decreasing the mitochondrial oxygen consumption in all states to a level that was 77% of the level without the presence of AuNRs (Figure 2a). It was also
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verified that AuNRs decreased the phosphorylation state (state 3) in a dose-dependent manner (6.25 – 100 µM Au) (Figure 2b). The mean oxygen consumption in state 3 reached the maximum of 9.3 (± 1.0) nmol/min/mg for the control mitochondrial suspension (absence of AuNRs) and the minimum of 2.6 (± 0.5) nmol/min/mg for the mitochondrial suspension with a higher concentration of AuNRs (100 µM Au). The phosphorylation state occurs when ADP is added to a mitochondrial suspension, which 10
activates ATP synthase, thus inducing a proton influx into the mitochondrial matrix through rotational catalysis. The entry of protons into the matrix reduces the electrochemical potential, which increases the activity of the complexes in the electron transport chain, thus increasing the consumption of oxygen, to restore the mitochondrial potential (Nicholls and Ferguson, 2013). The AuNRs did not cause significant changes
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in oxygen consumption after ADP was consumed in the resting state (state 4) or in the
presence of the ATP synthase inhibitor oligomycin (Figure 2b). However, after the
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addition of the chemical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), the oxygen consumption was decreased in the presence of AuNRs (maximal
state) (Figure 2b). At the 25 µM Au concentration, the oxygen consumption in the
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maximal state decreased to 64% of the level without AuNRs. The chemical uncoupler
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induces proton entry that is independent of ATP synthase, leading to an increase in
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oxygen consumption. It was reported that 50 nm gold nanospheres (AuNSs) caused a
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similar effect in the muscle mitochondria of contaminated zebrafish; this change
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reduced the oxygen consumption in state 3 and the maximal state of this organelle (Bourdineaud et. al., 2013). The decrease in state 3 and the maximal state that was
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induced by AuNRs may be associated with their interaction with the complexes of the electrons transport chain because even with protons in the mitochondrial matrix
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(induced by CCCP), the oxygen consumption decreased, suggesting some lack of function in the electron transport chain. We also verified the effect of non-coated BSA
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AuNRs in the mitochondria (Figure S7). In the absence of protein, the AuNRs were able to decrease the oxygen consumption to lowest values (nearly 80%) at the minimum concentration of gold used (6.25 µM). This result indicates that without the protein shell, AuNRs are more toxic. This increase in the toxicity can be related to the CTAB residues present on the AuNRs. Indeed, it has been reported that the complete removal
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of the CTAB, without the substitution by other capping agents, compromise the stabilization of the colloidal system (Daniel and Astruc, 2004). The respiratory control ratio (RCR) was also investigated for all of the different concentrations of AuNRs. The RCR can be calculated from the ratio between the
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oxygen consumption in the phosphorylation state (state 3) and the resting state (state 4). This ratio is one of the most accepted parameters for describing mitochondrial coupling
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because RCR measures the coupling between the electron flux and ATP synthesis
(Brand and Nicholls, 2011; Rustin et. al, 1994). Our data demonstrated that RCR decreased in a dose-dependent manner, corroborating the previous results (Figure 2c).
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Surprisingly, the mitochondrial suspension incubated with 100 µM Au showed behavior
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similar to that of the control. . However, in this particular concentration, the profile of
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the oxygen consumption clearly demonstrates that some lack of function in the
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mitochondria occurs, once the total oxygen consumed is extremely low. Other studies have also demonstrated the decreasing of the RCR when the mitochondria are submitted
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to an insult. An example, Andrzejewski and coworkers reported that metformin was able to decrease the respiration in isolated mitochondria, which was similar to the
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AuNR effects. According to the researchers, metformin decreased the RCR values due
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to a large decrease in state 3 that was attributed to metformin exerting an inhibitory effect on complex I of the electron transport chain (Andrzejewski et. al., 2014).
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The maintenance of the mitochondrial membrane potential is essential for the
synthesis of ATP and for mitochondrial viability. The oxidation processes of carbohydrates, lipids, and amino acids converge to the synthesis of reduced coenzymes (NADH and FADH2), which tend to generate electron flow to oxygen in the electron transport chain. The energy released during this process pumps protons out of the matrix space, thus generating an electrochemical gradient. The electrochemical potential 12
created by this gradient drives protons back to the matrix through ATP synthase. Consequently, the enzymatic activity of ATP synthase produces ATP from ADP due to the proton-motive force according to Mitchell’s chemiosmosis theory (Nicholls and Ferguson, 2013). Thus, the loss of the membrane potential may cause the death of the cell. In such a context, we investigated the effect of AuNRs on the mitochondrial
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membrane electric potential by monitoring the fluorescence of the probe safranin O in
the presence of Ca2+ (10 µM), in which an increase in fluorescence is associated with a
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decrease in the electric potential due to the dependence of its absorption and fluorescence on mitochondrial energization. The safranin O is a positively charged dye
that changes fluorescence in a linearly manner proportional to the mitochondrial
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membrane potential. The safranin O is attracted to the mitochondrial membrane
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potential and it accumulates in the intermembrane space. When the mitochondrial
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membrane potential disruption occurs, the safranin O dye is released in the solution
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emitting fluorescence (Åkerman & Wikström, 1976; Zanotti and Azzone, 1980; Vercesi
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et al, 1991). Thus, Ca2+ ions were added to the mitochondrial suspension to stimulate the opening of the mitochondrial permeability transition pore, allowing the entry of
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molecules with sizes of approximately 1.5 kD and protons (Skulachev, 1998), thus stimulating a disruption in the potential (Starkov et. al., 2002). Our results show that the
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AuNRs were able to generate a quick membrane potential disruption in a dosedependent manner (Figure 3a). The AuNRs at concentrations of 50 and 100 µM Au
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caused a membrane potential disruption after 2.6 ± 0.7 and 2.2 ± 1.0 minutes, respectively (Figure 3b). This finding indicates that the AuNRs accelerated the potential disruption by approximately 54% (see Figure 3b). Additionally, the AuNRs induced a rapid potential disruption even in the presence of the membrane transition pore inhibitor cyclosporine A (Kowaltowski and Vercesi, 1999), a classic inhibitor of the
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mitochondrial permeability transition pore (Figure S4). This rapid loss may be related to the action of AuNRs in the electron transport chain complexes, which affects the proton pumping dynamic that is essential for the electric potential of the mitochondrial membrane.
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It has been reported that gold (Pradhan et. al., 2016), silver (Ma et. al., 2015), and chitosan (Qi et. al., 2005) nanoparticles were able to decrease the mitochondrial
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membrane potential in different cells. Gold nanoparticles synthesized using indole-3carbinol stimulated a substantial reduction in the mitochondrial potential of Ehrlich ascites carcinoma cells (Pradhan et. al., 2016). In addition, AuNRs caused a similar
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effect upon exposure to A549 cancer cells, and they suggested that the AuNRs were
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able to induce a decrease in the mitochondrial membrane potential (Wang et al., 2011).
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The interaction between AuNRs and the mitochondrial complexes may be capable of
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interrupting the electron flux through the complexes of the electron transport chain, thus reducing their ability to pump protons to the intermembrane space and leading to a
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disruption in the mitochondrial membrane potential.
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Gradual permeabilization of mitochondrial membranes can be generated by an increase in the concentration of Ca2+ ions in the medium. Indeed, high levels of these
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ions induce the formation of reactive oxygen species (ROS) in the mitochondrial matrix, causing mitochondrial swelling due to opening of the mitochondrial permeability
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transition pore via the oxidation of the thiol groups present in the pore proteins. This process allows the internalization of water, ions and other molecules (Figueira et. al., 2013). Therefore, we evaluated the effect of AuNRs on the Ca2+-sensitive mitochondrial swelling by monitoring the absorbance of a mitochondrial suspension at 520 nm (Wilson et. al., 2005). In this experiment, the mitochondrial suspension with AuNRs showed a higher absorption than that of the control (suspension without AuNRs) 14
(Figure 4a and 4b). This result indicates that the mitochondrial swelling triggered by Ca2+ was delayed by the AuNRs. The mitochondrial suspension incubated with the higher concentration of AuNRs (100 µM Au) presented similar behavior to that of the control in the presence of cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (Figure S5). A similar effect was observed by Marín-Prida and
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coworkers when they evaluated the effect of 1,4-dihydropyridine derivates (VE-3N) on
mitochondria. Under standard conditions, VE-3N was able to inhibit the swelling
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process in a concentration-dependent manner (Marín-Prida et. al., 2017). The reduction in the mitochondrial swelling stimulated by AuNRs may be associated with the decrease
in ROS production that led to protection of the mitochondrial permeability transition
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progress. To investigate this possibility, we verified the effect of AuNRs on the
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production of ROS by monitoring the fluorescence of 2’,7’-dichlorofluorescin (DCF)
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(LeBel et. al., 1992). Curiously, the AuNRs induced a decrease in ROS production
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(Figure 4c and 4d): 18.1 ± 5.7 nM for the mitochondrial suspension without AuNRs and
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2.8 ± 2.2 nM with 100 µM Au. The reduction in ROS production caused by AuNRs may explain the delay in mitochondrial swelling, since low ROS levels reduce the
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oxidation of the mitochondrial permeability transition pore, thus decreasing the entry of fluids into the mitochondria. These results imply that AuNRs may have a protective
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effect that causes a delay in mitochondrial swelling as result of the decrease in ROS levels. Notably, studies involving cells and gold nanoparticles found an increase in the
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ROS level (Tang et. al., 2015; Wan et. al., 2015; Hwang et. al., 2012); on the other hand, our results showed that when AuNRs were exposed to isolated mitochondria, the AuNRs stimulated a decrease in ROS production. Moreover, our results suggest that the AuNRs interact with the respiratory chain complexes, thus affecting the redox cascade, consequently interrupting the flow of electrons between the complexes, and decreasing
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the reduction of O2 to H2O, ultimately leading to a low respiration rate. It is worth mentioning, here, that gold nanoparticles have already been shown to act as good catalysts for the reduction of H2O2 (Jv et. al., 2010; Navalon et. al., 2011) that also attest such a hypothesis. Nevertheless, in order to confirm the ability of AuNRs to reduce ROS, we proposed an experiment, using the Amplex-Red probe (Zhou et. al., 1997),
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that confirmed that the addition of AuNRs caused a depletion in H2O2 production
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(Figure S8).
Since some of the results showed a potential interaction between AuNRs and respiratory chain complexes, we conducted TEM analysis of the mitochondria to verify
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the presence or absence of AuNRs in the organelle. All details related to the sample
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preparation for TEM analysis can be found in the Supporting Information. It is
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important to mention that for this analysis, the mitochondrial suspensions incubated
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with AuNRs were washed with 4% cacodylate buffer (pH 7.2) at least 5 times to remove all residual reagents and AuNRs that did not effectively interact with the
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mitochondria. AuNRs were not found in the mitochondria or on their surfaces in any of the TEM images. Although AuNRs were not detected in TEM images, the AuNRs
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caused significant damage to the mitochondrial morphology (Figure 5). The
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mitochondria incubated with 100 µM Au exhibit disorganized crests and intermembrane separation (Figure 5b, arrows). The same effect was observed by Cambier and coworkers in the muscle mitochondria of zebrafish contaminated with 13 µg of CH3Hg
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(Cambiera et. al., 2009). Fanni and coworkers also observed disorganized mitochondrial cristae in Kupffer cells and hepatocytes treated with high concentrations of iron and cupper ions (Fanni et. al., 2014). Our study suggests that AuNRs were removed in the washing steps, indicating that the AuNRs interacted with the mitochondria via their external surface. Wang and 16
coworkers reported that after internalization into cancer cells, AuNRs were located at the edges of the mitochondria or around their inner membranes (Wang et al., 2011). We measured the diameter of mitochondria treated with 100 µM Au; the mitochondria had a mean size of 740 nm (± 36), while the mitochondria treated in the
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absence of AuNRs had a mean size of 862 nm (± 30) (Figure S9). The mitochondria incubated with AuNRs had a smaller diameter than that of mitochondria without
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AuNRs, confirming that the mitochondrial swelling delay was caused by AuNRs. The
potential interaction of AuNRs with the external surface of mitochondria along with the decrease in mitochondrial diameter implies that the AuNRs might interact with the
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mitochondria via electron transport chain complexes.
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4. Discussion
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Mitochondria do not have large pores. Both the outer and inner membranes present barriers that prevent the entry of AuNRs into the matrix (Kodiha et. al., 2015).
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Salnikov and coworkers reported that in heart cells, 3 nm gold nanoparticles, but not 6 nm nanoparticles, translocated across the outer mitochondrial membrane (Salnikov et.
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al., 2007). In the inner mitochondrial membrane, protein import channels provide openings of ca. 2 nm3 (Szabo and Zoratti, 2014). Thus, there seems to be a restriction preventing the incorporation of AuNRs into the organelle matrix. However, the
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translocation barrier in the mitochondrial membrane against AuNRs does not preclude the AuNRs from damaging the mitochondria. Our findings suggest that AuNRs interact with electron transport chain complexes through the external surface of the mitochondria, thus disrupting the electron transport through the complexes and stimulating a series of events, including the following: i) decrease in the oxygen 17
consumption; ii) depletion of proton pumping that leads to a rapid decrease in the membrane potential; iii) reduction in ROS production leading to a delay in mitochondrial swelling; and iv) morphological changes in the mitochondrial cristae and external membrane.
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We found that AuNRs with a mean length of 42 nm and aspect ratio of 3.7 that were treated with BSA presented a bathochromic shift of their longitudinal surface
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plasmon resonance, which indicated the anchorage of this protein on their surface. The
studies related to the effect of AuNRs on isolated rat liver mitochondria strongly indicate a potential mechanism of tumor cell death caused by AuNRs (Wang et al.,
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2011). The impact of AuNRs on the mitochondrial respiration and membrane electric
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potential suggests an interaction with the electron transport chain complexes. The
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decrease in the ROS levels in the presence of AuNRs also indicates that the electron
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flow between the complexes is interrupted, which reduces the production of ROS. The mitochondrial swelling delay and the absence of AuNRs in the mitochondria, as seen in
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TEM images, corroborates the hypothesis. The TEM images also demonstrate that at 100 µM concentration, the AuNRs caused disorganized mitochondrial crests and
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detachment of the mitochondrial membranes. The elucidation of the behavior of AuNRs
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in mitochondria is the first step in understanding their mechanism in the cellular environment. This finding thus provides new evidence of how AuNRs interact with
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mitochondria.
Acknowledgements Authors gratefully acknowledge the Brazilian research founding agencies, National Counsel of Technological and Scientific Development (CNPq), Coordination 18
for the Improvement of Higher Level Education (Capes), and Alagoas Research Support Foundation (Fapeal) for financial support. A.M.N, K.R.M.S., C.M.S.C., A.C.R.L. and M.R.M. thank CNPq for their research fellowships.
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Apendix A. Suplementary Data
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Suplementary material related to this data can be found in the online version
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Figure Captions Figure 1. (a) UV-Vis absorption spectra of AuNRs before and after incubation with 10% BSA in phosphate-buffered saline (PBS); (b) TEM images showing the size and
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shape of AuNRs.
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Figure 2. Effect of the amount of AuNRs on the mitochondrial oxygen consumption.
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(a) Oxygen consumption, (b) oxygen consumption in different states – the phosphorylation state (PS), the resting state (RS) and the maximal state (MS) - and (c)
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the respiratory control ratio (RCR). RLM (0.5 mg protein/mL were incubated in buffer solution at 28 ᵒC (5 mM complex I substrates, 200 µM EGTA, 250 µM ADP, 1 µg/mL
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oligomycin and 1µM CCCP). All of the concentrations are expressed in µM of Au. The data represent three separate experiments performed in duplicate. Mean values ±
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standard deviation, n = 3 (*p < 0.05).
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Figure 3. Effect of AuNRs at various concentrations on the mitochondrial membrane potential. (a) membrane potential disruption monitored by safranin O fluorescence and (b) mean potential disruption time. RLM (0.5 mg protein/mL) were incubated in buffer
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solution at 28 ᵒC (5 mM complex I substrates, 5 µM safranin O, and 10 µM Ca2+). All of the concentrations are expressed in Au. The data represent three separate experiments performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05).
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Figure 4. Effect of AuNR concentration on mitochondrial integrity. (a) Effect on
mitochondrial swelling and (b) Final mitochondria suspension turbidity mean: RLM
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(0.5 mg protein/mL) were incubated in buffer solution at 28 ºC (5 mM complex I
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substrates and 10 µM Ca2+); (c) effect of AuNRs on ROS production in time and (d)
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Mean of ROS level production: RLM (0.5 mg protein/mL) were incubated in buffer
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solution at 28 ºC (5 mM complex I substrates, 1 µM H2DCFDA and 10 µM Ca2+). All the concentrations are expressed in Au. The data represent three separated experiments
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performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05).
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Figure 5. Effect of AuNRs on mitochondrial morphology. TEM images of mitochondria in the absence (a, b) and presence of 100 µM Au (c, d). Scale: 500 nm.
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Arrows indicate intermembrane separations.
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Figure 6. Mechanistic scheme. Mitochondrial dynamic scheme in the absence (a) and
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presence (b) of AuNRs. The four mitochondrial complexes are labeled as I, II, III and IV. In the scheme, Mn-superoxide dismutase (MnSOD), glutathione peroxidase (GPx),
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peroxiredoxin (Prx), thioredoxin (TSH), glutathione (GSH), glutathione reductase (GR),
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thioredoxin reductase (TR), ubiquinone (Q), cytochrome c (CyT) and the mitochondrial
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permeability transition pore (MPT) are shown.
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S0300-483X(18)30245-2 https://doi.org/10.1016/j.tox.2018.12.002 TOX 52141
To appear in:
Toxicology
Received date: Revised date: Accepted date:
14 August 2018 22 November 2018 5 December 2018
´ Please cite this article as: Nunes AM, da Silva KRM, Calado CMS, Saraiva KLA, Q. Figueiredo RCB, Leite ACR, Meneghetti MR, Evaluation of gold nanorods toxicity on isolated mitochondria, Toxicology (2018), https://doi.org/10.1016/j.tox.2018.12.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Evaluation of gold nanorods toxicity on isolated mitochondria
Ábner M. Nunes1, Kleyton R. M. da Silva2, Claudia M. S. Calado1, Karina L. A.
1
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Saraiva3, Regina C. B. Q. Figueiredo3, Ana Catarina R. Leite2*, Mario R. Meneghetti1*.
Grupo de Catálise e Reatividade Química, Instituto de Química e Biotecnologia,
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Universidade Federal de Alagoas, Av. Lourival de Melo Mota, CEP 57072-970, Maceió, Alagoas, Brazil 2
Laboratório de Bioenergética, Instituto de Química e Biotecnologia, Universidade
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Federal de Alagoas, Av. Lourival de Melo Mota, CEP 57072-970, Maceió, Alagoas,
Laboratório de Biologia Celular de Patógenos, Departamento de Microbiologia,
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3
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Brazil
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Instituto Aggeu Magalhães, Fundação Oswaldo Cruz, Cidade Universitária, CEP
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50670-420, Recife, Pernambuco, Brazil
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*Corresponding Authors details:
E-mail: [email protected] (M.R.M)
Phone: +55 82 32141344 Phone: +55 82 998104445
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E-mail: [email protected] (A.C.R.L.)
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Graphical abstract
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Abstract
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Gold nanorods (AuNRs) have been studied extensively in biomedicine due to their
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biocompatibility and their unique properties. Some studies reported that AuNRs
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selectively accumulate on cancer cell mitochondria causing its death. However, the immediate effects of this accumulation needed further investigations. In this context, we
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evaluated the effect of AuNRs on the mitochondrial integrity of isolated rat liver mitochondria. We verified that AuNRs decreased the mitochondrial respiratory ratio by
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decreasing the phosphorylation and maximal states. Additionally, AuNRs caused a
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decrease in the production of mitochondrial ROS and a delay in mitochondrial swelling. Moreover, even with cyclosporine A treatment, AuNRs disrupted the mitochondrial potential. With the highest concentration of AuNRs studied, disorganized mitochondrial
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crests and intermembrane separation were observed in TEM images. These results indicate that AuNRs can interact with mitochondria, disrupting the electron transport chain. This study provides new evidence of the immediate effects of AuNRs on mitochondrial bioenergetics.
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Abbreviations: AuNRs, gold nanorods; AuNPs, gold nanoparticles; AuNSs, gold nanospheres;
BSA,
bovine
serum
protein;
CCCP,
carbonyl
cyanide
m-
chlorophenylhydrazone; CsA, cyclosporine A; CTAB, hexadecyltrimethylammonium bromide; MS, maximal state; OLIGO, oligomycin; PS, phosphorylation state; RCP, respiratory control rate; RLM, rat liver mitochondria; ROS, reactive oxygen species;
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RS, the resting state; SPR, surface plasmon resonance; TEM, transmission electron
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Keywords: gold nanorods; mitochondria; bioenergetics
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microscope.
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1. Introduction
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Due to their singular physicochemical properties, gold nanorods (AuNRs) are
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promising nanomaterials for a wide range of applications. AuNRs can be applied as
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catalysts for important chemical transformations (Priecel et al., 2016) in the biomedical field (Bobo et. al., 2016; Versiani et al., 2016 ). In this last area in particular, AuNRs
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have been used in the development of biosensors (Beuwer et. al., 2015), optical imaging (Zerda et. al., 2015; Qu et. al., 2016), gene (Nakatsuji et. al., 2017) and drug (Ali et. al.,
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2016; Feng et. al., 2015 ) delivery, photothermal therapy (Ali et. al., 2017), and a wide range of strategies for developing cancer treatments. It is worth mentioning here that
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many studies have reported the low toxicity of gold nanoparticles (AuNPs) (Patra et. al., 2007; Peng et al., 2009), nevertheless, their toxicity can be tuned by their surface modifications (Locatelli et. al., 2015; Wan et. al., 2015 ) and dimensions (size and shape) (Li et. al., 2014; Pietro et. al., 2016; Truong et. al., 2015).
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Due to the singular biological proprieties and features of AuNRs have been directly applied for therapeutic purposes in antitumor therapy through subcellular targeting, (Kodiha et. al., 2015; Zhang, 2015) with a strong focus on the nucleus and mitochondria. Mitochondria provide cancer cells with altered fuel availability, bioenergetics, oxidative stress, and cell death susceptibility, allowing them to survive in
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the face of adverse environmental conditions, such as starvation and cancer treatments. These features show the potential of mitochondria as a target for cancer therapy (Vyas
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et. al., 2016; Weinberg and Chandel, 2015). Thus, improvements based on the concept that AuNPs can target specific organelles will maximize their impact on tumor cells
(Kodiha et. al., 2015; Zhou et al., 2017). AuNRs often impair mitochondrial functions
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and thereby induce cell death (Zhang et. al., 2016). Among other things, CTAB-capped
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AuNRs were able to damage lipid bilayers and facilitate the permeabilization of
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membranes because AuNRs were capable of targeting mitochondria via their negative
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mitochondrial membrane potential (Kodiha et. al., 2015). It has been reported that
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AuNRs treated with fetal bovine serum albumin (BSA) caused cancer cell death with minimal injury to healthy cells (Wang et al., 2011). Both healthy and cancer cells were
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able to endocytose protein-coated AuNRs. Nevertheless, lysosome disruption was observed only in cancer cells and led to high AuNR accumulation in the mitochondria.
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AuNRs are able to induce cancer cell apoptosis and necrosis via lysosome- and mitochondria-mediated routes (Zhang et. al., 2017). Wang and coworkers demonstrated
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that AuNRs selectively accumulate in cancer cell mitochondria (Wang et al., 2011). The authors suggested that AuNR accumulation reduces the mitochondrial membrane potential and that the resulting dysfunction causes cell death. These works show again how important is the role of mitochondria in cancer cells for the development of new cancer therapies. For that reason, we decided to 4
investigate the effects of protein-coated AuNRs on the bioenergetics of isolated rat liver mitochondria (RLM) to obtain more information about how the mitochondriananoparticle interaction occurs and the effects on the organelle. Part of our studies are presented in this work.
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We would like to mention that despite of their interesting properties and applications of nanoparticulated materials, the risk of exposure to these materials for the
environment and/or humans must be subject of further studies (Ye et al. 2011;
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Kunzmann et al. 2011).
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2. Methods
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2.1. Chemicals
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All chemicals were obtained from commercial suppliers and used without
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further purification. Gold(III) chloride trihydrate (HAuCl4.3H2O, ≥ 99.9%), sodium borohydride (NaBH4, > 98%), hexadecyltrimethylammonium bromide (CTAB, ≥ 99%),
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bovine serum albumin (BSA, ≥ 96%), ethylene glycol-bis(2-aminoethylether)N,N,N′,N′-tetraacetic
acid
(EGTA,
≥
97%),
4-(2-hydroxyethyl)piperazine-1-
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ethanesulfonic acid, N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES, 99.5%), magnesium chloride (MgCl2, ≥ 98%), potassium phosphate monobasic
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(KH2PO4, ≥ 98%), dibasic potassium phosphate (K2HPO4, ≥ 98%), sodium pyruvate (≥ 99.0%), D-malic acid (≥ 97%), α-ketoglutaric acid (≥ 98.5%), aspartic acid (≥ 98%), succinic acid (≥ 99.0%), rotenone (≥ 95%), oligomycin, adenosine 5′-diphosphate sodium salt (ADP ≥ 95%), safranin O (≥ 85%), L-ascorbic acid p.a. (≥ 99%), H2O2 solution (30 wt % H2O), potassium chloride (KCl, ≥ 99%), carbonyl cyanide m-
5
chlorophenyl hydrazone (CCCP) and horseradish peroxidase (HRP) were purchased from
Sigma-Aldrich
(Saint
Louis,
MO,
USA).
Amplex-Red®
and
2’,7’-
dichlorodihydrofluorescein diacetate (H2DCFDA) were purchased from Thermo Fisher (Waltham, MA, USA). Sucrose, silver nitrate (AgNO3, > 99%), and ethyl alcohol (99%)
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were purchased Dinâmica (Diadema, SP, Brazil). 2.2. Gold nanorods preparation
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AuNRs were prepared by the seed-mediated methodology (Nikoobakht and ElSayed, 2003; Silva et al., 2013). Briefly, in a 20 mL cylindrical flask, gold seeds were prepared, beginning with the addition of 2.5 mL of an aqueous solution of CTAB (0.2
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M) and 5.0 mL of an aqueous solution of HAuCl4 (0.5 mM). Then, 0.6 mL of ice-cold
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NaBH4 solution (0.01 M) was added to the mixture with rapid stirring. The mixture was
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stirred for 2 minutes, and the final solution was left undisturbed at 27 °C for 2 h. The
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seed particles had a diameter of ca. 4 nm. In another flask, a mixture of aqueous
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solutions of 5.0 mL of HAuCl4 (1 mM), 2.5 mL of CTAB (0.2 M), and 0.15 mL of AgNO3 (4 mM) was prepared. Then, 70 µL of freshly prepared ascorbic acid aqueous
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solution (0.08 M) was added. A change in the color of the solution from light yellowish to colorless was observed. In the final step, 60 µL of seed solution was added to the
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growth solution, rapidly stirred for 10 seconds and allowed to age (4 hours) at 27 °C. After synthesis, the AuNRs were washed at least 3 times by centrifugation (13,500 rpm,
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15 minutes) and resuspended in a 10% bovine serum albumin (BSA) phosphatebuffered saline (PBS). AuNRs thus obtained were characterized by UV-vis spectroscopy and transmission electron microscope. 2.3. Gold nanorods characterization
6
The extinction spectra of the colloidal solutions containing AuNRs were recorded in a Shimadzu UV-3600 spectrophotometer with photomultiplier tube (PMT) and InGaAs detectors (slit 2 nm). The transmission electron microscopy (TEM) analyses were performed on a FEI Tecnai 20 model electron microscope (FEI, Hillsboro, OR, USA) at an accelerating voltage of 200 kV, and the samples were
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prepared by the addition of a drop of the gold colloidal solution on a copper grid coated with a porous carbon film. For particle size analysis, the image processing software
extract the mean size of the AuNRs (length and width).
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2.4. Mitochondrial Assay
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developed ImageJ Version 1.48v was used and more than 200 particles were analyzed to
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Rat liver mitochondria (RLM) were isolated from male Wistar rats by
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conventional differential centrifugation at 4 °C (Schneider and Hogeboom, 1950) and
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exposed to AuNRs in different concentrations (6.25 to 100 µM). The mitochondrial
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content was calculated in terms of proteins by the Bradford method, and all experiments were performed in a standard medium (125 mM sucrose, 60 mM KCl, 1 mM MgCl 2, 2
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mM K2HPO4 and 10 mM HEPES) at pH 7.2. All concentrations of AuNRs are expressed in μM of gold atoms and the data represent three separate experiments
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performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05). All mitochondrial assay have been carried out in the presence of BSA-coated AuNRs,
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excepted as indicated. The authors state that they have obtained appropriate institutional review board
approval. Wistar rats (age, 60 days; weight, 250 g; housing, 5 per cage) were obtained from the vivarium of the Universidade Federal de Alagoas. All procedures were carried
7
out in strict with the animal care guidelines of the Committee of National Research Council. 2.5. Oxygen consumption The oxygen consumed by the mitochondria was measured by using an OXIGY
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Oxygraph electrode (Hansatech Instrument, USA) in a 1.0 mL glass chamber with magnetic stirring (Chance and Williams, 1955; Robinson and Cooper, 1970). The initial
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concentration was fixed at 225 nmol/mL O2, and the sample was kept at 28 °C during the experiments. For the oxygen consumption evaluation, mitochondria (0.5 mg protein/mL) were incubated in the standard medium supplemented with 5 mM
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pyruvate/malate/glutamate/α-ketoglutarate and 200 µM EGTA. The phosphorylation
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state was induced by the addition of 250 µM ADP. After all of the ADP was consumed,
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the resting state began, and after several minutes, 1 µg/mL oligomycin was added to
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force this state to change. The maximal state was induced by the addition of 1 µM
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CCCP. In the experiments performed with AuNRs, the colloidal solution of AuNRs were added just after the mitochondria were added.
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2.6. Mitochondrial electrochemical membrane potential determination
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The mitochondrial membrane potential was evaluated by monitoring the
fluorescence of safranin O (Palmeira and Moreno, 2012) with the Shimadzu spectrofluorometer (RF, 5301PC, Japan) with an excitation wavelength of 495 nm and
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emission wavelength of 586 nm (slit: 5 nm). The mitochondrial suspension (0.5 mg protein/mL) was incubated with 5 mM pyruvate/malate/glutamate/α-ketoglutarate and 5 µM safranin O. The different amounts of AuNRs were added immediately after safranin O was added.
8
2.7. Mitochondrial swelling The mitochondrial swelling was evaluated by monitoring the absorbance of the mitochondrial suspension in a UV-Vis spectrophotometer (Shimadzu UV-3600) at 520 nm. The mitochondria (0.5 mg protein/mL) were added to the standard medium, and the
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absorbance was recorded every 60 seconds for 10 minutes at 27 °C. 2.8. Mitochondrial reactive oxygen species production
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The ROS production was estimated by monitoring the fluorescence of
H2DCFDA (Oliveira et. al., 2004) and the H2O2-specific probe Amplex® Red.
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H2DCFDA (1 µM) is chemically converted to fluorescein (Ex/Em wavelength: 488/525
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nm, slit width: 3 nm) by reactive oxygen species. On the other hand, in the presence of
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horseradish peroxidase (HRP, 1 U/mL) and H2O2, Amplex® Red is converted to the
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2.9. Statistical analysis
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fluorescent product resorufin (Ex/Em wavelengths: 563/587 nm, slit width: 3 nm).
The results of all experiments are expressed as the mean of at least three
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experiments, which were each performed in duplicate. Data were analyzed by one-way
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analysis of variance (ANOVA) followed by the Dunnett post hoc test, and p < 0.05 was
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considered statistically significant.
3. Results 3.1. Colloids and nanoparticles characterization
9
The colloidal solution of AuNRs was characterized by UV-Vis spectroscopy, and the morphology of the nanoparticles was analyzed by transmission electron microscopy (TEM). The UV-Vis absorption spectra showed two typical bands; the longitudinal and transverse surface plasmon resonances (SPR), see Figure 1a. The TEM images revealed AuNRs with a mean length of 42 nm and an aspect ratio of 3.7 (Figure
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1b). The CTAB-capped AuNRs were isolated and washed with deionized water (3x, 10
mL each) to remove the residual CTAB and were incubated with a 10% BSA in buffer
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solution (10 mL) at pH 7.4. The presence of BSA induced a 20 nm redshift of the
longitudinal SPR of the AuNRs (Figure 1c) (Wang et. al., 2007). In addition, adsorption of proteins by AuNRs was confirmed by the reduction of the molecular fluorescence of
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the BSA solution (Santos et al., 2018) after incubation and ablation of AuNR (Figure
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S1). The protein can bond to the AuNR via electrostatic attraction, hydrophobic
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interaction between an imperfect residual CTAB coating, and the buried hydrophobic
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residues of the protein (Alam and Mukhopadhyay, 2014), as well as covalent linkages
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through at least 12 Au-S bonds (Wang et. al., 2013).
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3.2. Evaluation of the mitochondria-AuNR interaction Our results indicate that the exposure of the mitochondrial suspension to 100 µM
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Au was capable of decreasing the mitochondrial oxygen consumption in all states to a level that was 77% of the level without the presence of AuNRs (Figure 2a). It was also
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verified that AuNRs decreased the phosphorylation state (state 3) in a dose-dependent manner (6.25 – 100 µM Au) (Figure 2b). The mean oxygen consumption in state 3 reached the maximum of 9.3 (± 1.0) nmol/min/mg for the control mitochondrial suspension (absence of AuNRs) and the minimum of 2.6 (± 0.5) nmol/min/mg for the mitochondrial suspension with a higher concentration of AuNRs (100 µM Au). The phosphorylation state occurs when ADP is added to a mitochondrial suspension, which 10
activates ATP synthase, thus inducing a proton influx into the mitochondrial matrix through rotational catalysis. The entry of protons into the matrix reduces the electrochemical potential, which increases the activity of the complexes in the electron transport chain, thus increasing the consumption of oxygen, to restore the mitochondrial potential (Nicholls and Ferguson, 2013). The AuNRs did not cause significant changes
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in oxygen consumption after ADP was consumed in the resting state (state 4) or in the
presence of the ATP synthase inhibitor oligomycin (Figure 2b). However, after the
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addition of the chemical uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), the oxygen consumption was decreased in the presence of AuNRs (maximal
state) (Figure 2b). At the 25 µM Au concentration, the oxygen consumption in the
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maximal state decreased to 64% of the level without AuNRs. The chemical uncoupler
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induces proton entry that is independent of ATP synthase, leading to an increase in
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oxygen consumption. It was reported that 50 nm gold nanospheres (AuNSs) caused a
M
similar effect in the muscle mitochondria of contaminated zebrafish; this change
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reduced the oxygen consumption in state 3 and the maximal state of this organelle (Bourdineaud et. al., 2013). The decrease in state 3 and the maximal state that was
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induced by AuNRs may be associated with their interaction with the complexes of the electrons transport chain because even with protons in the mitochondrial matrix
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(induced by CCCP), the oxygen consumption decreased, suggesting some lack of function in the electron transport chain. We also verified the effect of non-coated BSA
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AuNRs in the mitochondria (Figure S7). In the absence of protein, the AuNRs were able to decrease the oxygen consumption to lowest values (nearly 80%) at the minimum concentration of gold used (6.25 µM). This result indicates that without the protein shell, AuNRs are more toxic. This increase in the toxicity can be related to the CTAB residues present on the AuNRs. Indeed, it has been reported that the complete removal
11
of the CTAB, without the substitution by other capping agents, compromise the stabilization of the colloidal system (Daniel and Astruc, 2004). The respiratory control ratio (RCR) was also investigated for all of the different concentrations of AuNRs. The RCR can be calculated from the ratio between the
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oxygen consumption in the phosphorylation state (state 3) and the resting state (state 4). This ratio is one of the most accepted parameters for describing mitochondrial coupling
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because RCR measures the coupling between the electron flux and ATP synthesis
(Brand and Nicholls, 2011; Rustin et. al, 1994). Our data demonstrated that RCR decreased in a dose-dependent manner, corroborating the previous results (Figure 2c).
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Surprisingly, the mitochondrial suspension incubated with 100 µM Au showed behavior
N
similar to that of the control. . However, in this particular concentration, the profile of
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the oxygen consumption clearly demonstrates that some lack of function in the
M
mitochondria occurs, once the total oxygen consumed is extremely low. Other studies have also demonstrated the decreasing of the RCR when the mitochondria are submitted
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to an insult. An example, Andrzejewski and coworkers reported that metformin was able to decrease the respiration in isolated mitochondria, which was similar to the
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AuNR effects. According to the researchers, metformin decreased the RCR values due
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to a large decrease in state 3 that was attributed to metformin exerting an inhibitory effect on complex I of the electron transport chain (Andrzejewski et. al., 2014).
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The maintenance of the mitochondrial membrane potential is essential for the
synthesis of ATP and for mitochondrial viability. The oxidation processes of carbohydrates, lipids, and amino acids converge to the synthesis of reduced coenzymes (NADH and FADH2), which tend to generate electron flow to oxygen in the electron transport chain. The energy released during this process pumps protons out of the matrix space, thus generating an electrochemical gradient. The electrochemical potential 12
created by this gradient drives protons back to the matrix through ATP synthase. Consequently, the enzymatic activity of ATP synthase produces ATP from ADP due to the proton-motive force according to Mitchell’s chemiosmosis theory (Nicholls and Ferguson, 2013). Thus, the loss of the membrane potential may cause the death of the cell. In such a context, we investigated the effect of AuNRs on the mitochondrial
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membrane electric potential by monitoring the fluorescence of the probe safranin O in
the presence of Ca2+ (10 µM), in which an increase in fluorescence is associated with a
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decrease in the electric potential due to the dependence of its absorption and fluorescence on mitochondrial energization. The safranin O is a positively charged dye
that changes fluorescence in a linearly manner proportional to the mitochondrial
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membrane potential. The safranin O is attracted to the mitochondrial membrane
N
potential and it accumulates in the intermembrane space. When the mitochondrial
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membrane potential disruption occurs, the safranin O dye is released in the solution
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emitting fluorescence (Åkerman & Wikström, 1976; Zanotti and Azzone, 1980; Vercesi
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et al, 1991). Thus, Ca2+ ions were added to the mitochondrial suspension to stimulate the opening of the mitochondrial permeability transition pore, allowing the entry of
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molecules with sizes of approximately 1.5 kD and protons (Skulachev, 1998), thus stimulating a disruption in the potential (Starkov et. al., 2002). Our results show that the
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AuNRs were able to generate a quick membrane potential disruption in a dosedependent manner (Figure 3a). The AuNRs at concentrations of 50 and 100 µM Au
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caused a membrane potential disruption after 2.6 ± 0.7 and 2.2 ± 1.0 minutes, respectively (Figure 3b). This finding indicates that the AuNRs accelerated the potential disruption by approximately 54% (see Figure 3b). Additionally, the AuNRs induced a rapid potential disruption even in the presence of the membrane transition pore inhibitor cyclosporine A (Kowaltowski and Vercesi, 1999), a classic inhibitor of the
13
mitochondrial permeability transition pore (Figure S4). This rapid loss may be related to the action of AuNRs in the electron transport chain complexes, which affects the proton pumping dynamic that is essential for the electric potential of the mitochondrial membrane.
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It has been reported that gold (Pradhan et. al., 2016), silver (Ma et. al., 2015), and chitosan (Qi et. al., 2005) nanoparticles were able to decrease the mitochondrial
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membrane potential in different cells. Gold nanoparticles synthesized using indole-3carbinol stimulated a substantial reduction in the mitochondrial potential of Ehrlich ascites carcinoma cells (Pradhan et. al., 2016). In addition, AuNRs caused a similar
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effect upon exposure to A549 cancer cells, and they suggested that the AuNRs were
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able to induce a decrease in the mitochondrial membrane potential (Wang et al., 2011).
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The interaction between AuNRs and the mitochondrial complexes may be capable of
M
interrupting the electron flux through the complexes of the electron transport chain, thus reducing their ability to pump protons to the intermembrane space and leading to a
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disruption in the mitochondrial membrane potential.
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Gradual permeabilization of mitochondrial membranes can be generated by an increase in the concentration of Ca2+ ions in the medium. Indeed, high levels of these
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ions induce the formation of reactive oxygen species (ROS) in the mitochondrial matrix, causing mitochondrial swelling due to opening of the mitochondrial permeability
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transition pore via the oxidation of the thiol groups present in the pore proteins. This process allows the internalization of water, ions and other molecules (Figueira et. al., 2013). Therefore, we evaluated the effect of AuNRs on the Ca2+-sensitive mitochondrial swelling by monitoring the absorbance of a mitochondrial suspension at 520 nm (Wilson et. al., 2005). In this experiment, the mitochondrial suspension with AuNRs showed a higher absorption than that of the control (suspension without AuNRs) 14
(Figure 4a and 4b). This result indicates that the mitochondrial swelling triggered by Ca2+ was delayed by the AuNRs. The mitochondrial suspension incubated with the higher concentration of AuNRs (100 µM Au) presented similar behavior to that of the control in the presence of cyclosporine A, an inhibitor of the mitochondrial permeability transition pore (Figure S5). A similar effect was observed by Marín-Prida and
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coworkers when they evaluated the effect of 1,4-dihydropyridine derivates (VE-3N) on
mitochondria. Under standard conditions, VE-3N was able to inhibit the swelling
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process in a concentration-dependent manner (Marín-Prida et. al., 2017). The reduction in the mitochondrial swelling stimulated by AuNRs may be associated with the decrease
in ROS production that led to protection of the mitochondrial permeability transition
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progress. To investigate this possibility, we verified the effect of AuNRs on the
N
production of ROS by monitoring the fluorescence of 2’,7’-dichlorofluorescin (DCF)
A
(LeBel et. al., 1992). Curiously, the AuNRs induced a decrease in ROS production
M
(Figure 4c and 4d): 18.1 ± 5.7 nM for the mitochondrial suspension without AuNRs and
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2.8 ± 2.2 nM with 100 µM Au. The reduction in ROS production caused by AuNRs may explain the delay in mitochondrial swelling, since low ROS levels reduce the
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oxidation of the mitochondrial permeability transition pore, thus decreasing the entry of fluids into the mitochondria. These results imply that AuNRs may have a protective
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effect that causes a delay in mitochondrial swelling as result of the decrease in ROS levels. Notably, studies involving cells and gold nanoparticles found an increase in the
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ROS level (Tang et. al., 2015; Wan et. al., 2015; Hwang et. al., 2012); on the other hand, our results showed that when AuNRs were exposed to isolated mitochondria, the AuNRs stimulated a decrease in ROS production. Moreover, our results suggest that the AuNRs interact with the respiratory chain complexes, thus affecting the redox cascade, consequently interrupting the flow of electrons between the complexes, and decreasing
15
the reduction of O2 to H2O, ultimately leading to a low respiration rate. It is worth mentioning, here, that gold nanoparticles have already been shown to act as good catalysts for the reduction of H2O2 (Jv et. al., 2010; Navalon et. al., 2011) that also attest such a hypothesis. Nevertheless, in order to confirm the ability of AuNRs to reduce ROS, we proposed an experiment, using the Amplex-Red probe (Zhou et. al., 1997),
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that confirmed that the addition of AuNRs caused a depletion in H2O2 production
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(Figure S8).
Since some of the results showed a potential interaction between AuNRs and respiratory chain complexes, we conducted TEM analysis of the mitochondria to verify
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the presence or absence of AuNRs in the organelle. All details related to the sample
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preparation for TEM analysis can be found in the Supporting Information. It is
A
important to mention that for this analysis, the mitochondrial suspensions incubated
M
with AuNRs were washed with 4% cacodylate buffer (pH 7.2) at least 5 times to remove all residual reagents and AuNRs that did not effectively interact with the
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mitochondria. AuNRs were not found in the mitochondria or on their surfaces in any of the TEM images. Although AuNRs were not detected in TEM images, the AuNRs
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caused significant damage to the mitochondrial morphology (Figure 5). The
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mitochondria incubated with 100 µM Au exhibit disorganized crests and intermembrane separation (Figure 5b, arrows). The same effect was observed by Cambier and coworkers in the muscle mitochondria of zebrafish contaminated with 13 µg of CH3Hg
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(Cambiera et. al., 2009). Fanni and coworkers also observed disorganized mitochondrial cristae in Kupffer cells and hepatocytes treated with high concentrations of iron and cupper ions (Fanni et. al., 2014). Our study suggests that AuNRs were removed in the washing steps, indicating that the AuNRs interacted with the mitochondria via their external surface. Wang and 16
coworkers reported that after internalization into cancer cells, AuNRs were located at the edges of the mitochondria or around their inner membranes (Wang et al., 2011). We measured the diameter of mitochondria treated with 100 µM Au; the mitochondria had a mean size of 740 nm (± 36), while the mitochondria treated in the
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absence of AuNRs had a mean size of 862 nm (± 30) (Figure S9). The mitochondria incubated with AuNRs had a smaller diameter than that of mitochondria without
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AuNRs, confirming that the mitochondrial swelling delay was caused by AuNRs. The
potential interaction of AuNRs with the external surface of mitochondria along with the decrease in mitochondrial diameter implies that the AuNRs might interact with the
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N
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mitochondria via electron transport chain complexes.
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4. Discussion
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Mitochondria do not have large pores. Both the outer and inner membranes present barriers that prevent the entry of AuNRs into the matrix (Kodiha et. al., 2015).
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Salnikov and coworkers reported that in heart cells, 3 nm gold nanoparticles, but not 6 nm nanoparticles, translocated across the outer mitochondrial membrane (Salnikov et.
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al., 2007). In the inner mitochondrial membrane, protein import channels provide openings of ca. 2 nm3 (Szabo and Zoratti, 2014). Thus, there seems to be a restriction preventing the incorporation of AuNRs into the organelle matrix. However, the
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translocation barrier in the mitochondrial membrane against AuNRs does not preclude the AuNRs from damaging the mitochondria. Our findings suggest that AuNRs interact with electron transport chain complexes through the external surface of the mitochondria, thus disrupting the electron transport through the complexes and stimulating a series of events, including the following: i) decrease in the oxygen 17
consumption; ii) depletion of proton pumping that leads to a rapid decrease in the membrane potential; iii) reduction in ROS production leading to a delay in mitochondrial swelling; and iv) morphological changes in the mitochondrial cristae and external membrane.
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We found that AuNRs with a mean length of 42 nm and aspect ratio of 3.7 that were treated with BSA presented a bathochromic shift of their longitudinal surface
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plasmon resonance, which indicated the anchorage of this protein on their surface. The
studies related to the effect of AuNRs on isolated rat liver mitochondria strongly indicate a potential mechanism of tumor cell death caused by AuNRs (Wang et al.,
U
2011). The impact of AuNRs on the mitochondrial respiration and membrane electric
N
potential suggests an interaction with the electron transport chain complexes. The
A
decrease in the ROS levels in the presence of AuNRs also indicates that the electron
M
flow between the complexes is interrupted, which reduces the production of ROS. The mitochondrial swelling delay and the absence of AuNRs in the mitochondria, as seen in
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TEM images, corroborates the hypothesis. The TEM images also demonstrate that at 100 µM concentration, the AuNRs caused disorganized mitochondrial crests and
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detachment of the mitochondrial membranes. The elucidation of the behavior of AuNRs
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in mitochondria is the first step in understanding their mechanism in the cellular environment. This finding thus provides new evidence of how AuNRs interact with
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mitochondria.
Acknowledgements Authors gratefully acknowledge the Brazilian research founding agencies, National Counsel of Technological and Scientific Development (CNPq), Coordination 18
for the Improvement of Higher Level Education (Capes), and Alagoas Research Support Foundation (Fapeal) for financial support. A.M.N, K.R.M.S., C.M.S.C., A.C.R.L. and M.R.M. thank CNPq for their research fellowships.
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Apendix A. Suplementary Data
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Suplementary material related to this data can be found in the online version
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Figure Captions Figure 1. (a) UV-Vis absorption spectra of AuNRs before and after incubation with 10% BSA in phosphate-buffered saline (PBS); (b) TEM images showing the size and
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shape of AuNRs.
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Figure 2. Effect of the amount of AuNRs on the mitochondrial oxygen consumption.
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(a) Oxygen consumption, (b) oxygen consumption in different states – the phosphorylation state (PS), the resting state (RS) and the maximal state (MS) - and (c)
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the respiratory control ratio (RCR). RLM (0.5 mg protein/mL were incubated in buffer solution at 28 ᵒC (5 mM complex I substrates, 200 µM EGTA, 250 µM ADP, 1 µg/mL
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oligomycin and 1µM CCCP). All of the concentrations are expressed in µM of Au. The data represent three separate experiments performed in duplicate. Mean values ±
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standard deviation, n = 3 (*p < 0.05).
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Figure 3. Effect of AuNRs at various concentrations on the mitochondrial membrane potential. (a) membrane potential disruption monitored by safranin O fluorescence and (b) mean potential disruption time. RLM (0.5 mg protein/mL) were incubated in buffer
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solution at 28 ᵒC (5 mM complex I substrates, 5 µM safranin O, and 10 µM Ca2+). All of the concentrations are expressed in Au. The data represent three separate experiments performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05).
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Figure 4. Effect of AuNR concentration on mitochondrial integrity. (a) Effect on
mitochondrial swelling and (b) Final mitochondria suspension turbidity mean: RLM
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(0.5 mg protein/mL) were incubated in buffer solution at 28 ºC (5 mM complex I
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substrates and 10 µM Ca2+); (c) effect of AuNRs on ROS production in time and (d)
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Mean of ROS level production: RLM (0.5 mg protein/mL) were incubated in buffer
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solution at 28 ºC (5 mM complex I substrates, 1 µM H2DCFDA and 10 µM Ca2+). All the concentrations are expressed in Au. The data represent three separated experiments
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performed in duplicate. Mean values ± standard deviation, n = 3 (*p < 0.05).
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Figure 5. Effect of AuNRs on mitochondrial morphology. TEM images of mitochondria in the absence (a, b) and presence of 100 µM Au (c, d). Scale: 500 nm.
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Arrows indicate intermembrane separations.
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Figure 6. Mechanistic scheme. Mitochondrial dynamic scheme in the absence (a) and
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presence (b) of AuNRs. The four mitochondrial complexes are labeled as I, II, III and IV. In the scheme, Mn-superoxide dismutase (MnSOD), glutathione peroxidase (GPx),
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peroxiredoxin (Prx), thioredoxin (TSH), glutathione (GSH), glutathione reductase (GR),
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thioredoxin reductase (TR), ubiquinone (Q), cytochrome c (CyT) and the mitochondrial
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permeability transition pore (MPT) are shown.
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