For purpose of this review, silver nanomaterials include silver nanoparticles, stabilized silver salts, silver–dendrimer, polymer and metal oxide composites, and silver-impregnated zeoli
Trang 1R E V I E W P A P E R
A review of the antibacterial effects of silver nanomaterials
and potential implications for human health
and the environment
Catalina Marambio-Jones•Eric M V Hoek
Received: 29 July 2009 / Accepted: 6 March 2010 / Published online: 23 March 2010
Ó Springer Science+Business Media B.V 2010
Abstract Here, we present a review of the
antibac-terial effects of silver nanomaantibac-terials, including
pro-posed antibacterial mechanisms and possible toxicity
to higher organisms For purpose of this review, silver
nanomaterials include silver nanoparticles, stabilized
silver salts, silver–dendrimer, polymer and metal oxide
composites, and silver-impregnated zeolite and
acti-vated carbon materials While there is some evidence
that silver nanoparticles can directly damage bacteria
cell membranes, silver nanomaterials appear to exert
bacteriocidal activity predominantly through release of
silver ions followed (individually or in combination)
by increased membrane permeability, loss of the
proton motive force, inducing de-energization of the
cells and efflux of phosphate, leakage of cellular
content, and disruption DNA replication Eukaryotic
cells could be similarly impacted by most of these
mechanisms and, indeed, a small but growing body of
literature supports this concern Most antimicrobial
studies are performed in simple aquatic media or cell
culture media without proper characterization of silver
nanomaterial stability (aggregation, dissolution, and
re-precipitation) Silver nanoparticle stability is
gov-erned by particle size, shape, and capping agents as
well as solution pH, ionic strength, specific ions andligands, and organic macromolecules—all of whichinfluence silver nanoparticle stability and bioavailabil-ity Although none of the studies reviewed definitivelyproved any immediate impacts to human health or theenvironment by a silver nanomaterial containingproduct, the entirety of the science reviewed suggestssome caution and further research are warranted giventhe already widespread and rapidly growing use ofsilver nanomaterials
Keywords Silver Nanoparticle Antimicrobial Antibacterial Nanotechnology Nanotoxicology Safety EHS
IntroductionThe broad-spectrum antimicrobial properties of silverencourage its use in biomedical applications, waterand air purification, food production, cosmetics,clothing, and numerous household products Withthe rapid development of nanotechnology, applica-tions have been extended further and now silver is theengineered nanomaterial most commonly used inconsumer products (Rejeski 2009) Clothing, respi-rators, household water filters, contraconceptives,antibacterial sprays, cosmetics, detergent, dietarysupplements, cutting boards, sox, shoes, cell phones,laptop keyboards, and children’s toys are among the
C Marambio-Jones E M V Hoek (&)
Department of Civil and Environmental Engineering,
California NanoSystems Institute, University
of California, Los Angeles, 5732G Boelter Hall,
PO Box 951593, Los Angeles, CA 90095-1593, USA
e-mail: emvhoek@ucla.edu
DOI 10.1007/s11051-010-9900-y
Trang 2retail products that purportedly exploit the
antimi-crobial properties of silver nanomaterials
Different forms of silver nanomaterials already in
such products include: metallic silver nanoparticles
(Arora et al.2008; Chi et al.2009; Choi et al.2008;
Hwang et al.2008; Kim et al.2007,2008a,b; Kvitek
et al.2008; Lok et al.2006; Raffi et al.2008; Schrand
et al.2008; Sondi and Salopek-Sondi2004; Vertelov
et al 2008), silver chloride particles (Choi et al
2008), silver-impregnated zeolite powders and
acti-vated carbon materials (Cowan et al 2003; Inoue
et al 2002; Yoon et al 2008a, b), dendrimer–silver
complexes and composites (Balogh et al 2001;
Lesniak et al 2005; Zhang et al 2008),
polymer-silver nanoparticle composites (Bajpai et al 2007;
Damm and Munstedt2008; Damm et al.2008; Hlidek
et al 2008; Jin et al 2007; Kim et al 2009a, b;
Kvitek et al 2008; Naidu et al 2008; Nita 2008;
Sambhy and Sen 2008; Sanpui et al.2008; Xu et al
2006), silver-titanium dioxide composite
nanopow-ders (Yeo and Kang 2008), and silver nanoparticles
coated onto polymers like polyurethane (Jain and
Pradeep 2005) While all of these forms of silver
exert antimicrobial activity to some extent through
release of silver ions, silver nanoparticles might
exhibit additional antimicrobial capabilities not
exerted by bulk or ionic silver (Chen and Schluesener
2008)
Already, silver nanoparticles have been shown to
be effective biocides against: (a) bacteria such as
Escherichia coli, Staphylococcus aureus,
Staphylo-coccus epidermis, Leuconostoc mesenteroides,
Bacil-lus subtilis, Klebsiella mobilis, and Klebsiella
pneumonia among others (Benn and Westerhoff
2008; Chen and Chiang 2008; Falletta et al 2008;
Hernandez-Sierra et al.2008; Ingle et al.2008; Jung
et al 2009; Kim 2007; Kim et al 2007, 2009a, b;
Kvitek et al.2008; Raffi et al.2008; Ruparelia et al
2008; Smetana et al.2008; Sondi and Salopek-Sondi
2004; Vertelov et al 2008; Yang et al 2009; Yoon
et al.2008a,b); (b) fungi such as Aspergillus niger,
Candida albicans, Saccharomyces cerevisia,
Tricho-phyton mentagrophytes, and Penicillium citrinum
(Kim et al 2007, 2008a, b, 2009a, b; Roe et al
2008; Vertelov et al.2008; Zhang et al.2008); and (c)
virii such as Hepatitis B, HIV-1, syncytial virus
(Elechiguerra et al.2005; Lu et al 2008; Sun et al
2008; Zodrow et al 2009) Hybrid silver
nanocom-posites with dendrimers and polymers have been
shown effective for S aureus, Pseudomonas ginosa, E coli, B subtilis, and K mobilis (Balogh
aeru-et al 2001; Zhang et al 2008) Furthermore, silverloaded in nanoporous materials such as silver-exchanged zeolites exhibit antibacterial effects forPseudomonas putida, E coli, B subtilis, S aureus,and P aeruginosa (Cowan et al 2003; Inoue et al
2002; Lind et al.2009; McDonnell et al.2005).Despite the vast number of papers touting thebeneficial antimicrobial effects of silver nanomateri-als, a relatively modest number of studies haveattempted to elucidate the mechanisms by whichsilver nanomaterials exert this antimicrobial activity
As a result, the mechanisms are not widely stood or agreed upon For bacteria, commonlyproposed mechanisms in the literature begin withthe release of silver ions (Hwang et al.2008; Smetana
under-et al 2008) followed by generation of reactiveoxygen species (ROS) (Hwang et al 2008; Kim
et al 2007) and cell membrane damage (Choi et al
2008; Raffi et al 2008; Smetana et al 2008; Sondiand Salopek-Sondi 2004), but there are manycontradictory findings reported
The more widespread our use of silver terials becomes the more widespread will become thepotential for human and ecosystem exposure Silvernanoparticles may be released to the environmentfrom discharges at the point of production, fromerosion of engineered materials in household prod-ucts (e.g., antibacterial coatings and silver-impreg-nated water filters), and from washing or disposal ofsilver-containing products (Benn and Westerhoff
nanoma-2008) Silver released to both natural and engineeredsystems will likely impact the lowest trophic levelsfirst, i.e., bacteria However, little is known abouttrophic transfer of silver and impacts to higherorganisms Indeed, silver nanoparticles have alreadybeen proven toxic to both aerobic and anaerobicbacteria isolated from wastewater treatment plants(Choi and Hu 2008), which we speculate could lead
to severe disruption of this critical environmentalinfrastructure if the load of silver into wastewatertreatment plants increases significantly
Given the vast number of products leveraging thebenefits of silver, it seems prudent to assess thepotential human and ecosystem hazards associatedwith its increased utilization The main routes ofhuman exposure would be the respiratory system,gastrointestinal system, and skin, which are interfaces
Trang 3between the internal systems of the human body and
the external environment (Chen and Schluesener
2008) For example, silver nanomaterials may enter
through the respiratory tract due to inhalation of dust
or fumes containing silver nanomaterials at the point
of manufacture, it may be ingested from water,
children’s toys, or food containers treated with silver,
or it may penetrate the skin via silver-containing
textiles and cosmetics Additionally, other potential
entryways could include the female genital tract (due
to incorporation of silver nanoparticles into numerous
female hygienic products), via systemic
administra-tion as it is used for some imaging and therapeutic
purposes (Chen and Schluesener2008; Schrand et al
2008; West and Halas2003), or by incorporation into
medical implants, catheters, and wound dressings
(Furno et al 2004; Galiano et al 2008; Maneerung
et al.2008; Roe et al.2008)
In addition to broad-spectrum antimicrobial
effects, silver nanoparticles have produced toxic
effects in higher cell lines like zebra fish, clams,
rats, and humans (Arora et al.2008, 2009; Asharani
et al 2008; Braydich-Stolle et al 2005; Hsin et al
2008; Hussain et al.2005; Kim et al.2008a,b; Sung
et al 2008; Yeo and Kang 2008; Yeo and Yoon
2009) Evidence in rodents shows that after entering
into the body silver nanoparticles can accumulate
and, in some cases, damage tissues such as the liver,
lungs, and olfactory bulbs, or penetrate the blood–
brain barrier (Arora et al.2009; Braydich-Stolle et al
2005; Hussain et al.2005; Sung et al.2008) A study
in human cells concluded that silver can be genotoxic
(Asharani et al.2009) Additionally, a release of ionic
silver led to the sterility of Macoma balthica clams in
the San Francisco Bay during the 1980s (Brown et al
2003) If silver nanomaterials exhibit similar or
stronger reactivity, the impacts of this isolated event
in San Francisco may foreshadow potential
ecosys-tem impacts of silver nanomaterials
The various forms of silver nanomaterials are
among the most promising antimicrobial agents being
developed from nanotechnology, but the preliminary
evidence of effects on higher organisms alerts us to
remain cautious of its widespread utilization This
cautiousness demands additional research to
deter-mine how to safely design, use, and dispose products
containing silver nanomaterials without creating new
risks to humans or the environment Consequently,
the goal of this article is to provide a critical review
of the state-of-knowledge about silver nanomaterialantibacterial effects with insights toward betterunderstanding potential implications for humanhealth and the environment
Typical forms of silver nanomaterialsHerein, the term ‘‘silver nanomaterials’’ refers to anysilver-containing materials with enhanced activitydue to their nanoscale features In some cases,commercial products containing metallic silver nano-particles in the range of 5–50 nm or ionic silver aregiven the name ‘nanosilver’ (Panyala et al 2008).Silver nanoparticles are nanoscale clusters of metallicsilver atoms, Ag0, engineered for some practicalpurpose—most typically antimicrobial and sterileapplications
The most common method of producing silvernanoparticles is chemical reduction of a silver saltdissolved in water with a reducing compound such asNaBH4, citrate, glucose, hydrazine, and ascorbate(Gulrajani et al.2008; Martinez-Castanon et al.2009;Panacek et al.2006; Pillai and Kamat 2004) Strongreductants lead to small monodisperse particles,while generating larger sizes can be difficult tocontrol Weaker reductants produce slower reductionreactions, but the nanoparticles obtained tend to bemore polydisperse in size In order to generate silvernanoparticles with controlled sizes, a two-stepmethod is usually utilized In this method, nucleiparticles are prepared using a strong reducing agentand they are enlarged by a weak reducing agent(Schneider et al.1994; Shirtcliffe et al.1999) Sincereducing agents for silver nanoparticle synthesis areoften considered toxic or hazardous, the use of greensynthesis methods is becoming a priority (Panacek
et al 2006) A recent review of green synthesismethods for silver nanoparticles discussed the use ofpolysaccharides, polyphenols, Tollens agent, irradia-tion, biological reduction, and polyoxometalate(Sharma et al 2009)
Polysaccharides and polyphenols are typicallyused as capping agents during silver nanoparticlesynthesis, but they may also contribute to reduction
of silver ions through as yet poorly understoodmechanisms For polysaccharides, the reduction ofsilver may be linked to the oxidation of aldehydegroups to carboxylic acid groups (Manzi and van
Trang 4Halbeek 1999) Typical polysaccharides used are
glucose, starch, and heparin (Batabyal et al 2007;
Huang and Yang 2004; Manno et al 2008; Singh
et al 2009; Venediktov and Padokhin2008) In the
Tollens method, a silver ammoniacal solution is
reduced by an aldehyde forming silver nanoparticles
This method can be altered (i.e., ‘‘modified Tollens
method’’) by reducing Ag?using saccharides in the
presence of ammonia resulting in films with
nano-particles sizes ranging from 50 to 200 nm and silver
hydrosols ranging from 20 to 50 nm (Panacek et al
2006; Saito et al 2003; Yu and Yam 2004) Silver
nanoparticles can also be synthesized by irradiating
silver salts solutions containing reducing and capping
agents Different sources of irradiation have been
used such as laser, microwave, ionization radiation,
and radiolysis (Abid et al 2002; Ha et al.2006; Li
et al.2006; Long et al.2007; Mahapatra et al.2007;
Pillai and Kamat 2004; Sharma et al 2007, 2008;
Yanagihara et al 2001; Yin et al.2004; Zeng et al
2007)
Biological methods involve the production of
silver nanoparticles utilizing extracts from
bio-organ-isms as reductant, capping agents or both (Li et al
2007; Sanghi and Verma 2009) Such extracts can
include proteins, amino acids, polysaccharides, and
vitamins (Eby et al.2009; Sharma et al.2009) Plant
extracts such as apiin (a glucoside compound) and
leaf extract from magnolia, Persimmon, geranium,
and Pine leaf have also been used as reducing agents
of Ag? to produce silver nanoparticles (Kasthuri
et al.2009; Shankar et al.2003; Song and Kim2009)
Additionally, silver nanoparticles can be synthesized
by several microorganisms such as the bacterial
strains Bacillus licheniformis, K pneumonia, and
fungi strains such as Verticillium and Fusarium
oxysporum, Aspergillus flavus (Ahmad et al 2003;
Kalishwaralal et al 2008; Mokhtari et al 2009;
Mukherjee et al 2001; Senapati et al 2004;
Vig-neshwaran et al 2007) It is not clear if these
microbes are impacted (favorably or unfavorably) by
exposure to engineered forms of nano-scaled silver,
but their seemingly favorable interactions with silver
suggest resistance may be fairly widespread
Another procedure utilized to synthesize silver
nanoparticles is the solvated metal atom dispersion
(SMAD) method (Stoeva et al.2002) In this method,
a metal is co-vaporized with a solvent onto a liquid
nitrogen cooled surface, as liquid nitrogen is removed
the metal atoms and solvent warms causing theaggregation of metal atoms SMAD can be performed
in conjunction with digestive ripening, in this way thenanoparticles resulting from SMAD method arefurther refined by heating them in inert atmosphere
in the presence of selected ligands that encourage theparticles to reach a narrow size range As a result,monodisperse spherical particles are obtained (Sme-tana et al.2005,2008)
The use of silver ions as antimicrobial agents islimited by the solubility of silver ions in biologicaland environmental media containing Cl-, becauseAgCl has a very low solubility and rapidly precip-itates out of solution In some cases, silver salts arestabilized with hyperbranched polymers or dendri-mers that act as nanoreactors, wherein silver ionsare initially complexed with a specific moiety in thepolymeric structure and then reduced to form silvernanoparticles within the polymeric matrix (Fig.1)(Lesniak et al 2005; Zhang et al 2008) Dendri-mer–silver complexes prevent silver ions fromprecipitating and keep silver dispersed in the medialong enough to be delivered where it is desired(Balogh et al 2001; Lesniak et al 2005; Zhang
et al 2008) Silver ions can also be stabilized inzeolite channels (Fig 2) or deposited in activatedcarbon fibers (Inoue et al 2002; Ogden et al 1999;Pal et al 2009)
Composites of silver coatings over titanium ide nanoparticles are used in products such as babybottles and blood-clotting agents to produce antibac-terial activity (Yeo and Kang 2008) Other hybridsilver nanomaterials may include silver nanoparticlescoated onto polyurethane and silver–magnetite com-posite nanoparticles (Fe3O4@Ag); both of thesehybrids are utilized for water disinfection (Gong
diox-et al 2007; Jain and Pradeep 2005) One of thechallenges of using silver (or any) nanoparticles forwater treatment is recovering the particles after thetreatment process Silver–magnetite nanoparticlesoffer the potential advantage of being removed by amagnet, avoiding release to the environment, andmaking possible direct reuse without additionalseparation processes For example, in relatedresearch, magnetite particles proved effective forremoval of arsenic from water (Mayo et al 2007;Yavuz et al.2006) However, for silver materials, anadditional concern is controlling the release of metalions into the final produce water
Trang 5Evidence of silver toxicity in microbes
and higher organisms
Here, toxicity refers to any deleterious effects on an
organism upon exposure to silver Obviously, if the
practical intent is to disinfect or sterilize a specific
type of organism, then toxicity may be interpreted
as a positive outcome (e.g., antibacterial, antiviral,
etc.) However, if the same material exerts
unin-tended or undesired impacts to other organisms, then
such toxicity may be interpreted as a potential
hazard
Evidence of toxicity to bacteriaTable 1 presents a concise summary of silver nano-material antibacterial studies Kim et al reported that13.4-nm silver nanoparticles prepared by reduction ofsilver nitrate with sodium borohydride show mini-mum inhibitory concentration (MIC) against E colibelow 6.6 nM and above 33 nM for S aureus (Kim
et al 2007) In another study, 16-nm silver ticles generated by gas condensation were able tocompletely inhibit colony forming units (CFU)ability of E coli at 60 lg/mL (Raffi et al 2008)
nanopar-Fig 1 General formation
Terminal groups on the
surface are marked as
–CH2CH2–CO–Z Silver
ions (represented by M?)
can be pre-organize and
subsequently contained in
the form of solubilized and
stabilize, high-surface area
silver domains Redrawn
based on (Balogh et al.
2001 )
Fig 2 Silver ions
stabilized in zeolite
channels and ionic
exchange of silver ions with
other cations in the media.
Left picture shows zeolite
type A and right picture
shows zeolite type X.
Adapted from (Auerbach
2003 )
Trang 6Commercially available silver nanopowders at a
concentration of 300 lg/mL and SMAD-produced
silver nanoparticles at a concentration of 30 lg/mL
were able to reduce (after 10 min contact time)
colony forming units of E coli and S aureus from
2 9 104CFU/mL to 0 and \20, respectively (Smetana
et al.2008)
Additionally, MICs ranging from 13.5 to 1.69 lg/mL
were reported for bacterial strains such as S aureus
CCM 3953, Enterococcus faecalis CCM 4224, E coli
CCM 3954, and P aeruginosa CCM 3955, and for
strains isolated from human clinical material like
P aeruginosa, methicillin-susceptible S epidermidis,
methicillin-resistant S epidermidis, methicillin-resistant
S aureus, vancomycin-resistant Enterococcusfaecium, and K pneumonia when exposed to26-nm silver nanoparticles prepared by the reduc-tion of [Ag(NH3)2]? with D-maltose (Kvitek et al
2008)
Silver nanoparticles also produced 76% reduction
of B subtilis CFU after applying silver nanoparticleswith a size distribution from 14 to 710 nm in anaerosol form (Yoon et al.2008a,b) Likewise, silvernanoparticles are effective against E coli, S aureus,and L mesenteroides Also, 10-nm MyramistinÒstabilized silver nanoparticles inhibit growth of
E coli and S aureus at 2.5 lg/mL and L teroides at 5 lg/mL (Vertelov et al.2008)
mesen-Table 1 Bactericidal activity of nano-scaled silver and silver loaded in zeolite
Silver form Size data Bacterial strain Key aspects References
Silver nanoparticles/
nano-sized silver
powders
13.4 nm b E coli, S aureus Minimal inhibition concentration against
E coli was lower than 6.6 nM and higher than 33 nM for S aureus
Minimal inhibition concentration from 1.69 to 13.5 lg/mL
14.1–710 nmc B subtilis 76% CFU reduction by applying silver
nanoparticles aerosol on B subtilis aerosol
(Yoon et al 2008a ,
b ) Silver nanoparticles
or chloride ions
(Balogh et al 2001 )
Silver zeolite E coli CFU reduced by 7 log units in 5 min (Inoue et al 2002 ) Zeolite containing
silver and zinc
E coli Minimal bactericidal concentration of
78 lg/mL (as Ag?) for bacteria grown is Luria- Bertani broth
Note: Size measured byadynamic light scattering,bTEM images, andcscanning mobility particle sizer
Trang 7Dendrimer–silver nanocomposites have also been
proven effective antibacterials For example,
poly(amidoamine) dendrimer–silver composites have
been used against S aureus, P aeruginosa, E coli,
B subtilis, and K mobilis (Balogh et al.2001; Zhang
et al.2008) Additionally, silver ions loaded in zeolites
elicit antibacterial properties Two recent studies
demonstrated 7 log reduction in CFUs for E coli,
from an initial concentration of 107CFU/cm3, after
5 min of contact time with 333.3 lg/mL of Ag-loaded
zeolite (Inoue et al.2002), and MICs of 78 lgAg?/mLfor E coli and 39 lgAg?/mL for S aureus and
P aeruginosa, plus some bactericidal activity againstListeria monocytogenes (Cowan et al.2003)
Evidence of toxicity to other microorganismsSilver nanoparticles also inactivate fungi, virii, andalgae (Table2) For example, silver nanoparticles ofsizes ranging from 1.4 to 7.1 nm and stabilized in
Table 2 Summary of silver nanoparticles toxicity to other microorganisms
Strain Silver nanoparticles Size (nm) Key aspects Reference
1.4–7.1 b Formation of inhibition zones
around silver nanoparticles inoculated spots in agar plates
(Zhang et al 2008 )
S cerevisiae MyramistinÒstabilized
silver nanoparticles
10 a MIC were found to be 5 mg/L (Vertelov et al 2008 )
T mentagrophytes Silver nanoparticles 3 b IC80between 1 and 4 mg/L (Kim et al 2008a , b )
C Albanicas Silver nanoparticles 3 b Silver nanoparticles inhibited
micelial formation, which is responsible for pathogenicity
(Kim et al 2008a , b )
Silver nanoparticles 3b Antifungal activity may be exerted
by cell membrane structure disruption and inhibition of normal budding process
(Kim et al 2009a , b )
Silver nanoparticles coated on plastic catheters
3–18b Catheter coated with silver
nanoparticles inhibited growth and biofilm formation.
1.4–7.1b Formation of inhibition zones
around silver nanoparticles inoculated spots in agar plates
(Zhang et al 2008 )
Viruses
Hepatitis B virus Silver nanoparticles 10b Inhibition of virus replication (Lu et al 2008 )
HIV-1 Silver nanoparticles 16.19 ± 8.68b Only 1–10 nm nanoparticles
attached to virus restraining virus from attaching to host cells.
(Elechiguerra et al 2005 )
Syncitial virus Silver nanoparticles 44% inhibition of Syncitial virus
infection
(Sun et al 2008 ) Algae
C reinhardtii Silver nanoparticles 10–200a EC50for the photosynthetic yield
was found in 0.35 mg/L of total silver content after 1 h of exposure
(Navarro et al 2008 )
Note: Size measured byadynamic light scattering andbTEM images
Trang 8hyperbranched polymers, and silver nanoparticles
stabilized with MyramistinÒ (size 10 nm) inhibited
the growth of A niger (Tomsic et al.2009; Vertelov
et al.2008; Zhang et al.2008); the same MyramistinÒ
stabilized particles were found toxic toward S
cere-visiae showing a MIC of 5 mg/L Further, 3-nm silver
nanoparticles showed IC80values from 1 to 4 lg/mL
against T mentagrophytes and 2 to 4 lg/mL for
C albanicans (Kim et al.2008a,b); while in other study,
it was reported that the antifungal activity of silver
nanoparticles against C albanicans could be exerted
by cell membrane structure disruption leading to
reproduction inhibition (Kim et al 2009a, b)
Addi-tionally, silver nanoparticles (sizes ranging from 3 to
18 nm) coated in catheters were able to inhibit growth
of C albicans Although, in this case, no analysis of
the antifungal molecular mechanism was done, the
authors speculate that silver ions (Ag?) released from
the matrix were the antifungal agents (Kim et al
2008a, b, 2009a, b; Roe et al 2008) Furthermore,
silver nanoparticles suppress yeast growth and show
MIC between 6.6 and 13.2 nM (Kim et al 2007)
Silver nanoparticles of sizes from 1.4 to 7.1 nm and
stabilized in hyperbranched polymers
(HPAMAM-N(CH3)2/AgNPs composite) inhibit P citrinum
growth (Zhang et al 2008) Although information
about toxicity for algae is limited, silver nanoparticles
reduced the photosynthetic yield of Chlamydomonas
reinhardtii; in this case, the observed toxicity was
attributed to Ag?ions (Navarro et al.2008)
Evidence of virus inactivation is also reported in
literature For example, silver nanoparticles of 10 nm
are able to inhibit hepatitis B virus replication (Lu
et al 2008) Additionally, PVP-coated silver particles in the range 1–10 nm attach to HIV-1 virus,inhibiting the virus from attaching to host cells(Elechiguerra et al.2005) In other study, PVP-coatedsilver nanoparticles reduced respiratory syncytialvirus infection by 44% (Sun et al.2008) Polysulfoneultrafiltration membranes impregnated with silvernanoparticles of sizes ranging from 1 to 70 nmshowed enhanced virus removal, thus improvingwater disinfection via low pressure membrane filtra-tion (Zodrow et al 2009)
nano-Evidence of toxicity for mammalian cellsSignificant evidence has been reported in relation tothe toxicity of silver nanoparticles to higher organ-isms It has been shown toxic to fish such as zebrafish(Asharani et al.2008; Yeo and Kang2008; Yeo andYoon 2009), Diptera species such as Drosophilamelanogaster, known asfruit fly (Ahamed et al.2010)and different mammalian cell lines of mice (Brayd-ich-Stolle et al.2005; Hussain et al.2005), rats (Kim
et al 2008a, b; Sung et al 2008), and also humans(Asharani et al 2009; Braydich-Stolle et al 2005;Hsin et al 2008; Hussain et al 2005) This reviewpresents only a few, brief examples of silver nano-material toxicity for mammalian cells (Table 3).More detailed, focused reviews on this topic areavailable elsewhere (Chen and Schluesener 2008;Panyala et al.2008)
Table 3 Evidence of nano-scaled silver toxicity for mammalian cells
Rat lung cells Reduction in lung function and inflammatory lesions (Sung et al 2008 )
Sprague-Dawley rats Silver nanoparticles accumulation in olfactory bulb
and subsequent translocation to the brain
(Kim et al 2008a , b ) Mouse stem cells Cell leakage and reduction of mitochondrial function (Braydich-Stolle et al 2005 ) Rat liver cells Cell leakage and reduction of mitochondrial function (Hussain et al 2005 ) Human fibrosarcoma
and human skin/carcinoma
Oxidative stress Low doses produced apoptosis and higher dose necrosis
(Arora et al 2008 ) Mouse fibroblast 50 lg/mL induced apoptosis to 43.4% of cells (Arora et al 2009 )
Human colon cancer 100 lg/mL produced necrosis to 40.2% of cells
Human glioblastoma Silver nanoparticles were found cytotoxic,
genotoxic and antiproliferative
(Asharani et al 2009 ) Human fibroblast Silver nanoparticles were found cytotoxic,
genotoxic and antiproliferative
(Asharani et al 2009 )
Trang 9Evidence of silver nanoparticle toxicity for
mam-malian cells was presented in the in vivo studies
performed by Sung et al (2008) and Kim et al
(2008a,b) In the former, a 90-day inhalation study in
rats showed that silver nanoparticles reduce lung
function and produce inflammatory lesions in the
lungs In the later, silver nanoparticles accumulated
in the olfactory bulbs of Sprague-Dawley rats and
also accumulated in the brain
Evidence from in vitro studies is also available in the
literature For example, silver nanoparticles have been
shown to reduce mitochondrial function and to
increase membrane leakage of mouse spermatogonial
stem cell and rat liver cells (Braydich-Stolle et al.2005;
Hussain et al 2005) Studies performed on human
fibrosarcoma and human skin/carcinoma cells with
silver nanoparticles used in a topical antimicrobial
agent concluded that in the presence of the
nanopar-ticles the cellular levels of glutathione are reduced,
indicating oxidative stress, that results in cellular
damage and lipid peroxidation (Arora et al.2008)
However, in the study performed by Arora et al the
dose required to induce apoptosis (0.78–1.56 lg/mL)
was much smaller than that required to produce
necrosis (12.5 lg/mL) in both cell types Therefore,
the authors concluded that, after the required in vivo
studies, it would be possible to define a safe range for
the application of silver nanoparticles as a topical
antimicrobial agent Similar differences of the required
concentration to cause apoptosis or necrosis were
found in a second study by the same authors in mouse
fibroblasts and liver cells (Arora et al.2009) In this
second article, it was suggested that although silver
nanoparticles may enter into the cells, the cellular
antioxidant mechanisms would limit oxidative stress
Mechanistic studies of silver nanoparticle toxicity
in mammalian cells have considered mouse fibroblast
and human colon cancer cells (Hsin et al 2008) In
this study, silver nanoparticle doses of 50 lg/mL
induced apoptosis to 43.4% of fibroblast cells, while
100 lg/mL produced necrosis to 40.2% of the cancer
cells The authors concluded that the apoptotic
mechanisms in fibrosblast cells are a mitochondrial
mediated pathway including the generation of ROS in
the cell, which activate the apoptosis regulators JNK
and p53 proteins inducing protein Bax to migrate to
the surface of the mitochondria That subsequently
induces cytochrome C release from mitochondria and
cleavage of PARP Additionally, in a study done by
Asharani, a possible mechanism of toxicity to humancells was proposed (Asharani et al 2009) Silvernanoparticles would affect the mitochondrial respira-tory chain, causing ROS generation and affecting theproduction of ATP, which subsequently leads to DNAdamage In this study, the authors also concluded that
‘‘even and small dose of Ag-NP (silver nanoparticles)has the potential to cause toxicity’’ and that silvernanoparticles are cytotoxic, genotoxic, and antipro-liferative, being as toxic for human glioblastoma asfor normal human fibroblasts cells
Mechanisms of silver’s antibacterial propertiesAlthough the mechanisms behind the activity ofnano-scaled silver on bacteria are not yet fullyelucidated, the three most common mechanisms oftoxicity proposed to date are: (1) uptake of free silverions followed by disruption of ATP production andDNA replication, (2) silver nanoparticle and silverion generation of ROS, and (3) silver nanoparticledirect damage to cell membranes The variousobserved and hypothesized interactions betweensilver nanomaterials and bacteria cells are conceptu-ally illustrated in Fig.3
Fig 3 Diagram summarizing nano-scaled silver interaction with bacterial cells Nano-scaled silver may (1) release silver ions and generate ROS; (2) interact with membrane proteins affecting their correct function; (3) accumulate in the cell membrane affecting membrane permeability; and (4) enter into the cell where it can generate ROS, release silver ions, and affect DNA Generated ROS may also affect DNA, cell membrane, and membrane proteins, and silver ion release will likely affect DNA and membrane proteins Similar pictures have been published in (Damm et al 2008 ; Neal 2008 )
Trang 10Free silver ion uptake
Silver nanoparticles have been reported to dissolve
generating silver ions and it is thought that in vivo this
release would be product of reactions of silver
nano-particles with H2O2(Asharani et al.2009) Asharani has
proposed the following reaction as a possible
mecha-nism for silver nanoparticle oxidative dissolution
2Agþ H2O2þ 2Hþ! 2Agþþ 2H2O E0¼ 0:17 V:
Asharani suggests that in eukaryotic cells this
reaction could occur in the mitochondria, where
exists an important concentration of H? Similarly,
we hypothesized that a similar mechanism could
occur in the bacterial cell membrane where proton
motive force takes place
Another possible mechanism for the oxidative
dissolution of silver nanoparticles has been reported
by Choi et al., in this case silver is oxidized in the
presence of oxygen Choi et al speculated that the
observed changed of color of their silver
nanoparti-cles suspensions, over a week period, would be
attributed to this mechanism
4Agþ O2þ 2H2O$ 4Agþþ 4OH:
The amount of free silver ion measured in this case
was approximately 2.2% of the total silver content in
the silver nanoparticle suspension (Choi et al.2008)
In other article, a 0.1% content of the total silver in
partially oxidized silver nanoparticles suspensions
was attributed to silver ions (Lok et al.2007)
Ionic silver has known antibacterial properties;
thus, it is expected that eluted ions from silver
nanoparticles are responsible for at least a part of
their antibacterial properties At sub-micromolar
concentrations, Ag? interacts with enzymes of the
respiratory chain reaction such as NADH
dehydro-genase resulting in the uncoupling of respiration from
ATP synthesis Silver ions also bind with transport
proteins leading to proton leakage, inducing collapse
of the proton motive force (Dibrov et al.2002; Holt
and Bard 2005; Lok et al.2006) Silver inhibits the
uptake of phosphate and causes the efflux of
intra-cellular phosphate (Schreurs and Rosenberg 1982)
The interaction with respiratory and transport
pro-teins is due to the high affinity of Ag? with thiol
groups present in the cysteine residues of those
proteins (Holt and Bard 2005; Liau et al 1997;Petering1976) Additionally, it has been reported that
Ag? increases DNA mutation frequencies duringpolymerase chain reactions (Yang et al.2009).Bacterial cells exposed to milli-molar Ag? dosessuffer morphological changes such as cytoplasm shrink-age and detachment of cell wall membrane, DNAcondensation and localization in an electron-light region
in the center of the cell, and cell membrane degradationallowing leakage of intracellular contents (Feng et al
2000; Jung et al.2008) Physiological changes occurtogether with the morphological changes Bacterial cellsenter an active, but non-culturable state in whichphysiological levels can be measured but bacteria arenot able to growth and replicate
Several studies have linked the toxicity of silvernanoparticles to the release of silver ions For example,Smetana et al observed that silver ions eroded fromhigh-surface area silver powders prepared by SMADmethod interacted and destroyed bacterial cells (Sme-tana et al 2008) In the same study, a secondpreparation of silver nanoparticles using water-solubleligands was used to obtain silver nanoparticles withhigher surface area to improve their antibacterialefficacy However, the second preparation of silvernanoparticles showed lower toxicity toward bacterialcells than uncoated powders, suggesting the ligandsprevented silver ion erosion; thus, diminishing theresulting toxicity Another possible reason for thisresult is that the surface coatings prevented adhesion
of silver nanoparticles to the bacterial cell surface, butthe authors did not explore this option
Hwang et al observed that Ag?induced the sameeffect in bioluminescence bacteria sensitive to mem-brane protein damage and slightly less effect in a strainsensitive to superoxides compared to silver nanopar-ticles (Hwang et al.2008) The authors suggested thatsilver nanoparticles produce silver ions that moveinside the cell producing ROS through redox reactionswith oxygen In other research, bacterial activity ofactivated carbon fiber supported silver was attributed
to the synergistic action of silver ions, superoxides,and hydrogen peroxide (Le Pape et al.2004).Generation of reactive oxygen speciesReactive oxygen species (ROS) are natural byprod-ucts of the metabolism of respiring organisms While