2013

http://informahealthcare.com/txmISSN: 1537-6516 (print), 1537-6524 (electronic)

Toxicol Mech Methods, 2013; 23(3): 161–167! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/15376516.2013.764950

RESEARCH ARTICLE

Toxic effects of repeated oral exposure of silver nanoparticles on smallintestine mucosa of mice

Brigesh Shahare*, Madhu Yashpal, and Gajendra Singh

Department of Anatomy, Electron Microscope Facility, Institute of Medical Sciences, Banaras Hindu University, Varanasi, Uttar Pradesh, India

Abstract

As the use of silver nanoparticles (AgNPs) is increasing fast in industry, food, medicines, etc.,exposure to AgNPs is increasing in quantity day by day. So, it is imperative to know the adverseeffects of AgNPs in man. In this study, we selected mice as an animal model and observed theeffect of AgNPs on small intestinal mucosa. AgNPs ranging from 3 to 20 nm were administeredorally at a dose of 5, 10, 15 and 20 mg/kg body weight to the Swiss-albino male mice for 21 d.There was a significant decrease (p50.05) in the body weight of mice in all the AgNPs-treatedgroups. Mice treated at a dose of 10 mg/kg showed the maximum weight loss. Effects werenoted by using light microscopy as well as transmission electron microscopy. It was foundthat AgNPs damage the epithelial cell microvilli as well as intestinal glands. It may behypothesized that loss of microvilli reduced absorptive capacity of intestinal epithelium andhence weight loss.

Keywords

Microvilli, mitotic figure, silver nanoparticles,transmission electron microscope

History

Received 4 November 2012Revised 3 January 2013Accepted 7 January 2013Published online 19 February 2013

Introduction

Since ancient civilizations, man has used silver in medicine,

eating utensils, ornaments, coins, clothes and as a disinfectant

for water and for treating human wounds (Castellano et al.,

2007; Richard et al., 2002). Now a day’s, new forms of silver

having particle size less than 100 nm, i.e. silver nanoparticles

(AgNPs) comes into focus due to its anti-bacterial activity

(Alt et al., 2004; Martinez-Gutierrez et al., 2010; Nersisyan

et al., 2003; Panacek et al., 2006; Shrivastava et al., 2007;

Sondi & Salopek-Sondi, 2004; Xing et al., 2011), anti-viral

and anti-tumor activity (Huang et al., 2011) as well as anti-

platelet property (Shrivastava et al., 2009). AgNPs are use in

daily life such as in medicine, toothpastes, paints, washing

machines, food materials, water purification and shampoos.

Due to these widespread uses, AgNPs released into the

environment are bound to spread either in the air, water or

soil. Already, It has been shown that AgNPs can enter the

human body through several ports, and there have been

several excellent reviews regarding intestinal uptake of

particles (Florence & Hussain, 2001; Hussain et al., 2001;

Jeong et al., 2010). There are reasons to be sure that exposure

of gastrointestinal tract to the AgNPs must be taking place

consciously or unconsciously. Nanomaterials can be ingested

directly via water, food, cosmetics, drugs, drug delivery

devices, etc. (Oberdorster et al., 2005; Peters et al., 1997).

Beside its widespread use, toxicity study of AgNPs needs

further investigation. Present work was conducted to know the

effect of AgNPs on small intestine using light microscopy and

transmission electron microscope (TEM).

Methods

Animal model for in vivo study

A total of 50, 8–10 weeks-old Swiss albino male mice were

obtained from animal house of the Department of Anatomy,

Institute of Medical Sciences, Banaras Hindu University,

Varanasi, India. After being checked for infections of any kind

for 1 week, all 50 mice were weighed. Animals with an

average weight of 23� 5 g were used for the study.

Commercial mice food (Pashu Aahar Kendra, Varanasi,

India) along with water was given ad libitum. Mice were

housed individually in plastic cages with a bed lined with

absorbent material, i.e. rice husk providing nesting material.

The cages were placed in a conventional room and bedding

changed daily. The room was air conditioned at 23 �C and

40–60% humidity with a light/dark cycle of 12 h each. All the

work performed on animals was in accordance with and

approved by the Animal Ethics Committee of Institute of

Medical Sciences, Banaras Hindu University (Varanasi,

India). The animals were treated with utmost humane care

and all aseptic precautions were observed.

Address for correspondence: Prof. Gajendra Singh, Department ofAnatomy, Electron Microscope Facility, Institute of MedicalSciences, Banaras Hindu University, Varanasi 221005, Uttar Pradesh,India. Tel: (O) þ91 542 – 2367568; (R) þ91 542 – 2369341. Fax: þ91542 – 2368174. Mob: þ91 9415223588. E-mail: gajendrasinghims@gmail.com

*Present address: Department of Anatomy, Gajara Raja Medical College,Gwalior, Madhya Pradesh, India.

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

Synthesis of AgNPs

AgNPs were synthesized essentially as described by

Shrivastava et al. (2007). Briefly, AgNO3 (0.017 g) was

dissolved in deionized water (100 ml) along with sodium

hydroxide (0.01 M) to form a solution of stable soluble

complex of silver ions. During this process, liquid ammonia

(30%) was added drop wise. Further, a mixture of D-glucose

(0.01 M) and hydrazine (0.01 M) were also added to the

solution of silver ions with continuous stirring. This ensures the

complete reduction of the silver ions at a final concentration of

0.005 M. The pH of the solution was adjusted to 7.4 with citric

acid (1 M). The brown solutions AgNPs were stored in closed

glass vials at 4 �C for future experiments. Before each

experiment, the solution that contains nanoparticles was

sonicated (Labsonic 2000, B. Braun, Apeldoorn, The

Netherlands) for about 2 min and passed through filters of

0.2mm pore size (Sartorius) in order to remove solid insoluble,

larger nanoparticles or particle agglomerates, if any.

Characterization of AgNPs

The size, morphology and dispersal of AgNPs were

characterized using a TEM model Technai 12 G2, FEI of

the Department of Anatomy, BHU, Varanasi. For TEM, the

sample were prepared by placing a drop of AgNPs on copper

grids (TAAB, England, UK) coated with 2% Collodion in

Amyl acetate (TAAB, England, UK) and subsequently dried

in air, before transferring it to the microscope operated at an

accelerated voltage of 120 kV.

Treatment of animals with AgNPs

Six to eight week old mice were identified by an ‘‘Ear Notch

Code’’ and subsequently divided into five groups (n¼ 5 in

each group). Each group was treated with or without AgNP

solution as follows: first group (Control) received deionised

water as vehicle; second group (Group A) received 5 mg/kg

body weight (b.w.)/d AgNPs solution; third group (Group B)

received 10 mg/kg b.w./d AgNPs solution; fourth group

(Group C) received 15 mg/kg b.w./d AgNPs solution and

fifth group (Group D) received 20 mg/kg b.w./d AgNPs

solution. All mice were exposed to oral gabbage administra-

tion of vehicle and AgNP solution for 21 d. Body weight was

recorded weekly i.e. on day 0, 7, 14 and 21. Each mouse from

the experimental sets of study was carefully observed for any

signs of toxicity or ill health throughout the experiment and

was sacrificed on day 21 of AgNP treatment.

Collection of organ from mice

Body weight of all animals was recorded before killing. All

animals were sacrificed by the cervical dislocation.

Abdominal cavities of mice were cut open by a midline

abdominal incision to collect small intestine.

Histopathology of small intestine

Small intestine was fixed in aqueous Bouin’s fluid (Bouin,

1897) for histological studies. Thereafter, the organs were

embedded in paraffin, stained with hematoxylin and eosin,

and examined under light microscopy. Observations

were made on a binocular microscope. The results were

recorded using a digital camera system attached to

microscope.

TEM of AgNPs treated cells

Different intestinal samples, either with or without nanopar-

ticle pretreatment, were fixed in Karnovsky’s fixative fol-

lowed by post-fixation in osmium tetraoxide. The tissues were

dehydrated through an ascending series of acetone concen-

trations, cleared in toluene and embedded in resin (Araldite

CY212) to prepare the blocks for TEM. Ultra-thin sections

(70 nm thick) were cut with an ultra-microtome (Leica EM

UC6, Leica Microsystems, Wetzlar, Germany). Samples were

mounted on Formvar-coated grids followed by staining with

uranyl acetate and lead citrate, and examined under a

Technai-12 (FEI, Eindhoven, The Netherlands) electron

microscope equipped with SIS Mega View III CCD camera

(FEI, Eindhoven, The Netherlands) at 120 KV. Measurements

were done using AnalySIS software (SIS, Muenster,

Germany).

Statistical analysis

The experiment was terminated at the end of day 21 of

AgNP’s treatment and repeated three times to check the

reproducibility of the results obtained in the first experiment.

The data represented is the mean�SEM of one experiment.

All the data were analyzed by one-way analysis of variance

followed by post-hoc test (Student Newman Keul’s test). The

level of significance was tested at p50.05 and confidence

limit 95% using SPSS software (Version 16.0; DST Centre for

Interdisciplinary Mathematical Sciences, Faculty of Science,

Banaras Hindu University, Varanasi, India).

Results

Characterization of AgNPs

TEM imaging of the AgNPs were performed to assess the

range of primary particle size, obtain the size distribution and

observe the general morphology of the particles. The size of

particles ranged from 3 to 20 nm (average diameter of AgNP

in the solution¼ 10.15 nm) (Figure 1) and the shape of the

particles, in general, was either oval or circular (Figure 2).

Body weight of mice and food consumption

The most important finding observed during the course of

treatment was decrease in the body weight of mice. A

significant decrease in the body weight of mice was observed

in all the treated groups compared to the control on day 14

and 21 during the experiment (Figure 3). Among treated

groups, there was no significant change in the body weight

for the first 7 d, i.e. from day 0. However, after day 7, a

significant decrease in the body weight of mice was observed

on day 14 and 21 of the experiment. It is noteworthy that,

despite decrease in the body weight of mice in all AgNPs

treated groups, no significant difference was observed in the

food consumption (food supplied after due measurement) of

mice between the treated and the control groups.

The maximum decrease in the body weight was observed

in the group B which received AgNPs at the dose of 10 mg/kg

body weight for 21 d. Hence, this dose was considered as an

162 B. Shahare et al. Toxicol Mech Methods, 2013; 23(3): 161–167

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

optimum dose and further description is on mice treated with

AgNPs at a dose of 10 mg/kg of body weight of mice for 21 d.

Histopathology of small intestine

Histological observation under light microscope revealed that

in control mice the villi of small intestine were lined by simple

columnar epithelium containing the tall absorptive cells with

striated border due to microvilli, sometimes called terminal

bar (Figure 4A). A few goblet cells were also observed

between the columnar epithelial cells. Lamina propria was

present in the core of each villus (Figure 4A). Intestinal glands

were observed in the lamina propria with mitotic figures

(Figure 5A). After oral administration of AgNPs, the following

changes were observed in the small intestine.

(a) The striated border (terminal bar) was not demarkable as

the microvilli were lost (damaged) (Figure 4B).

(b) Increased number of inflammatory cells in the lamina

propria of each villus leading to the widening of lamina

propria zone (Figure 4B).

(c) Increased number of mitotic figure in the intestinal

glands (Figure 5B and C). This increased number of

mitotic figure indicates that AgNPs damages the intes-

tinal epithelial cell and to replace these damaged

absorptive cells, stem cells in intestinal glands starts

proliferating and show high mitotic activity.

TEM of small intestine

In control mice, striated border of enterocytes shows layer of

densely packed microvilli (Figure 6A) which are nothing but

the cylindrical protrusion of the apical cytoplasm to amplify

area for absorption covered by plasmalema (cell wall).

However, after oral administration of AgNPs in mice, the

Figure 1. Histogram showing average diam-eter and percentage of different sizes ofAgNPs. The distribution pattern shows mostof the particles in the size range of 5–10 nm.

Figure 2. Transmission electron photomicrograph of AgNPs showing circular and mono-dispersed particles (Scale bar-200 nm).

DOI: 10.3109/15376516.2013.764950 Effects of silver nanoparticles on small intestine mucosa of mice 163

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

microvilli were totally disrupted. A few distorted one were still

present in the area with shrunken lumen, which showed bead-

like swelling probably because of AgNPs inside lumen. Small

fragments of microvilli could be seen scattered in the area, i.e.

lumen of intestine. AgNPs were present in various strata of

small intestine including broken microvilli (Figure 6B).

Discussion

In the present work, we have attempted to investigate the

adverse effects of repeated sustained oral exposure of AgNPs

on the mucosa of the small intestine in mice. Intestine is lined

by simple columnar epithelium. On the luminal aspect of each

epithelial cell, microvilli were projected which are nothing

but the cytoplasmic extension of enterocytes. These microvilli

give the border a striated appearance in light microscopy,

which is also known as ‘‘terminal bar’’.

In the present study, we observed that after administration

of AgNPs, this terminal bar got lost which clearly indicated

the damage to the microvilli. TEM also revealed that these

microvilli were completely disrupted and broken, which

indicates that AgNPs can cross the protective barrier of the

small intestine i.e. the intestinal mucins and glycoproteins and

cause impairment to the microvilli.

From the above histological finding, we can explain the

significant decrease in body weight of mice in AgNPs treated

mice. The main function of small intestine is absorption. Due

to the loss of microvilli of enterocytes, the absorptive surface

area of the intestine was markedly reduced which led to

decrease in the absorption of nutrient materials resulting in a

reduction in the body weight of mice.

There are certain evidences that after subcutaneous

injection, AgNPs are translocated to the blood circulation

and distributed throughout the main organs, especially in the

kidney, liver, spleen, brain and lung in the form of particles

(Tang et al., 2009). Tang et al. postulates that AgNPs crossed

the BBB and by transcytosis of capillary endothelial cells

these particles get accumulated in the brain micro-vessels

vascular endothelial cells of rat (Tang et al., 2010). They also

proposed that AgNPs induce degenerative changes in the

some endothelial cells, which lead to the loosening of tight

junction between the endothelial cells and AgNPs cross

through these crevices. In our study, we observed the

structural changes in the enterocytes, i.e. loss of microvilli,

which may give passage to AgNPs for entering into the

intestinal wall and then hence into the portal circulation and

systemic circulation.

Ultra-structural studies have shown that activated platelets,

when exposed to AgNPs in a concentration-dependent

manner, lost their characteristic well-developed hyaloplasmic

processes, pseudopods (Shrivastava et al., 2009). In addition,

AgNPs were also found to be accumulated within platelet

granules. After 28 d of repeated oral administration of AgNPs,

a dose-dependent increased accumulation of AgNPs was also

observed in the lamina propria in both the small and large

intestine of Sprague–Dawley rats (Jeong et al., 2010). Jeong

et al. (2010) also reported frequent cell shedding at the tip of

the villi in intestine. Dose-dependent increase in the pigmen-

tation of the villi observed after 90 days oral exposure of

AgNPs, which was apparent treatment related effects (Kim

et al., 2010). More recently, Loeschner et al. (2011) also

reported presence of silver granules in the intestinal system of

rats after 28 d repeated oral exposure of AgNPs or to silver

acetate to rats (Loeschner et al., 2011). Similarly, in the

present study, AgNPs were present in the various strata of

small intestine including broken microvilli. A few distorted

Figure 3. Histogram representing change in the body weight in control and AgNP treated groups of mice. Control group received deionised watera vehicle; Group A received 5 mg/kg body weight (b.w.)/d AgNPs solution; Group B received 10 mg/kg b.w./d AgNPs solution; Group C received15 mg/kg b.w./d AgNPs solution and Group D received 20 mg/kg b.w./d AgNP solution. Values are represented as mean� SEM. *Denotes the level ofsignificance (p50.05) from the control group.

164 B. Shahare et al. Toxicol Mech Methods, 2013; 23(3): 161–167

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

microvilli were still present in the area with shrunken lumen,

which showed bead-like swelling probably because of AgNPs

inside lumen. Furthermore, in AgNPs treated mice, the

microvilli on the intestinal absorptive cells were found to be

badly damaged and broken (Figure 4B). It is possible that the

nanoparticles are taken up across the intestinal barrier since

particles with diameters less than 1 mm are particularly

susceptible to absorption by the intestinal lymphatic system

(Florence et al., 1997).

We may speculate that AgNPs somehow interact with the

structural elements of the microvillus of the intestinal

absorptive cells causing structural changes, and finally

destruction of the microvilli. As evident from Figure 2(B),

the terminal bar was absent in AgNPs treated intestinal cells

and could be made out at light microscopy level with 1000X

magnification. This view corroborates with Sondi & Salopek-

Sondi (2004).

Sondi & Salopek-Sondi (2004) reported that AgNPs

treated E. coli show significant changes in bacterial mem-

branes. They also reported the formation of ‘‘pits’’ on the

surfaces of the bacterial cell wall and accumulation of AgNPs

in the bacterial membrane. A similar effect was also reported

in E. coli bacteria treated with highly reactive metal oxide

nanoparticles (Stoimenov et al., 2002). More recently,

Shrivastava et al. (2007) also reported AgNPs managing to

enter the gram-negative bacteria by making perforations in

the membrane and thus resulting in cell lysis. A bacterial

membrane with pits on the surfaces shows a considerable

increase in permeability, leaving the bacterial cells incompe-

tent of regulating transport through the plasma membrane

and, finally, resulting in cell death (Sondi & Salopek-Sondi,

2004). Sondi & Salopek-Sondi, (2004) hypothesized that the

change in membrane permeability is probably due to

Figure 5. Photomicrographs of cross-sections of small intestine ofcontrol (A) and AgNPs administered mice (B, C). (A) Showing intestinalgland cells with few numbers of mitotic figures (circles). Note thenarrow lumen of each intestinal gland. Inset showing a mitotic figure.H&E� 400. (B) Showing a large number of mitotic figures (circles)along with dilated intestinal glands (arrowheads). H&E� 400. (C) Sameas B. Inset showing mitotic figure at higher magnification. H&E� 400.

Figure 4. Photomicrographs of cross-sections of small intestine showingintestinal villi in control (A) and AgNPs administered mice (B). (A) Inthe control small intestine, each villous is lined by simple columnarepithelium containing absorptive cells with uniform and continuousstriated border of microvilli, i.e. the terminal bar (arrow) and laminapropria (asterisk) in the core of each villous. Inset showing terminal bar(arrowhead). H&E� 400. (B) Showing disrupted border of microvilli(arrow). Note the widening of lamina propria zone (asterisk). Insetshowing the loss of terminal bar (arrowhead). H&E� 400.

DOI: 10.3109/15376516.2013.764950 Effects of silver nanoparticles on small intestine mucosa of mice 165

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

progressive release of lipopolysaccharide molecules and

membrane proteins. It is well known that the outer membrane

of gram-negative bacterial cells is primarily made of tightly

packed lipopolysaccharide molecules, which impart an effect-

ive permeability barrier (Nikado et al., 1985; Raetz, 1990;

Silva et al., 1973).

The above supposition of Sondi & Salopek-Sondi (2004)

may possibly explain the destruction of hyaloplasmic

processes (pseudopods) of AgNPs-treated platelets

(Shrivastava et al., 2009) and microvilli of the intestinal

absorptive cells in mice in the present study. These AgNPs

also pass through a thick layer of glycocalyx, which is a

polysaccharide, covers the microvilli of the intestinal absorp-

tive cells and platelets too. The thick layer of glycocalyx

imparts protective function to microvilli from any corrosive

material. A similar layer of glycocalyx is also present on the

outer surface of the platelets.

At this point, we may speculate that AgNPs somehow

interact with this protective barrier and other structural

elements of the microvilli of the intestinal absorptive cells

causing structural changes, resulting in the membrane

permeability and finally destruction of the microvilli.

Subsequently, the epithelial cells of the gastro-intestinal

tract get destroyed. Therefore, it is conceivable that in order to

replace these damaged enterocytes, the neck cells of the crypt

of Lieberkuhn start mitosis at a faster rate. Hence, the AgNPs

administrated mice show larger number of mitotic figures as

compared to the control animals. If the normal cells do not

replace the damaged enterocytes, the site may result in the

formation of ulcer.

Furthermore, it could also be hypothesized that the AgNPs

may possibly interfere with the cell cycle kinetics without

inducing cell death. Because small intestine is an organ,

which affords a generous population of continuous dividing

cells, it may also be speculated that when AgNPs breach the

protective barrier of the intestinal cells, somehow these

nanoparticles trigger the regulators of the cell cycle and the

process of cell division is accelerated and cellular uptake of

AgNPs will be reduced over time due to augmented cell

division. Several studies have shown that the uptake of the

nanoparticles by cells is influenced by different phases of

their cell cycle and cell division can dilute the concentration

of nanoparticles in the cell population (Errington et al., 2010;

Kim et al., 2012; Summers, 2012; Summers et al., 2011).

Moreover, it may also be speculated that AgNPs affect the

cellular response at the molecular level and may increase the

expression of certain genes related to cell cycle pathway and

cause abnormal cell proliferation (Kawata et al., 2009).

Conclusion

Based on our results, it is postulated that AgNPs somehow

interact with the protective layer of the glycocalyx and other

structural elements of the microvilli of the intestinal absorp-

tive cells causing structural changes, resulting in the alteration

of membrane permeability and finally destruction of the

microvilli. Subsequently, the epithelial cells of gastro-

intestinal tract get destroyed and are the reason for the

decrease in body weight of mice. We thus conclude that

AgNPs destroy the mucosa of small intestine and impede its

function.

Declaration of interest

Authors have no conflict of interest. Madhu Yashpal is thankful toUniversity Grants Commission, New Delhi, India for financial support inthe form of Dr D.S. Kothari Postdoctoral Fellowship [F.4-2/2006 (B SR)/13-455/2011 (BSR)].

References

Alt V, Bechert T, Steinrucke P, et al. (2004). An in vitro assessment ofthe antibacterial properties and cytotoxicity of nanoparticulate silverbone cement. Biomaterials 25:4383–91.

Bouin P. (1897). Phenomenes Cytologique anoramaux dans.I’Histogenes, Nancy.

Castellano JJ, Shafii SM, Ko F, et al. (2007). Comparative evaluation ofsilver-containing antimicrobial dressings and drugs. Int wound J4:114–22.

Errington RJ, Brown MR, Silvestre OF, et al. (2010). Single cellnanoparticle tracking to model cell cycle dynamics and compartmen-tal inheritance. Cell Cycle 9:121–30.

Figure 6. Transmission electron photomicrograph of small intestine ofcontrol (A) and AgNPs administered mice (B). (A) The striated border ofan enterocyte showing a layer of densely packed microvilli (arrow)(Scale bar-500 nm). (B) Showing damaged and broken microvilli (arrow)due to prolonged exposure of AgNPs. Note the fragments of themicrovilli are scattered in the lumen of intestine. Hardly any microvillusis in its normal shape and site. The zone just below the microvilli showshomogenous lamina propria, which appears edematous (asterisk).Nanoparticles can be seen in the deeper layers and in the lumen of theintestinal villous. Note the presence of AgNPs in various strata of smallintestine (circles) (Scale bar-1mm).

166 B. Shahare et al. Toxicol Mech Methods, 2013; 23(3): 161–167

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.

Florence AT. (1997). The oral absorption of micro- and nanoparticulates:neither exceptional nor unusual. Pharmaceut Res 14:259–66.

Florence AT, Hussain N. (2001). Transcytosis of nanoparticle anddendrimer delivery systems: evolving vistas. Adv Drug Deliv Rev50:S69–89.

Huang Z, Jiang X, Guo D, Gu N. (2011). Controllable synthesis andbiomedical applications of silver nanomaterials. J NanosciNanotechnol 11:9395–408.

Hussain N, Jaitley V, Florence AT. (2001). Recent advances in theunderstanding of uptake of microparticulates across the gastrointes-tinal lymphatics. Adv Drug Deliv Rev 50:107–42.

Jeong GN, Jo UB, Ryu HY, et al. (2010). Histochemical study ofintestinal mucins after administration of silver nanoparticles inSprague–Dawley rats. Arch Toxicol 84:63–9.

Kawata K, Osawa M, Okabe S. (2009). In vitro toxicity of silvernanoparticles at noncytotoxic doses to HepG2 human hepatoma cells.Environ Sci Technol 43:6046–51.

Kim JA, Aberg C, Salvati A, Dawson KA. (2012). Role of cell cycle onthe cellular uptake and dilution of nanoparticles in a cell population.Nat Nanotechnol 7:62–8.

Kim YS, Song MY, Park JD, et al. (2010). Subchronic oral toxicity ofsilver nanoparticles. Part Fibre Toxicol 7:20–31.

Loeschner K, Hadrup N, Qvortrup K, et al. (2011). Distribution of silver inrats following 28 days of repeated oral exposure to silver nanoparticlesor silver acetate. Part Fibre Toxicol 8:18–42.

Martinez-Gutierrez F, Olive PL, Banuelos A, et al. (2010).Synthesis, characterization, and evaluation of antimicrobial andcytotoxic effect of silver and titanium nanoparticles. Nanomed-Nanotechnol 6:681–8.

Nersisyan HH, Lee JH, Son HT, et al. (2003). A new and effectivechemical reduction method for preparation of nanosized silver powderand colloid dispersion. Mater Res Bull 38:949–56.

Nikaido H, Vaara M. (1985). Molecular basis of bacterial outermembrane permeability. Microbiol Rev 49:1–32.

Oberdorster G, Maynard A, ILSI Research Foundation/Risk ScienceInstitute Nanomaterial Toxicity Screening Working Group, et al.(2005). Principles for characterizing the potential human health effectsfrom exposure to nanomaterials: elements of a screening strategy. PartFibre Toxicol 2:8.

Panacek A, Kvitek L, Prucek R, et al. (2006). Silver colloidnanoparticles: synthesis, characterization, and their antibacterialactivity. J Phys Chem 110:16248–53.

Peters A, Wichmann HE, Tuch T, et al. (1997). Respiratory effects areassociated with the number of ultrafine particles. Am J of Resp CritCare 155:1376–83.

Raetz CRH. (1990). Biochemistry of endotoxins. Annu Rev of Biochem59:129–70.

Richard III JW, Spencer BA, McCoy LF, et al. (2002). Acticoatversus silver ion: the truth. J Burns Wound Care (Renamed as ePlasty)1:11–20.

Shrivastava S, Bera T, Roy A, et al. (2007). Characterization of enhancedantibacterial effects of novel silver nanoparticles. Nanotechnology18:225103–12.

Shrivastava S, Bera T, Singh SK, et al. (2009). Characterization ofantiplatelet properties of silver nanoparticles. ACS Nano3:1357–64.

Silva MT, Sousa JCF. (1973). Ultrastructure of the cell wall andcytoplasmic membrane of gram-negative bacteria with differentfixation techniques. J Bacteriol 113:953–62.

Sondi I, Salopek-Sondi B. (2004). Silver nanoparticles as antimicrobialagent: a case study on E. coli as a model for Gram-negative bacteria.J Colloid Interface Sci 275:177–82.

Stoimenov PK, Klinger RL, Marchin GL, Klabunde KJ. (2002). Metaloxide nanoparticles as bactericidal agents. Langmuir 18:6679–86.

Summers HD. (2012). Nanoparticles in the life of a cell. NatNanotechnol 7:9–10.

Summers HD, Rees P, Holton MD, et al. (2011). Statistical analysis ofnanoparticle closing in a dynamic cellular system. Nat Nanotechnol6:170–4.

Tang J, Xiong L, Wang S, et al. (2009). Distribution, translocation andaccumulation of silver nanoparticles in rats. J Nanosci Nanotechnol9:4924–32.

Tang J, Xiong L, Zhou G, et al. (2010). Silver nanoparticles crossingthrough and distribution in the blood-brain barrier in vitro. J NanosciNanotechnol 10:6313–17.

Xing ZC, Chae WP, Huh MW, et al. (2011). In vitro anti-bacterial andcytotoxic properties of silver-containing poly(L-lactide-co-glycolide)nanofibrous scaffolds. J Nanosci Nanotechnol 11:61–5.

DOI: 10.3109/15376516.2013.764950 Effects of silver nanoparticles on small intestine mucosa of mice 167

Tox

icol

ogy

Mec

hani

sms

and

Met

hods

Dow

nloa

ded

from

info

rmah

ealth

care

.com

by

101.

61.1

27.2

6 on

03/

11/1

3Fo

r pe

rson

al u

se o

nly.