Appl Biochem Biotechnol (2013) 171:469487

DOI 10.1007/s12010-013-0357-1

Bactericidal Effect of Poly(Acrylamide/Itaconic Acid)Silver

Nanoparticles Synthesized by Gamma Irradiation
Against Pseudomonas Aeruginosa

M. Eid & E. Araby

Received: 12 April 2013 / Accepted: 17 June 2013 /

Published online: 16 July 2013
# Springer Science+Business Media New York 2013

Abstract Antimicrobial activity of silver nanoparticles is gaining importance due its broad
spectrum of targets in cell compared to conventional antimicrobial agents. In this context,
silver nanoparticles were synthesized by gamma irradiation-induced reduction method of
acrylamide and itaconic acid with irradiation dose up to 70 kGy. Silver nanoparticles were
examined by Fourier-transform infrared, scanning electron microscopic images (SEM), and
ultravioletvisible spectrophotometer. The particle size was determined by X-ray diffraction,
transmission electron microscopy (TEM), and dynamic light scattering. The antibacterial
effect was studied by disk diffusion method against some bacterial pathogenic strains. Silver
nanoparticles showed promising activity against Pseudomonas aeruginosa and slightly
active against Escherichia coli, methicillin-resistant Staphylococcus aureus, and Klebsiella
pneumonia. The bactericidal effect of silver nanoparticles was tested against P. aeruginosa.
The killing rate of P. aeruginosa was found to be 90 % of viability at (100 l/ml) of silver
nanoparticles. Exposure of P. aeruginosa cells to silver nanoparticles caused fast loss of
260 nm absorbing materials and release of potassium ions. The TEM and SEM observation
showed that silver nanoparticles may destroy the structure of bacterial cell membrane in
order to enter the bacterial cell resulting in the leakage of the cytoplasmic component and the
eventual death.

Keywords Silver nanoparticles . -Irradiation . Particle size . Bacterial cells . Antibacterial

activity . P. aeruginosa

Introduction

The growth of unwanted bacteria has for a long time been and is still a problem for the food
industry and in the medical field. Therefore, there is a need for methods to kill or slow down
the growth of unwanted bacteria. Resistance in human pathogens is a big challenge in fields
like pharmaceutical and biomedicine. Antibiotic resistance profiles lead to fear about the

M. Eid (*) : E. Araby

National Center for Radiation Research and Technology, Nasr City( P.O. Box 29( Cairo 11731, Egypt
e-mail: mona_eid2000@yahoo.com
470 Appl Biochem Biotechnol (2013) 171:469487

emergence and re-emergence of multidrug-resistant (MDR) pathogens and parasites [1].

Once an individual is infected with MDR bacteria, it is not possible to cure easily, and he/she
has to spend more time in the hospital and requires a multiple treatment of broad-spectrum
antibiotics, which are less effective, more toxic, and more expensive [2]. Therefore, devel-
opment of or modification in antimicrobial compounds to improve bactericidal potential is a
priority area of research in this modern era [3]. Nanotechnology provides a good platform to
modify and develop the important properties of metal in the form of nanoparticles having
promising applications in diagnostics, biomarkers, cell labeling, contrast agents for biolog-
ical imaging, antimicrobial agents, drug delivery systems, and nanodrugs for treatment of
various diseases [4, 5]. Hence, researchers are shifting towards nanoparticles in general and
silver nanoparticles in particular to solve the problem of emergence of MDR bacteria [6].
Silver has a strong antimicrobial potential, which has been used since the ancient times.
But with the advent of antibiotics progress, the medical applications of silver as antimicro-
bial were declined [7, 8]. Antimicrobial effects of silver can be increased by manipulating
their size at nanoscale. Because of their change in physiochemical properties, silver
nanoparticles have emerged as antimicrobial agents owing to their high surface area/volume
ratio and the unique chemical and physical properties [9]. Silver nanoparticles having size in
the range of 10100 nm showed strong bactericidal potential against both Gram-positive and
Gram-negative bacteria [10]. The bactericidal activity of silver nanoparticles against the
pathogenic, MDR as well as multidrug-susceptible strains of bacteria was studied by many
scientists, and it was proved that the silver nanoparticles are the powerful weapons against
the MDR bacteria such as Pseudomonas aeruginosa, ampicillin-resistant Escherichia coli,
erythromycin-resistant Streptococcus pyogenes, methicillin-resistant Staphylococcus aureus
(MRSA), and vancomycin-resistant Staphylococcus aureus (VRSA).
Many approaches have been tested for the preparation of highly stable silver nanoparticle
dispersions, since the stabilization of nanoparticles is one of the key problems to applications.
Stabilization of nanoparticle dispersions is difficult due to the high surface area/volume ratio
and high surface energy of nanoparticles. Surfactants, polymers, micelles, and ligands are
widely used as stabilizers in order to obtain highly stable silver nanoparticles or generate a
specific property in the nanoparticles [11, 12]. Many of these commonly used stabilization
agents, such as polyvinyl alcohol and polyvinylpyrrolidone. In recent years, researchers have
begun to use biopolymers as stabilizers for the synthesis of biocompatible metal nanoparticles.
Because of the developing resistance of bacteria against bactericides and antibodies and
the irritant and toxic nature of some antimicrobial agent biomaterial, scientists have focused
their research on nanosized metal particles such as Ag, titanium dioxide, and copper. Silver,
in its many oxidation states (Ago, Ag+, Ag2+, and Ag3+), has been recognized as an element
with strong biocidal action against many bacterial strains and microorganisms [13].
The most common method for synthesis of Ag NPs is the chemical reduction of silver salt
solutions by reducing agents in different stabilizers such as polymers and surfactants [14]. In
addition, -irradiation is known as an effective way for fabrication of Ag nanoparticles in
aqueous media [15, 16]. Following our interest on nanostructures, we report a simple and
clean method for in situ fabrications of Ag nanoparticles by -irradiation reductions of silver
ions embedded polymeric composites [17].
The purpose of the present study was to elucidate the antimicrobial activity and mechanism of
silver nanoparticles incorporated into hydrogel against some pathogenic strains. Therefore, we
herein report a simple method to fabricate silver nanoparticles using gamma irradiation embedded
Poly(acrylamide/itaconic acid) hydrogels. The silver ions are in situ reduced by gamma radiation
and protected by the hydrogels. The resulting silver nanoparticles were evaluated using ultravi-
oletvisible (UV-Vis), Fourier-transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD),
Appl Biochem Biotechnol (2013) 171:469487 471

transmission electron microscope (TEM), dynamic light scattering (DLS), and scan
electron microscope (SEM). The bactericidal activity using silver nanoparticles was
carried out by determine bacterial killing kinetics, MIC and MBC determination,
release of cellular materials and finally the interaction between silver nanoparticles
and bacteria using SEM and TEM.

Materials and Methods

Chemicals

Acrylamide (AAm) of purity of 99.9 % (Merck, Germany) itaconic acid (IA) of purity
99.9 % (Aldrich, Germany), silver nitrate, and isopropyl alcohol were used in this study.

Preparation of P(AAm/IA)-Ag Nanoparticles

Aqueous solution of 1 g of AAm, 0.1 g of IA, and 5 mmol AgNO3 were dissolved in 5 ml distilled
water then added 0.15 ml isopropyl alcohol as scavenger of hydroxyl radical. The prepared
solutions placed in glass test tubes and gamma irradiated to 5, 20, 30, 40, 50, and 70 kGy at
ambient temperature using a Co60 gamma source. The prepared hydrogel embedded silver
nanoparticles obtained in cylindrical shapes were cut into 2 mm and dried for constant weight.

Characterization

Fourier-Transform Infrared Measurements

The dried hydrogels were ground to a fine powder, then mixed with dried KBr, and pressed
to a disk for IR analysis. FTIR spectra of hydrogels measured by FTIR spectrometer (6300,
JASCO Japan) in the range from 400 to 4,000 cm1.

UV-Vis Spectrophotometry

Preliminary characterization of the formed silver nanoparticles in the hydrogels was carried
out using UV-Vis spectroscopy. Usually, silver (Ag) nanoparticles exhibit unique and
tunable optical properties due to their surface plasmon resonance that are dependent on
shape, size, and the distribution of the nanoparticles [18]. UV-Vis absorption spectra of the
hydrogelsilver nanocomposites were recorded on JASCO V 560 UV/Vis spectrophotom-
eter with a scan range of 300600 nm. For this study, silver nanoparticles were extracted
from hydrogelsilver nanocomposites by soaked in distilled water (10 mg hydrogel/ml H2O)
over a period of a week at room temperature; the supernatant was used to measure the
absorption spectra. The distilled water was used as a blank solution.

XRD Investigation

The XRD method was used to identify the silver nanoparticles in the polymer
nanocomposites. These measurements were carried out on Shimadzu X-ray diffrac-
tometer (XRD-6000 model) equipped with X-ray tube [Cu target, 40 kV (voltage),
30 mA (current)]. The X-ray data were recorded in the range from 4 to 90 2 with
continuous scanning mode and scanning speed 8 /min. The average particle size of
472 Appl Biochem Biotechnol (2013) 171:469487

the nanoparticles was calculated from the full width at half maximum (FWHM) and
the peak position of XRD line broadened according to the Scherrer equation:
.
d K b cos

where d is the particle size, K=0.89 is the Scherrer constant related to the shape and index (hkl)
of the crystals, is the wavelength of the X-ray (Cu Ka, 1.54056), is the diffraction angle,
and is the corrected full width at half maximum (in radian) [19].

Dynamic Light Scattering

To measure the particle size by DLS, finely grounded hydrogels silver nanoparticles powder
(0.01 g) were extracted in 20 ml bidistilled water, stirred for 30 min until become homoge-
neous, filtered, and the supernatant was measure by Zeta Potential/Particle Sizer
NICOMPTM 380 ZLS Pss. NICOMP particle sizing systems Santa Barbara, CA, USA [20].

Transmission Electron Microscopy

Transmission electron microscopy (TEM) measurements were performed with a (JEOL,

JEM 100CX, Japan) operating at 80 kV. TEM was used to find out the size of silver
nanoparticles inside the hydrogel nanocomposites. To image the silver nanocomposites on
TEM, finely grounded hydrogels silver nanocomposites samples were dispersed in 1 ml of
ethanol followed by sonication to get a solution of silver nanoparticles. Approximately 10
20 l of this solution was dropped on a 3-mm copper grid, drying at room temperature. The
copper grid was inserted into a transmission electron microscope.

Scanning Electron Microscopy

A JEOL-JSM-5400 scanning electron microscope (Japan) was used for investigating the
pore structure of different hydrogels at high magnification and resolution by means of
energetic electron beam. The exchange Ag was characterized by EDX Oxford-ISIS attached
to SEM-JEOL-5400 with voltage of 20 keV. Scanning electron microscopy (SEM) investi-
gate the morphological changes of tested P. aeruginosa were treated by 50 l/ml of hydrogel
silver nanopartictes prepared by irradiation dose 20 kGy compared with untreated strain.
Strain were prepared by cutting the agar, fixed for a minimum of 3 h in 2.5 %v/v glutaral-
dehyde 100 mM phosphate buffer solution of pH 7.2 and then fixed in 1 %w/v osmium
tetraoxide for 1 h at 300 C. After each fixation, the cells were washed twice with phosphate-
buffered saline (PBS), dried, and then coated with gold coater for 5 min. The coated samples
were examined with SEM [21].

Applications

Microorganisms and Growth Conditions

The following human bacterial pathogens both Gram-positive MRSA and Gram-negative E.
coli, K. pneumonia, and P. aeruginosa as multidrug-resistant strains were purchased from
Microbiological Department in NCRRT Atomic Energy Authority Egypt. Cultures were
grown on nutrient agar plate and maintained in the nutrient agar slants at 4 C. Overnight
culture in the nutrient broth was used for the present experimental study.
Appl Biochem Biotechnol (2013) 171:469487 473

Antibacterial Activity of Silver Nanoparticles Against Pathogenic Bacteria

Disk diffusion assay was performed to determine the antibacterial activity of silver
nanoparticles in triplicates [22]. Overnight cultures of tested pathogens which contain
approximately 106107 colony forming unit were swabbed over the surface of sterile
MuellerHinton agar plates using sterile cotton swabs. Disks impregnated with silver
nanoparticles prepared with irradiation doses (20, 30, 40, 50, and 70 kGy) compared to
plain hydrogel were applied on the solid agar medium by pressing slightly and incubated at
322 C for 24 h. After incubation, the zone of inhibition was measured and expressed as
zone of sensitivity millimeter in diameter.

Bacterial Killing Kinetics using Silver Nanoparticles

To examine the bacterial killing kinetics of silver nanoparticles, a modified method de-
scribed by Pal et al. [23] was followed. P. aeruginosa was grown in 10 ml of nutrient broth
supplemented with different concentrations (10, 15, 25, 50, 75, and 100 l/ml) of silver
nanoparticles irradiated by 20 kGy at 32 C without agitation. Killing kinetic rates and
bacterial concentrations were determined by measuring the colony forming unit in the
nutrient agar plates. Percentage of bacterial growth inhibition was calculated according to
equation of Shahi et al. [24].
.
BGI% BCBT  100 BC

where BGI is the bacterial growth inhibition, BC is number of bacterial colonies in the
control, and BT is number of bacterial colonies in the treatment.

MIC and MBC Determination

For determining the minimum inhibitory concentration of silver nanoparticles required for
the inhibition of P. aeruginosa, broth dilution method was used. This method facilitates the
testing of inhibitory activity at various silver nanoparticle concentrations. Nutrient broth was
supplemented with different concentration of nanoparticles (5200 l/ml) and inoculated
with bacterial suspension (initial inoculums, 3108 CFU/ml). Control tube was maintained
without silver nanoparticles. The MIC was determined after 24 h of incubation at 37 C by
observing the visible turbidity and measuring the optical density of these culture broths at
600 nm [25]. The MIC is the lowest concentration of antimicrobial agents that completely
visually inhibits 99 % growth of the microorganisms. The MIC measurement was done in
triplicate to confirm the value of MIC for tested bacteria.

Minimal Bactericidal Concentration After MIC determination of the Ag nanoparticles

tested, aliquots of 50 l from all tubes in which no visible bacterial growth was observed
were seeded in MHA plates not supplemented with Ag nanoparticles and were incubated for
24 h at 37 C. The MBC endpoint is defined as the lowest concentration of antimicrobial
agent that kills 100 % of the initial bacterial population.

Potassium Ions Efflux Assay

The concentration of free potassium ions in bacterial suspension was measured after
exposure to different concentrations of silver nanoparticles 50 l, 100 l in sterile peptone
474 Appl Biochem Biotechnol (2013) 171:469487

water (0.1/100 ml) for 0, 30, 60, and 120 min. At each pre-established interval, the
extracellular potassium concentration was measured by photometer (Alfa Wassermann
Starlyte 111, Na+, K+, Cl analyzer). Control flasks without silver nanoparticles were
tested similarly. Results were expressed as amount of extracellular free potassium
(mmol/l) in the growth media in each interval of incubation [26].

Release of Cellular Material

The measure of the release of 260 nm absorbing material from P. aeruginosa cells
under study was carried out in aliquots of 2 ml of the bacterial inocula in sterile
peptone water added to different concentrations of silver nanoparticles (25, 75, and
100 l) at 32 C. After 120 min of treatment, cells were centrifuged at 3,500 rpm,
and the absorbance of the obtained supernatant was measured spectrophotometrically
using JASCO V 560 UV/VIS spectrophotometer at 260 nm to detect the bacterial cell
damage [26].

Scan Electron Microscopy and Transmission Electron Microscopy

In order to find out and elucidate the antibacterial mechanism of silver nanoparticles,
scanning electron microscopy and transmission electron microscopy techniques were used.
Cells of P. aeruginosa before and after treatment were fixed overnight with 2.5 % glutar-
aldehyde. For SEM, these cells were then fixed in 1 %w/v osmium tetra oxide for 1 h, at
300 C; after each fixation, the cells were washed twice with PBS, dried, and then examined
with SEM. For TEM, samples were postfixed in 2 % osmium tetraoxide, dehydrated in a
series of graded ethanol, infiltrated, and embedded in LR white resin. Then, ultrathin
sections (5070 nm thickness) were cut, stained with uranyl acetate and counter stained
with 4 % lead citrate. These sections were mounted on carbon coated copper grids and
inserted into TEM [21].

Results and Discussion

When monomer is subjected to gamma radiation in dilute aqueous solution, most of

the radiation energy is absorbed by water. As a result a short-lived reactive species
are formed as hydroxyl radicals, hydrated electrons and hydrogen atoms. The double
bond of C=C on acrylamide and CH bond of itaconic acid were broken by
ionizing radiation and free radicals are generated. These free radicals react with each
other and copolymeric poly (acrylamide/itaconic acid) hydrogel is produced as shown
in reactions 15 [27].

rays
H2 O RH:HO; e aqetc 1 radical formation

(2) initiation
Appl Biochem Biotechnol (2013) 171:469487 475

(3) Copolymerization

(4) macroradical
formation

(5) crosslinking

The solvated electrons e aq and H are strong reducing agents so that the silver ions
reduced into the zero-valet state:
Ag e aq  Ag 6

Ag H  Ag H 7
In contrast, hydroxyl radical (OH) is a powerful oxidizing species that able to oxidize the
ions or the atoms into a higher oxidation state, and thus to compensate the reduction reactions 6
and 7. For this reason, isopropyl alcohol as OH radical scavenger was added to this solution.
The OH radicals capable of abstracting hydrogen from the alcohol producing isopropyl radical,
which acts as a reducing agent to reduce silver ion (reactions 8 and 9) [28]
CH3 2 CHOH OH  CH3 2 C OH H2 O 8

Ag CH3 2 C OH  Ag CH3 2 CO H 9

Characterization

Fourier-Transform Infrared Measurements

A comparative description of FTIR spectra of P(AAm/IA) and P(AAm/IA)-Ag

nanoparticles hydrogels is shown in Fig. 1a and b. The characteristic features of the
476 Appl Biochem Biotechnol (2013) 171:469487

spectrum of Ag nanoparticlehydrogel composites are almost very similar to those of

the plain polymer.
Figure 1a shows a broad peak at 34003600 cm1 due to both the presence of carboxylic
groups of IA as well as NH stretching of amide groups present in AAm. A peak corre-
sponding to the carbonyl group (C=O) of the (CONH2) of the AAm unit is observed at
1,665 cm1, while a characteristic peak at 1,600 cm1 arises from the carbonyl group C=O of
the itaconic acid.
However, in Fig. 1b, the broad peak at 3,4003,600 cm1 almost decreases in the
spectrum of Ag nanoparticle hydrogel. In addition, the peak at 1,665 cm1, corresponding
to carbonyl groups of amide functionalities in a plain hydrogel, is shifted to
1616 cm1 in the spectrum of silver nanoparticle hydrogel. The interaction between
the nitrogen function of the polymer support and the metal atoms generally results in
a red shift of the band corresponding to the nitrogen functions in the FTIR spectra
thus indicating the binding of Ag with N and O moieties of these functional groups
[2931]

UV-Vis Spectra

The existence of silver nanoparticles in the hydrogel networks is tested by UV-Vis

spectral analysis. Figure 2 illustrates the absorption peaks for silver nanoparticle
hydrogel in the range of 380500 nm wavelength range in UV-Vis spectra that are
assigned to silver nanoparticles. To determine the reduction effect of irradiation dose
on the formation of silver nanoparticles in hydrogel networks by gamma radiation, we
have evaluated UV-Vis study for hydrogels at different irradiation doses. The results
show that increasing the irradiation dose would highly affect the diameter and size
distribution of silver nanoparticles [32].
Figure 2 shows the UV-Vis spectra of the P(AAm/IA)-Ag nanoparticles hydrogels
prepared at different irradiation doses. With the low irradiation dose, such as 5 kGy, it is a

Fig. 1 FTIR spectra of a P(AAm/IA) and b P(AAm/IA)-Ag nanoparticles hydrogels prepared at 30 kGy
Appl Biochem Biotechnol (2013) 171:469487 477

Fig. 2 UV-Vis spectra of the

P(AAm/IA)-Ag nanoparticles
hydrogels prepared at different ir-
radiation doses

single narrow peak in the UV-Vis spectra, which means the size distribution of the silver
nanoparticles is narrow. At higher irradiation dose, such as 20 and 30 kGy, the peak
intensities found to be higher (0.708 and 0.72) than that in 5 kGy (0.65) and that
means there is more yield of silver nanoparticles. The peaks at 20 and 30 kGy are
somewhat broader than that at 5 kGy, which means that the silver nanoparticles size
distribution is broadened. At a higher irradiation dose at 40 and 50 kGy, the peak is
the broadest and the intensity is the lowest (0.692 and 0.68). The reason can be
attributed to the degradation effect of gamma radiation on the prepared hydrogels.
It is known that, at low irradiation dose, the hydrogels are much cross-linked and less
degraded, and so the silver particles are well fixed by the amide and carboxylic group in the
hydrogel network and they less agglomerate. When much hydrogel are degrade into the
small fragments at high irradiation dose, some silver particles that cannot be enveloped in
the hydrogel network agglomerate into big particles. And so, the silver particles number
decrease and the size distribution is broadened. Figure 2 showed that the absorption peak for
5 kGy at 395 nm was red shifted to 435 nm for 20 and 30 kGy, and a further red shift to 445
and 465 nm for 40 and 50 kGy was observed. Furthermore, it clearly confirms that no
aggregations or cluster formation of silver particles because of complete absence of peaks or
tails at 560 nm.

X-ray Diffraction

In order to confirm the identity of the nanoparticles, X-ray diffraction technique was
employed. As shown in Fig. 3a, the observed peak around 2 value 20 corresponds
to the amorphous nature of hydrogels. Figure 3bf containing diffraction signals at 2
values of 37.7, 43.9, 64.3, and 77.4 attributed to the diffraction (111), (200),
(220), and (311) planes of the bulk Ag, respectively, which can be assigned to Ag
face cubic center structure silver nanoparticles confirming the presence of silver
nanoparticles in the hydrogel nanocomposites [33, 34]. Interestingly, the XRD patterns
do not exhibit any characteristic diffraction peaks of blank hydrogel (Fig. 3a).
478 Appl Biochem Biotechnol (2013) 171:469487

From the full width at half maximum, the particle size for the sample can be calculated.
According to the Scherrer formula,
.
d K b cos

where d is the particle size, K=0.89 is the Scherrer constant related to the shape and index
(hkl) of the crystals, is the wavelength of the X-ray (Cu Ka, 1.54056 ), is the diffraction

Fig. 3 XRD pattern of a P(AAm/IA) 30 kGy and P(AAm/IA)-Ag nanoparticles hydrogels prepared by
different irradiation dose of bf 20, 30, 40, 50, and 70 kGy, respectively
Appl Biochem Biotechnol (2013) 171:469487 479

angle, and is the corrected full width at half maximum (in radian). The average crystallite
size of different Ag nanoparticles was represented in Table 1. From the table, it can be notice
that the particle size decrease by increasing of irradiation dose up to 50 kGy then increase at
70 kGy which may be attributed to the increase of the degree of cross-linking by increasing
the irradiation dose. It is well known that the degrees of conversion and cross-linking greatly
depend on the irradiation dose.
The higher exposure dose means longer exposure time, which consequently prolongs the
propagation step of the process of copolymerization leading to higher degrees of conversion
and cross-linking. The decrease in the particle size by increasing of irradiation dose up to
50 kGy may be because of increasing the cross-linking percentage in the hydrogels, which
lead to reduce the free volume available for the formation of Ag nanoparticles by increasing
the tightness of the network structure. The results indicate that the higher irradiation dose
leads to the narrower phase transition, which may hinder the aggregations or cluster
formation of larger silver particle size.

Scanning Electron Microscope

Scanning electron microscopy images of plain P(AAm/IA) hydrogel and hydrogel

silver nanocomposite are depicted in Fig. 4. In Fig. 4a, it can be observed a clear and
flat surface of the plain hydrogel. On the other hand, silver particles are clearly
visible as bright shiny spherical particles shapes throughout the surface of the
hydrogelsilver nanocomposites (Fig. 4b).

Table 1 Average particle size

(nm) of Ag nanoparticles calculat- Dose (kGy) 2 () FWHM D () D (nm) Average particle
ed from XRD pattern of P(AAm/ size (nm)
IA)-Ag nanoparticles hydrogels
prepared by different irradiation 20 37.74 0.1436 578.1 57.81 50.6
dose 43.98 0.1706 496.6 49.66
64.35 0.1993 465.7 46.57
77.47 0.2083 483.5 48.35
30 37.77 0.1905 435.8 43.58 50.57
43.99 0.1583 535.2 53.52
64.37 0.1710 542.8 54.28
77.49 0.1979 508.9 50.89
40 37.70 0.1740 477.0 47.70 44.82
43.95 0.2000 423.5 42.35
64.32 0.1978 469.1 46.91
77.43 0.2380 423.0 42.30
50 37.71 0.2200 377.3 37.73 42.11
43.94 0.1906 444.4 44.44
64.31 0.2111 439.5 43.95
77.42 0.2379 423.2 42.32
70 37.72 0.1683 492.4 49.24 49.24
43.94 0.1626 520.9 52.09
64.30 0.1851 501.3 50.13
77.41 0.2213 454.9 45.49
480 Appl Biochem Biotechnol (2013) 171:469487

Fig. 4 SEM images of a P(AAm/IA) hydrogel and b P(AAm/IA)-silver nanocomposites at 50 kGy

Transmission Electron Microscope

Figure 5 shows TEM image and the particle size distribution of silver nanoparticles
stabilized by P(AAm/IA) hydrogel prepared by gamma irradiation at a total dose of
50 kGy with starting AgNO3 concentration of 5 mmol. It can be seen that the silver
nanoparticles formed in the cross-linked networks are spherical in shape and well dispersed
without aggregation. The size distributions were obtained by measuring the diameters of 37
particles in three images of an arbitrarily chosen area of TEM image (Fig. 5b). As can be
seen, a small particle size in the range of 2555 nm has been observed (mean
diameter=42.53 nm), and most of the nanoparticles are separated from each other due to
the protection by hydrogels. A small size of nanoparticles are seen in TEM micrographs
indicates a good stabilization of silver nanoparticles in P(AAm/IA) hydrogels.

Dynamic Light Scattering Measurements

Figure 6 shows the DLS measurements of P(AAm/IA)-Ag nanoparticles hydrogels prepared

with a total dose of 50 kGy and starting AgNO3 concentration of 5 mmol. The particle size

Fig. 5 Typical TEM images (a) and histogram of particle size distribution (b) from the as-made silver nanoparticle
samples prepared with a total dose of 50 kGy and starting AgNO3 concentration of 5 mM (bar scale, 500 nm)
Appl Biochem Biotechnol (2013) 171:469487 481

Fig. 6 DLS measurement of silver nanoparticle samples prepared with a total dose of 50 kGy and starting
AgNO3 concentration of 5 mmol

was found to be about 72.3 nm. The large Ag nanoparticles measured by DLS compared to
that measured by X-ray and TEM analysis was because this method measures hydrodynamic
diameter, which is greater due to the polymer attached to the particles and may be due to the
association and chance of aggregation into larger size via van der Waal's force or hydrogen
bond [35, 36]. The decrease in the Ag nanoparticle size under these conditions may be
because the Ag nanoparticles found to be far apart from each other and decrease their chance
for aggregation into larger size.

Applications

Continuous increase in resistance to drug/antibiotics in human pathogens leads to the

re-emergence of MDR pathogens and parasites. Infections caused by such pathogens
require a multiple treatment, containing broad-spectrum antibiotics. In fact, these
treatments are less effective, more toxic and also expensive. Nanotechnology provides
a good platform to overcome the problem of resistance, with the help of the silver
nanoparticles. Since the ancient time, antimicrobial efficacy of silver was reported.
The bactericidal potential can be increased by manipulating the size at nanolevel,
leading to increased surface area/volume ratio and also by changing the chemical and
physical properties. Silver nanoparticles of size of 10100 nm have strong bactericidal
potential against both Gram-positive and Gram-negative bacteria. Therefore, silver
nanoparticles having bactericidal potential would be used as powerful weapons against
the MDR bacteria such as P. aeruginosa, ampicillin-resistant E. coli, erythromycin-
resistant S. pyogenes, MRSA and VRSA [37]. In this study, antimicrobial activity of
silver nanoparticles has been investigated against different pathogenic strains, which
resemble large problem in its treatment with most antibiotics.

Bacterial Susceptibility to Silver Nanoparticles

Antimicrobial activities of silver nanoparticles are determining by measured Zone of

inhibition against tested organisms. The results represented in Fig. 7a indicates that
482 Appl Biochem Biotechnol (2013) 171:469487

the silver nanoparticles showed higher antibacterial action against Gram-negative

organism P. aeruginosa compared to that of the other tested organisms, where their
activity were limited. The ZOI for P. aeruginosa in Fig. 7b shows increase in zone
radius for silver nanoparticles prepared at irradiation dose 20 kGy followed by
70 kGy. The higher ZOI for hydrogel formed by 20 kGy was due to the lower
cross-linking at lower irradiation dose. In the other hand, the lower antibacterial action
of samples obtained with 30 kGy may be attributed to the formation of tightly cross-
linked hydrogel at this dose, as a result decrease in the concentration of extracted Ag
nanoparticles and lead to decrease of ZOI.
Silver nanoparticles of size in the range of 10100 nm showed strong bactericidal
potential against both Gram-positive and Gram-negative bacteria [10]. The bactericidal
activity of silver nanoparticles against the pathogenic, MDR as well as multidrug susceptible
strains of bacteria was studied by many scientists, and it was proved that the silver
nanoparticles are the powerful weapons against the MDR bacteria such as P. aeruginosa
[37]. A study of the antibacterial activity of silver nanoparticles against Gram-negative
bacteria was carried out by Morones et al. [10], which suggested that silver nanoparticles
disturb the cell function by attaching to the surface of the cell membrane, penetrating in
bacteria, followed by subsequent release of silver ions. Silver nanoparticles are the effective
killing agent of broad spectrum of Gram-negative bacteria such as Acinetobacter,
Escherichia, Pseudomonas, Salmonella, and Vibrio; Gram-positive bacteria such as Bacil-
lus, Clostridium, Enterococcus, Listeria, Staphylococcus, and Streptococcus; and antibiotic-
resistant bacteria such as methicillin- and vancomycin-resistant S. aureus (MRSA and
VRSA) and methicillin-resistant E. faecium by preventing biofilm formation. Biofilm is a
self-secreted extracellular polysaccharide matrix, made up of surface-attached aggregates of
microorganisms. Biofilm acts as an efficient barrier against antimicrobial agents and the host
immune system and protect the bacterial colony. It was observed that silver nanoparticles
inhibit the formation of biofilms [38].

Bacterial Killing Kinetics using Silver Nanoparticles

Antibacterial properties of silver nanoparticles were performed against P. aeruginosa

on nutrient agar plates containing different concentrations of silver nanoparticles

Fig. 7 a Zone of inhibition (ZOI) for tested pathogenic strains against silver nanoparticles at different
gamma irradiation doses and b photograph showing zone of inhibition for P. aeruginosa against silver
nanoparticles at different irradiation doses: 1 (20 kGy), 2 ( blain hydrogel), 3 (30 kGy), 4 (40 kGy), 5
(50 kGy), and 6 (70 kGy)
Appl Biochem Biotechnol (2013) 171:469487 483

ranging from 10 to 100 g/ml (Table 2). The presence of these particles at a
concentration 10 g/ml inhibits bacterial growth by 8.57%. By increasing the amount
of silver nanoparticles to 25, 50, 75, and 100 g/ml, the number of bacterial colonies,
grown on plates is gradually reduced. The silver nanoparticle concentration of
100 g/ml shows 90% inhibition of P. aeruginosa colonies growth on nutrient agar
medium. As a result, in comparison to antibacterial activity of silver nanoparticles
prepared by other authors [39], these particles have a relatively good biocidal effect
depending on the concentration of silver nanoparticles as well as on the CFU of the
bacteria used in the experiments.
Previous studies also showed that there are several mechanisms about the bactericidal
effect of silver nanoparticles. Silver nanoparticles may attach to the surface of the cell
membrane, interrupting permeability and metabolic pathways of the cell [40]. Silver
nanoparticles not only interact with the surface of the membrane but can also penetrate into
the bacterial cell membrane [41]. In addition, silver nanoparticles can bind to the DNA
inside the bacterial cells, preventing its replication or interaction with the bacterial ribosome
[42, 43]. It has been discovered that silver nanoparticles can damage the structure of the
bacterial cell membrane and reduce the activity of some membranous enzymes, which cause
E. coli bacteria to die eventually [44].
Silver nanoparticles were selected to study MIC and MBC using the broth dilution
method. MIC of silver nanoparticles against P. aeruginosa at a concentration 110 l/ml
showed bacteriostatic compound and the MBC or bactericidal effects against P.
aeruginosa, which was 125 l/ml. Silver nanoparticles exerted a bactericidal effect
against the bacterial strain under study because the MBC/MIC ratio values were lower
than 1.2 [3].

Loss of 260-nm Absorbing Material and Release of Potassium Ions

The results of loss 260-nm absorbing materials of P. aeruginosa cells treated with
silver nanoparticles at (25, 75 and 100 l/ml) and release of potassium ions at (50
and 100 l/ml) are shown in Figs. 8 and 9, respectively. The DO260 nm of the filtrate
of the cells exposed to silver nanoparticles revealed an increasing release of 260 nm
absorbing material according to the time exposure and silver nanoparticles concentra-
tion. The efflux of potassium ions from P. aeruginosa cells occurred immediately after
the addition of silver nanoparticles following a steady loss along the evaluated
intervals. No leakage of potassium ions was observed when the strain grew in media

Table 2 Bacterial growth and

killing kinetics (%) in the presence Nanosilver P. aeruginosa colony forming Killing
of silver nanoparticles concentrations (g/ml) unit (CFU/ml) (%)

0 280 0
10 256 8.57
15 215 23.2
25 169 39.6
50 100 64.3
75 65 76.8
100 29 90.0
484 Appl Biochem Biotechnol (2013) 171:469487

Fig. 8 UV-Vis spectra of bacteri-

al cell damage induced by silver
nanoparticles against P.
aeruginosa

without the silver nanoparticles. These results suggest that increased permeability of
membrane is a factor involved in the establishment of the antibacterial property of
tested silver nanoparticles.
Exposure of P. aeruginosa cells to silver nanoparticles caused fast loss of 260 nm
absorbing material and release of potassium ions, these findings assume that the accumula-
tion of silver nanoparticles in the cytoplasm membrane causing loss of their integrity and
become increasingly permeable to protons and ions. Marked leakage of cytoplasm material
is used as indicative of gross and irreversible damage of cytoplasm membrane and plasma
membrane [41]. Moreover, the observation that the amount of loss of 260 nm absorbing
material was as extensive as the leakage of potassium ions might indicate that the membrane
structural damage sustained by P. aeruginosa cells resulted in release of macromolecules
constituents.

Fig. 9 Leakage of potassium ions

from P. aeruginosa induced by
exposure to (50 l and 100 l/ml
silver nanoparticles) for 120 min
at 37 C
Appl Biochem Biotechnol (2013) 171:469487 485

Fig. 10 Scanning electron microphotography of P. aeruginosa: a normal cells and b cells after treatment with
silver nanoparticles

Interaction Between Silver Nanoparticles and Bacteria

In order to achieve an understanding of this effect, knowledge about the structure of bacteria is
needed. Morphology and structure of normal P. aeruginosa cells and treated by silver
nanoparticles for 18 h were observed using scan electron microscope. After treatments with
silver nanoparticles, structural changes in the morphology of bacteria were clearly observed in
the scanning electron micrographs as the surfaces of the cells were damaged (Fig. 10a and b).
These results are in concurrence with the earlier antibacterial studies carried out with silver
nanoparticles [41, 45]. In addition, these findings were further supported by transmission
electron micrographs. The TEM images given in Fig. 11a and b reveal that these nanoparticles
were attached to the surface of bacterial cell wall, permeated the cell membrane, and entered
into the cell interior. As a result, silver nanoparticles interact with the cell wall and membrane,
damage the cell membrane, increase the cell permeability, and leak the intracellular contents by
cell disruption. These observations confirm the earlier findings on the antibacterial mechanism
of silver nanoparticles [9, 10, 41, 46].

Fig. 11 Morphology and structure of bacterial cells under transmission electron microscopy: a normal P.
aeruginosa cells and b cells treated by silver nanoparticles
486 Appl Biochem Biotechnol (2013) 171:469487

Chen et al. [47] reported that the observation with TEM suggested that S-T-gel may
destroy the structure of bacterial cell membranes in order to enter the bacterial cell. S-T-gel
then condensed DNA and combined and coagulated with the cytoplasm of the damaged
bacteria, resulting in the leakage of the cytoplasmic component and the eventual death of
bacteria.

Conclusion

Our current study demonstrates the possible preparation of silver nanoparticles of different
sizes and morphologies using different irradiation doses. The degree of cross-links due to the
different irradiation doses appears to be the key factor in regulating the size of silver
nanoparticles. The prepared silver nanoparticles were evaluated using UV-Vis, XRD anal-
ysis, and DLS confirmed that the silver nanoparticles were formed in a nanoscale according
to the irradiation dose. TEM images showed that the silver nanoparticles are distributed in
the polymeric materials. The silver nanoparticles synthesized by gamma irradiation are
showing promising antibacterial activity on P. aeruginosa. These results indicate the poten-
tial of the synthesized nanoparticles towards the development of antibacterial coatings on
different material surfaces for various biomedical and environmental applications. These
formulations can be used for the treatment of drug resistant bacterial infections.

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