an approach to the mechanism of the cytotoxic effect of ... · the filamentous fungus was...
TRANSCRIPT
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An approach to the mechanism of the cytotoxic effect
of Silver and Zinc Oxide Nanoparticles
Arzate-Quintana, Carlos*1; Sánchez-Ramírez, Blanca*2; Infante-Ramírez, Rocío2; Piñón-
Castillo Hilda Amelia1; Montes-Fonseca, Silvia Lorena*1; Duarte- Moller, Alberto1; Luna-
Velasco, Antonia*1; Orrantia-Borunda, Erasmo1§.
1Centro de Investigación en Materiales Avanzados S.C., Ave. Miguel Cervantes
120, complejo industrial Chihuahua, CP 31109, Chihuahua, Chih. México
2Facultad de Ciencias Químicas, Universidad Autónoma de Chihuahua, Chih.,
Mexico.
*These authors contributed equally to this work
§Corresponding author
Carlos Arzate Quintana: [email protected]
Blanca Estela Sánchez-Ramírez: [email protected]
Rocío Infante-Ramírez: [email protected]
Hilda Amelia Piñón-Castillo: [email protected]
Silvia Lorena Montes Fonseca: [email protected]
Alberto Duarte Moller: [email protected]
Antonia Luna Velasco: [email protected]
Erasmo Orrantia Borunda: [email protected]
2
ABSTRACT
Background
Cytotoxicity of nanoparticles (NPs) has already been demonstrated in several studies.
However, their toxicity mechanisms are not completely understood. Additionally, those
mechanisms vary based on the type of NPs and cells exposed. The present work is
focused on establishing the toxicity and main cytotoxic mechanisms of silver (AgNP)
and zinc oxide (ZnONP) nanoparticles on representative pathogen microorganisms
groups.
Results
Based on inhibition assays, AgNP showed inhibitory effects to bacteria and fungi.
Assays in liquid media indicated growth inhibition of Staphylococcus aureus, finding
the minimal inhibitory concentration (MIC) and minimal bactericidal concentration
(MBC) of 5,5 and 6,5 g/mL of AgNP, respectively. In the case of Pseudomonas
aeruginosa, a MIC of 7,0 g/mL and MBC of 7,5 g/mL were obtained and Candida
albicans showed a 50% lethal dose (LD50) of 140 g/mL. In solid media, the MIC
values of 1, 2 and 60 g/mL of AgNP were found for P.aeruginosa, S.aureus and
C.albicans, respectively. Whereas, Coniphora eremophila was inhibited by AgNP
obtaining a LD50 of 30 g/mL. In relation to ZnONP, no inhibitory effect was noted
against bacteria at the maximum concentration tested (240 g/mL). Nevertheless, those
NPs caused inhibitory effects to fungi, finding LD50 values of 140 and 80 g/mL
against C.albicans in liquid and solid media, respectively. Lastly, the growth of C.
eremophila was inhibited 11, 6% at 80 g/mL of ZnONP. Since ZnONP tend to
agglomerate and precipitate in liquid media they showed higher activity in solid media.
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Taking all inhibition results, it was clearly noted that assays in liquid media were
somewhat less sensitive than assays in solid media. The minor inhibitory effect of NPs
in liquid media was associated with the low interaction of NPs with cells, caused by the
aggregation of NPs trapped in biofilms excreted by bacteria (confirmed by scanning
electron microscopic, SEM). SEM analysis also provided evidence of structural damage
of the bacteria P. aeruginosa exposed to AgNPs and the fungi C. albicans exposed to
AgNPs and ZnONP. However, No DNA fragmentation was observed through
electrophoresis analysis in all DNA extracts from microorganisms treated with NPs.
Catalytic activity of lipase and lactic dehydrogenase (LDH) enzymes was inhibited by
AgNP and considerably inhibited by silver ions (Ag+) in cell-free assays, which
indicates the potential of Ag+ from AgNP to inactivate membrane proteins and
respiratory enzymes. Therefore, that might be one of the main mechanisms of
membrane of membrane damage of pathogen microorganisms.
Conclusions
To our knowledge, this is the first report that indicates the toxicity of AgNP and ZnONP
to the pathogen fungi C. eremophila, as well as confirms the structural damage of yeast
cells exposed to both NPs (based on electronic microscopy).
KEYWORD
Silver, zinc oxide, nanoparticles, nanotoxicology, enzymatic inhibition, cell damage
BACKGROUND
Recently, interest in the design of new metallic nanoparticles has increased due to their
potential in nanotechnology applications, including nanomedicine, sensors, catalysis,
and electronics. In medical and industrial area, inorganic NPs (e.g. Ag, CuO, and ZnO)
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are being explored as bactericidal and fungicidal agents [1]. It is now known that
particle size and dispersion in aqueous media are important factors that affect the
antimicrobial properties of NPs. Then, different NPs formulations are being evaluated
and included in various base materials in order to increase their durability (e.g. textiles
resistant to degradation) and their resistance to pathogens (e.g. base materials used in
surgical instruments to lower the risk of nosocomial infections by contamination with
pathogens) [2, 3]. Silver was used to treat infectious wounds long time before
antibiotics were introduced in modern medicine. However, its use was relegated almost
entirely in medicine, not only because of its high toxicity, but also due to the practicality
of antibiotics. Currently, silver nanoparticles have captured the interest of the scientific
community as a potential antimicrobial agent [2, 3]. Zinc oxide is a compound that has
been used an important ingredient to prepare medical ointments and pigments. Recent
reports have demonstrated the antibacterial, antifungal and photo-catalytic activities of
ZnONP[1].
Even though, many articles have demonstrated the inhibitory and cytotoxic activity of
metallic nanoparticles, the explanations of how cellular damage is caused had not yet
reached a consensus. The main toxicity mechanisms related to NPs toxicity are the
generation of reactive oxygen species, release of toxic ions, absorptive capacity, etc.[4-
6]. In NPs toxicity studies with microorganisms, pathogens are not commonly
evaluated. In general, most NPs toxicity reports are for bacteria and considerably much
less are for yeast/fungi, being Saccharomyces cerevisiae the main yeast model used.
Until now there is only one report that evaluated the viability of the pathogenic yeast
Candida albicans exposed to ZnONPs [7]. Recently, the filamentous fungi Penicillium
expansum was also evaluated with ZnONPs [8].
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Here is presented the evaluation of the inhibitory effect of AgNP and ZnONP on
representative pathogen microorganisms groups, including P.aeruginosa, S.aureus,
C.albicans and C. eremophila. Additionally an important goal was to provide evidence
of the main mechanisms involved in the NPs toxicity to cells.
METHODOS
Nanomaterials
AgNP and ZnONP were kindly provided by the Mexican mining company Peñoles
(Torreón, Coahuila, México). Nanoparticle stock dispersions were prepared by adding a
known quantity of nanoparticle powder to a 50mL tube and then filled with 50mL of
deionized water. The tube was then exposed to sonication at room temperature until
complete homogenization (10-30 minutes). To avoid oxidation and transformation,
storage was performed at -20°C until further use.
Characterization of nanoparticles
Size and shape of AgNP and ZnONP were determined using scanning electron
microscopy (SEM) through a JOEL equipment model JSM-7401f. The chemical
composition was determined by energy dispersive spectrometry, which was coupled to
SEM. X-ray diffractometry (XRD) was used to confirm the crystallinity phase of NPs
[9].
Bacterial strains and culture conditions
All strains were obtained from the global biosource center ATCC. Bacteria were
selected due to their importance as human pathogens and in the case of the filamentous
fungus due to their industrial importance. Staphylococcus aureus ATCC 29213 and
Pseudomonas aeruginosa ATCC 15442 were used as models to evaluate damage in
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gram positive and gram negative bacteria. Candida albicans ATCC 10213 and
Coniophora eremophila ATCC 64458 were included as yeast and filamentous fungi
specimens, respectively. Bacteria models and C. albicans were grown in soy tripticase
broth and Sabouroud broth, respectively (at 37oC for12 h). C.eremophila was grown in
sabouraud agar at 25oC for 6 days. Inhibition assays were performed in minimum media
M9, minimum media Lee and minimum solid media Lee for bacteria, yeast and fungi
cells, respectively.
Minimal Inhibitory concentration (MIC) and Minimal Bactericidal Concentration
(MBC) assays in broth
MIC was determined by using the broth dilution method described by the Clinical and
Laboratory Standards Institute (CLSI), for bacteria that grow aerobically (M07-A8) [10]
and for antifungal activity (M27-A3)[11]. 96 well plates with round bottom were used
in all assays, with a final volume of 200 L for each well. AgNP and ZnONP were used
in several dilutions and suspended in M9 media for bacteria assays, and Lee media for
C.albicans. Both media solutions were added with 0,1% Pluronic F127 (Sigma P2443)
as a dispersant to avoid nanoparticle sedimentation. All wells had a final microorganism
concentration of 1x107 colony forming units (CFU). The lowest concentration of
nanoparticles that inhibited the growth of bacteria was considered as the MIC. MBC
was determined by the CFU drop method [12] in soy trypticase agar for bacteria and
Sabouraud media for C. albicans. For MBC assays, only wells with no observable
growth were used. The MBC was defined as the lowest concentration of nanoparticles
that completely prevented microbial growth, determined by visible inspection of the
wells. MBC assays were performed in triplicate.
MIC and MBC assays in solid media
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Due to the tendency of different nanoparticles to precipitate, assays in solid agar were
performed in M9 and Lee minimal solid media were used for bacteria and yeast
respectively [13]. Before solidification, both media were added with glucose 1M (990
l/50 mL), yeast extract (10 mg/50 mL), vitamins and mineral trace elements (90 l/50
mL) (ATCC) and with increasing concentrations of nanoparticles. Media without
nanoparticles were used as control and 200 CFU were inoculated in each plate in
triplicated assays. Plates were then incubated at 35oC for 24 hours and inhibition was
determined by CFU counting, comparing all the treatments with the non-treated
controls.
In assays with C. eremophila, Lee minimal media was added as described above and
increasing concentrations of nanoparticles as described for bacteria and yeast assays.
The filamentous fungus was inoculated in Lee media by placing a 5 mm diameter
sample from a previous culture in Sabouraud media. Fungus radial growth was
measured every 3 days during 9 days. Growth inhibition was determined comparing the
growth in plates exposed to nanoparticles with non-treated controls [14, 15].
DNA damage determination assays
To evaluate DNA fragmentation, S. aureus, P. aeruginosa and C. albicans were
exposed to AgNP and ZnONP. In order to obtain enough DNA, microorganisms were
growth in sterile 250 mL Erlenmeyer flasks containing a nanoparticle concentration
lower than MIC. In the case that MIC could not be determined due to resistance; a
concentration of 100 g/mL was used to ensure cellular stress and to avoid nanoparticle
sedimentation. The remaining volume was completed with minimal media and the
inoculum. Flasks were incubated for 24 hours at 37oC and 200 rpm. Samples of each
flask were deposited in 1.5 mL Eppendorf tubes and centrifuged at 10,000 rpm for 5
8
minutes. Supernatant was discarded and the pellet containing microorganisms was
stored at -20°C until use.
Genomic DNA was obtained from cell pellets using Wizard genomics DNA extraction
kit (Promega, WI, USA) according to manufacturer instructions [16]; DNA isolated was
run in 1% agarose gels with 0,1 mg/mL of ethidium bromide and 100mL TAE buffer
(40 mM TRIS-acetate and 1mM EDTA), Hyper ladder 1 (10,000 to 200 kD, Bioline)
was used as molecular weight marker. Electrophoresis was performed for 30 minutes at
100V. Gels were analyzed using an UV documentation system. The presence of
fragmented DNA was considered as evidence of damage in genetic material [17].
Scanning electron microscopy (SEM)
Cell pellets from each strain and treatment obtained as described above were
resuspended and fixed in glutaraldehyde at a concentration of 2,5 % during 2 hours and
washed using cacodilate buffer (0,1 M). Dehydration was then performed as follows:
samples were resuspended in 70 % ethanol and incubated during 15 minutes, then
samples were centrifuged at 4,000 rpm, this step was repeated increasing
concentrations of ethanol (85 %, 95 % and absolute). Finally, samples were resuspended
in absolute ethanol, transferred and wrapped in a 5cm diameter filter paper. Filters then
were processed in a CO2 critical point dryer. Cell powder was then placed in to a
specimen holder and covered in gold in order to observe sample morphology in SEM.
Catalytic activity of Lactic dehydrogenase exposed to AgNP Lactic dehydrogenase
(LDH) activity in presence of AgNP was measured using a colorimetric method based
on the concentration of enzymatic cofactor nicotinamide adenine dinucleotide (NADH,
Sigma Chemical Co., USA). 2,8mL of buffer Tris-HCl 0,2M and pH 7,3 was added to a
series of 10 mL test tubes. Then, 100L of 10 mM sodium pyruvate (Sigma Chemical
9
Co., USA) and 100L of 6,6 mM of NADH were added to the buffer and incubated at
room temperature for 5 minutes. To measure the activity of the enzyme in absence of
treatments, a control with 100 mL of 1mg/mL LDH (Sigma Chemical Co., USA) was
carried out in the experiments. Measurements were done at 340 nm after 1 minute of
incubation. To prove AgNP and AgNO3 (silver nitrate) inhibition, LDH was incubated
in presence of these treatments for ten minutes at room temperature before it was added
to the solution containing cofactor and substrate. LDH activity is inversely proportional
to the amount of NADH used in the reaction; therefore, the lowest concentration of
NADH indicates a higher enzymatic activity.
Catalytic activity of lipase exposed to AgNP
Catalytic activity of lipase exposed to AgNP in cell-free media was measured through
the determination of hydrolyzed triglycerides by means of a Randox manual
colorimetric method (West Virginia, USA). This method is based on the hydrolysis of
triglycerides by lipases, and quinoneimine produced by oxidases as an indicator. A
volume of 1 ml of reactive that includes both enzymes was exposed to AgNP (5 g/mL)
or Ag+ (0,001 mM/mL) for 15 min at 37 oC, and then 10L of triglyceride standard was
added. Enzymes not exposed to AgNP were included as control reference.
Quinoneimine production is proportional to the lipase activity and it was measured at an
OD of 500nm.
RESULTS AND DISCUSSION
Nanoparticle characterization
Based on SEM analysis, it was obtained that AgNP were in the size range of 15 to
25nm, whereas ZnONP had a slightly bigger size range of 20 to 100nm. Chemical
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composition was corroborated by X-ray diffraction. The composition of crystal phase of
NPs was confirmed by XRD analysis. Both, ZnONP and AgNP showed diffraction
patrons that correspond to those inorganic compounds, as is showed in Fig 1 and 2,
respectively.
Figure 1- X-ray powder diffraction patterns of AgNP (red line) validated with Ag pattern from
the JCPDS database (green lines). Analysis of silver nanoparticles
Figure 2- Xray diffraction analysis of zinc oxide nanoparticles
Inhibition of microorganisms exposed to nanoparticles
AgNP completely inhibited the growth of S.aureus, P.aeruginosa, however,
C.eremophila and C.albicans still showed growth in presence of treatments (Table 1).
The high toxicity of AgNP correlates with previous works including similar assays with
gram positive and gram negative bacteria [9, 18, 19], and filamentous fungi [20].
Nevertheless, size, dispersants, and chemical synthesis of the particles influence the
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inhibition activity resulting in different MIC for the same materials, as we observed
when the same nanoparticles were used in liquid and solid media, and showed different
MICs [9, 21-23]. ZnONP did not showed bactericidal effect, but they had a considerable
antifungal effect against C.eremophila. The low antimicrobial effect of ZnONP could be
due to the absence of UV-light exposure in assays. ZnONP have shown to have a photo-
catalytic effect [8, 24], which is related their effectiveness as inhibitor of bacteria and
fungi.
Table 1. Values of MIC, MBC and LD 50 obtained for nanoparticle in the
microorganisms tested.
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Table 1. - MIC and MBC of nanoparticle treatments against the tested microorganisms
Mechanisms related to NPs exposure
In inhibition assays of bacteria with AgNPs, it was noted the presence of biofilms (in
both, liquid and solid media). S. aureus and P. aeruginosa usually produce extracellular
excretions (exo-polysaccharides). Although, when bacteria were exposed to NPs, they
tended to produce more exo-polysaccharides as a defense mechanism against NPs. In
fact, it was clear that AgNP were trapped by the exo-polysaccharides excreted by
bacteria causing a considerable agglomeration effect of NPs in liquid media. In order to
confirm the presence of AgNP aggregates with biofilms, SEM analysis coupled to EDS
were performed. SEM images clearly showed that AgNP were trapped in the biofilms
Microorganism
Aqueous media Solid media
AgNP ZnONP AgNP ZnONP
C. albicans
LD50
140 g/mL
LD50
140 g/mL
LD50
60 g/mL
MIC
140 g/mL
LD50
80 g/mL
P. aeruginosa
MIC
7,0 g/mL
MBC
7,5 g/mL
Resistant
MIC
1,0 g/mL
Resistant
S. aureus
MIC
5,5 g/mL
MBC
6,5 g/mL
Resistant
MIC
2,0 g/mL
Resistant
C. eremophila ND ND
LD50
30 g/mL
11,6% inhibition at
80 g/mL
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(organic material) (Figure 3). Additionally, elemental analysis by EDS confirmed the
presence of silver in aggregates with biofilms, which evidenced the trapping of AgNPs.
The trapping and agglomeration effect of NPs in liquid media decreased the contact of
NPs with microorganism in inhibitory assays, which correlates with the lower inhibition
of AgNPs in liquid media compared with values in solid media. The excretion of exo-
polysaccharides by bacteria is a well-known mechanism used to decrease the
concentration of heavy metals in aqueous environments and survive under those
conditions [25, 26].
Figure 3- SEM analysis of exo-polisaccharides obtained after exposure of bacteria to AgNP
Toxicity assays with AgNP have reported that one of the toxicity mechanisms is the
release of the toxic ion Ag+ [27]. In order to know the contribution of Ag+ ions in MIC
assays, microorganisms were exposed to various AgNO3 concentrations, obtaining that
AgNO3 had MICs values in the order 0.107 g/mL of Ag+ for all microorganisms. We
can assume that inhibition is caused by silver ions [6], and not by the nanoparticles as
seen on tissue cells of mammalians in wish it has been confirmed that both, silver ions
and the process of phagocytosis of nanoparticles cause cell damage [21, 28-30]. Also,
higher toxicity of silver ions can be related to lower positive charge [31] that makes
bacteria more susceptible to silver toxicity than other metallic ions such as zinc.
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DNA damage of cells exposed to AgNP or ZnONP was evaluated through
electrophoretic analysis in agarose gels. DNA fragmentation was not detected in DNA
extracts from all microorganism treated with NPs (compared with DNA extracts from
microorganisms not exposed to NPs). The genotoxicity of AgNPs has been previously
demonstrated in human cells and tissues as well as in plants [32], mice [30] and worms
[5]. However, no DNA damage has been exhibited for bacteria (e.g. Salmonella
Typhimurium) exposed to AgNP [33] and a weak DNA damage was observed in
bacteria treated with ZnONP (e.g. S. Typhimurium and E. Coli) [34]. In the case of
metallic ions (such as Ag+), it has been reported that ions interact with DNA and RNA
under in vitro conditions without causing a significant DNA change in its structure
conformation [35].
In the other hand, the structural damage of cells non-treated and treated with ZnONP or
AgNP (at concentrations lower that MIC) was evaluated by electron microscopy. A
clearly structural damage was noted in some cells, observing fragmentation of the
cellular wall of C.albicans with either, AgNP or ZnONP (Fig 4).
Figure 4- Cell wall damage of C.albicans exposed to nanoparticles. Left: Untreated
control; center: AgNP; right: ZnONP.
However, the higher structural damaged was noted for yeast cells treated with AgNP.
To our knowledge, this is the first report that presents the structural damage of yeast
cells exposed to NPs (AgNP and ZnONP) by electronic microscopy. Recently, It was
reported the susceptibility of the membrane integrity of the yeast Saccharomyces
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cerevisiae treated with some inorganic NPs (AgNP not included), using flow cytometry
[36]. Where, Mn2O3 NPs and Fe NPs caused a significant membrane damage of yeast
cells. In relation with bacteria, it was observed some deep invaginations in cell
membrane of the gram negative bacteria P.aeruginosa exposed to AgNP (Figure 5).
Figure 5- Cell membrane damage of P.aeruginosa exposed to silver nanoparticles. Left:
Untreated control; right: Ag nanoparticles.
Structural alteration of membrane cells have been previously reported for E. coli, S.
Typhimurium and S. aereus exposed to NPs (including AgNP) [34, 37]. There, the gram
negative bacteria S. Typhimurium treated with AgNP showed membrane invaginations
and other structural damage (outgrowths and deformations. Even though S. aureus and
C. eremophila showed inhibition in the presence of AgNP, no visible structural damage
was observed in SEM micrographs (data not shown). Previous reports have shown
structural damage of S. aureus cells exposed to AgNP [37]. However, the smaller size
of AgNP (8-10 nm) used in that work might cause a different structural impact on cells.
Additionally, it was reported that S. aereus (gam positive) presented late and small
changes in shape and membrane structure compared with structural changes for S.
Typhimurium (gram negative) treated with same NPs. The literature indicates that the
interaction of AgNP with cells could be by the adhesion of NPs on the surface of
membranes (or via release of Ag+ [38]. In this case, it was not observed the adsorption
of NPs on the surface of cells (based on SEM), which probably was due to the washing
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steps during embedding process. The interaction of Ag+ with sulfur groups of
membrane proteins could result in the inactivation of respiratory enzymes and then
causing structural damage of cells exposed to AgNP [39]. These results agree with
Díaz-Visurraga et al, published in 2011[40].
In order to determine the potential of AgNP and Ag+ to inactivate enzymes, the
inactivation of LDH and lipase enzymes was measured after their exposure with AgNP
and Ag+ in cell free experiments. Those enzymes were selected because of their
constitutive presence in microorganisms. LDH uses NADH as cofactor and the activity
of the enzyme is related with a decrease in the NADH concentration. As is showed in
fig 6, the NADH measured in enzyme treatments with AgNP or Ag+ was higher
compared with controls (not exposed to AgNP or Ag+). Noting that enzyme treatments
exposed to Ag+ had the highest NADH values. Those results indicate that both, Ag+ and
AgNP inactivated LDH enzyme. However, Ag+ exhibited a greatest inactivation of the
LDH enzyme. The inactivation of lipase enzyme treated with AgNP and Ag+ was also
determined. A colorimetric method was used to measure the amount of hydrolyzed
triglycerides, which was proportional to the enzyme activity. The amount of hydrolyzed
triglycerides in enzyme treatments with AgNP and Ag+ was significantly lower
compared with values obtained in controls not treated with AgNP or Ag+ (Figure 7).
Similar than for LDH enzyme, lipase enzyme was inactivated by AgNP and Ag+,
showing the highest inactivation of the enzyme by Ag+. ZnONP did not show inhibition
of the catalytic activity of enzymes (Data not shown), which could be due to the assay
were in absence of UV light and those NPs have a photocatalytic nature.
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Fig. 6- Activity of lactic dehydrogenase exposed to silver nanoparticles by optical
density measurement
Fig. 7- Measurement of lipase activity exposed to silver nanoparticles using a
commercial colorimetric method.
CONCLUSIONS
AgNP were toxic to pathogens bacteria and fungi cells tested, whereas ZnONP only
affected fungi/yeast cells at higher doses than AgNP. Damage of cell structure was
observed through electron microscopy in yeast and bacterial cells treated with NPs
concentration lower than MIC values. To our knowledge, this is the first report that
indicates the toxicity of AgNPs and ZnONP to the pathogen fungi C. eremophila, as
well as confirms the structural damage of yeast cells exposed to both NPs (based on
electronic microscopy). A defense mechanism of microorganisms against nanoparticle
toxicity was the excretion of exopolysaccharides, where NPs were trapped, decreasing
the interaction of cells with NPs. Catalytic activity of enzymes in cell-free media (lipase
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and LDH) was considerable inhibited by AgNPs and its ions in the followed order, Ag+
> AgNP, which indicates the potential of Ag+ from AgNP to inactivate membrane
proteins and respiratory enzymes. Therefore, that might be one of the main mechanisms
of membrane damage of pathogen microorganisms during their interaction with AgNP.
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LIST OF ABBREVIATIONS
AgNP: Silver nanoparticles
CLSI: Clinical and laboratory standards
institute
LD50: 50% lethal dose
LDH: Lactic dehydrogenase
MBC: Minimal bactericide
concentration
MIC: Minimal Inhibitory concentration
NADH: Nicotinamide adenine
dinucleotide
NPs: Nanoparticles
OD: Optical density
SEM: Scanning electron microscopy
TAE: Tris-acetate-EDTA
TEM: Transmission electron
microscopy
ZnONP: Zinc Oxide nanoparticles