Chitosan and silver nanoparticles as pudding with raisins with

antimicrobial properties
M. Carmen Rodrguez-Argelles
a
, Carmen Sieiro
b
, Roberto Cao
c,
, Lucia Nasi
d
a
Departamento de Qumica Inorgnica, Universidade de Vigo, 36310 Vigo, Spain
b
Departamento de Biologa Funcional y Ciencias de la Salud, Area de Microbiologa, Universidade de Vigo, 36310 Vigo, Spain
c
Laboratorio de Bioinorgnica, Facultad de Qumica, Universidad de La Habana, La Habana 10400, Cuba
d
IMEM-CNR, Parco Area delle Scienze 37/A, I-43124 Parma, Italy
a r t i c l e i n f o
Article history:
Received 17 May 2011
Accepted 3 August 2011
Available online 17 August 2011
Keywords:
Antimicrobial
Chitosan
Nanoparticle
Nanocomposite
Silver
TEM
a b s t r a c t
Chitosan nanoparticles (CS-NP) containing small silver nanoparticles are reported (Ag@CS-NP). CS-NP
was synthesized using tripolyphosphate (TPP) as a polyanionic template. TPP also served to electrostat-
ically attract Ag
+
inside CS-NP, where it was reduced by the terminal glucosamine units of the biopoly-
mer. This procedure is environmental friendly, inexpensive, and permits the synthesis of very small AgNP
(0.931.7 nm), with only a discrete dependence from the amount of silver nitrate used (5200 mg). The
obtained hybrid nanocomposites Ag@CS-NP were characterized by DLS, HRTEM, and HAADFSTEM pre-
senting a mean hydrodynamic diameter of 78 nm. The antimicrobial activity of Ag@CS-NP against Can-
dida glabrata, Sacharomyces cerevisiae, Escherichia coli, Klebsiella pneumoniae, Salmonella, Staphylococcus
aureus, and Bacillus cereus corresponded to MIC values lower than for AgNO
3
.
2011 Elsevier Inc. All rights reserved.
1. Introduction
Chitosan is a biopolymer of (1 ?4)-2-amino-2-deoxy-b-D-glu-
can and (1 ?4)-2-acetamido-2-deoxy-b-D-glucan units, generally
predominating in the former units [1]. At pH < 6, the polymer dis-
solves due to protonation of the amine groups, as represented in
Fig. 1.
Chitosan is a non-toxic, inexpensive, and biocompatible poly-
mer, biodegradable by different hydrolytic enzymes [2]. This bio-
polymer presents very important biological properties among
which antimicrobial, anti-inammatory, antioxidant, and antitu-
moral can be cited [3]. Chitosan has been widely used in the regen-
eration of different types of tissues, especially skin [4,5] and bones
[6] and in many other biomedical and pharmaceutical applications
[1,7,8].
The polycationic nature of chitosan in acidic medium favors a
strong electrostatic interaction with polyanions, as tripolyphos-
phate (TPP), which permits the formation of chitosan nanoparticles
(CS-NP), already reported several years ago [9,10].
CS-NP is water soluble and presents a structure that permits the
inclusion (entrapment) of different types of compounds making it
able to efciently function as bionanocarriers [1114]. Such prop-
erty will permit the development of a wide variety of systems with
important biomedical and pharmaceutical applications.
Chitosan has served to obtain different types of metal nanopar-
ticle-polymer composites. Using chitosan as a polymeric matrix
and Na[BH
4
] as reducing agent, relatively small silver nanoparticles
(AgNP) have recently been reported [15,16]. These AgNP presented
surface plasmon resonance (SPR) maxima within 410420 nm,
assuming diameters lower than 5 nm. The position of the SPR band
depended on the proportions in which the reagents were mixed.
Generally, AgNP are synthesized with diameters of 10 nmor lar-
ger, since smaller ones are difcult to obtain [17]. Different green
methods (no use of Na[BH
4
] nor other contaminating reducing
agents) have been reported to synthesize sub10-nmAgNP, but gen-
erally of 3 nm or higher diameters [1821]. For example, 35 nm
AgNP were obtained using polyphosphonate and H
2
as reducing
agent [18]. A similar procedure, but using phosphonated calixe-
renes, gave 2.115 nm AgNP, where the size depended on the
amount of AgNO
3
and type of calixarene used [19]. H
2
is a clean
reducing agent, but must be used with caution. Starch has also
been used as a clean reducing agent of AgNO
3
, but AgNP no smaller
than 5.3 nm have been reported [20,21].
The main goal of the present report consists in obtaining
small AgNP entrapped in CS-NP (Ag@CS-NP) using a highly
friendly and inexpensive procedure, since CS is used as both
reducing agent and stabilizer. CS-NP would behave as a bionano-
carrier of AgNP. This was because the resulting system could
serve for biological applications combining the interesting prop-
erties of both components, including the antimicrobial properties
of AgNP [22,23].
0021-9797/$ - see front matter 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jcis.2011.08.006

Corresponding author.
E-mail address: caov@fq.uh.cu (R. Cao).
Journal of Colloid and Interface Science 364 (2011) 8084
Contents lists available at SciVerse ScienceDirect
Journal of Colloid and Interface Science
www. el sevi er . com/ l ocat e/ j ci s
2. Materials and methods
2.1. Materials
Low molecular weight chitosan (85% deacetylation) and sodium
tripolyphosphate (TPP) were purchased from Aldrich; AgNO
3
was
from Scharlau; Mili-X water was used in all cases.
2.2. Spectroscopy
A Cary 50 Conc (Varian) UVVis spectrophotomer was used for
the determination of the SPR bands of AgNP.
High resolution transmission electron microscopy (HRTEM), as
well as high angle annular dark eld (HAADF) in scanning mode
(STEM), was carried out by using a JEOL 2200FS microscope work-
ing at 200 kV.
Dynamic light scattering (DLS) measurements of obtained CS-
NP and Ag@CS-NP aqueous solutions were performed on a Nano-
trac Particle Analyzer PMX 200C (Microtrac Inc.).
The amount of Ag
+
that remained unreduced was determined
by a Perkin Elmer ICP-OES Optima 4300DV spectrometer with pre-
vious centrifugation of the solutions at 5000 rpm.
2.3. Synthesis of chitosan nanoparticles (CS-NP)
An aqueous solution of TPP (5.4 mL, 1 mg/mL) was added drop
wise to a solution of chitosan (10 mg) dissolved in acetic acid
(2%, 10 mL) with constant agitation. A white discrete opalescence
was observed, and the formation of the nanoparticles was con-
rmed by the Tyndall effect. This system was maintained under
constant agitation for no less than 1 h.
2.4. Synthesis of silver nanoparticles inside CS-NP (Ag@CS-NP)
The above solution of ChNP was heated to boiling (under reux)
and then an aqueous solution of AgNO
3
(5200 mg in about 0.6
1 mL) was added drop wise to it. A yellow color appeared after
4080 min of agitation of the boiling solution and was maintained
other 56 h under the same conditions.
2.5. Enzymatic treatment of samples
The enzymatic treatment of the Ag@CS-NP samples was carried
out using a chitosanase fromStreptomyces griseus (Sigma). Chitosan
degradation was evaluated by measuring the reducing power of
the samples [24,25] and expressed as micrograms of glucosamine
(or its equivalent in reducing power) released. The pH of the sam-
ples was adjusted to 5.5 using 1 M Tris buffer pH 7. Reactions con-
taining 1.5 mL of sample with different amounts of chitosanase
(ranging from 0.03 to 0.45 U/mL) were incubated at 35 C for 12 h.
2.6. Strains and culture conditions
Escherichia coli (E. coli) CECT 101, Klebsiella pneumoniae (K. pneu-
moniae) CECT 143, Salmonella sp, Staphylococcus aureus (S. aureus)
CECT 4439, and Bacillus cereus (B. cereus) CECT 193 were incubated
in MuellerHinton broth (Cultimed) at 35 C. Candida glabrata (C.
glabrata) CECT 1448 and Sacharomyces cerevisiae UV30 (S. cerevisi-
ae) were incubated in Saboureaud broth (Cultimed) at 26 and
30 C, respectively. S. cerevisiae and Salmonella sp. are wild type
strains from our laboratory. CECT: Spanish Type Culture Collection.
The visual turbidity of the tubes was noted both before and after
incubation. The media were solidied, when necessary, with 1.5%
agar (Cultimed).
2.7. Minimal inhibitory and microbicidal concentration
The antimicrobial properties for the samples were determined
using the twofold broth dilution technique [26]. All determinations
were performed in duplicate. The samples were used as prepared
and tested at nal concentrations (of silver) of 24, 12, 6, 3, 1.5,
0.75, and 0.37 lg Ag/mL. Inocula of 5 10
4
bacteria/mL and
1 10
3
yeast/mL were used. The minimal inhibitory concentration
(MIC, lg/mL) was dened as the lowest concentration of com-
pound inhibiting the growth of each strain. The tubes were incu-
bated at the appropriate temperature for 24 h. Growth was read
by the visual turbidity of the tubes noted both before and after
incubation. Media and positive growth controls were also run
simultaneously. The minimal bactericidal concentrations (MBC,
lg/mL) and the minimal fungicidal concentrations (MFC, lg/mL)
were measured by subculturing 100 lL of each sample remaining
clear in tubes containing 1 mL of fresh medium.
3. Results and discussion
The reported method for the synthesis of CS-NP based on the
use of TPP [9,10] was adjusted by the determination of the opti-
mum amounts of each reagent. The formation of CS-NP was con-
trolled by observing the presence of opalescence and Tyndall
effect (using a Laser pointer), a more precise procedure.
TPP played three important roles in the synthetic procedure
used, all of electrostatic nature. In the formation of CS-NP, the
polyanion TPP served to agglutinate chitosan units as a template,
and once CS-NP was formed, it attracted Ag
+
ions inside the
nanoparticle enhancing the diffusion process. Additionally, the
electrostatic attraction of Ag
+
by TPP assisted the regulation of
the size of the formed AgNP. Once Ag
+
diffused inside the CS-
-
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-
Ag
+
-
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-
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-
-
-
-
-
-
-
-
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
Ag
+
-
-
-
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-
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-

(100 C)
-
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-
Fig. 2. Schematic representation of the formation of AgNP inside CS-NP, where the
negatively charged semicircular sections correspond to TPP.
NH
3
+
OH
CH
2
OH
O
(
(
n
Fig. 1. Schematic representation of chitosan through its predominating protonated
(1 ?4)-2-amino-2-deoxy-b-D-glucan units.
M.C. Rodrguez-Argelles et al. / Journal of Colloid and Interface Science 364 (2011) 8084 81
NP matrix and the system was heated up to 100 C, glucosamine
units of chitosan served as reducing agent to form AgNP inside
CS-NP (Ag@CS-NP). Therefore, both CS-NP and TPP ruled the syn-
thesis of Ag@CS-NP and should have also regulated the size of
AgNP. The formation of Ag@CS-NP is schematically represented
in Fig. 2, where the negatively charged semicircular sections cor-
respond to TPP.
According to the schematic representation given in Fig. 2, the
resulting Ag@CS-NP system could be considered similar to a pud-
ding with raisins. Such aspect was conrmed when the system
was studied by HRTEM. In Fig. 3, two HAADFSTEM images of
Ag@CS obtained using 30 mg of AgNO
3
are presented. The mea-
sured average size of these AgNP was of 2.0 (0.64) nm according
to the size distribution histogram (Fig. 4a). The HRTEM images
and the Fast Fourier transform analysis (inset in Fig. 3b) indicate
the formation of AgNP with a fcc structure, which is not the pre-
dominant structure expected.
Other amounts of AgNO
3
(from 5 to 200 mg) were also used to
obtain Ag@CS-NP (TEM images are not given), and in all cases, the
average sizes of the obtained AgNP were below 2 nm. For example,
with 5 mg of AgNO
3
, the average diameter of AgNP was of 0.95
(0.30) nm (Fig. 4b), while when using 200 mg of AgNO
3
, the ob-
tained diameter was of 1.7 (0.32) nm (Fig. 4c). Therefore, only
small variations were observed in the diameters of the obtained
AgNP, with an insignicant dependence on the amount of AgNO
3
used. This result was unexpected considering that the concentra-
tion of AgNO
3
is a determining factor in all the methods reported
on the synthesis of AgNP [27].
Fig. 3. HAADFSTEM image of Ag@CS-NP (two different magnications) obtained from 30 mg of AgNO
3
. Inset of (b) is the Fast Fourier transform indicating a crystalline fcc
structure of AgNP.
Fig. 4. Size distribution diagrams of AgNP (in Ag@CS-NP) formed: (a) with 30 mg of AgNO
3
; (b) 5 mg; (c) 200 mg; (d) also AgNP formed in CS with 5 mg of AgNO
3
.
82 M.C. Rodrguez-Argelles et al. / Journal of Colloid and Interface Science 364 (2011) 8084
AgNP was also synthesized in CS (dissolved in acetic acid) using
a similar procedure to that reported here for Ag@CS-NP, but with-
out the presence of TPP and, therefore, without the formation of
CS-NP. Under such conditions, larger AgNP were obtained, with
an average diameter of 5.4 (1.9) nm when only 5 mg of AgNO
3
was added (Fig. 4d). As a comparison, it is important to mention
that in a recent report on the synthesis of AgNP in free chitosan,
the sizes of the nanoparticles varied within 5 and 15 nm [28].
Once compared the sizes of AgNP obtained in chitosan as nano-
particles (CS-NP) with those using free CS, it results evident that
CS-NP plays an important role in the regulation of the size of AgNP.
The formation of very small AgNP inside CS-NP can be attrib-
uted to two main factors: (1) CS-NP behaves as a template in the
formation of AgNP and (2) CS is a soft reducing agent able to mod-
ulate the size of AgNP.
As mentioned in the Introduction, with phosphonated calixare-
nes, relatively small AgNP have been reported [19], where the for-
mer plays the role of a template, mainly through the participation
of the phosphonate groups. In the case of CS-NP, this tridimensional
system should participate as a whole in the complete regulation of
the size of AgNP with the assistance of negatively charged TPP.
On the other hand, CS-NP behaves as a soft reducing agent
through the participation of its terminal glucosamine groups. An
expression of the low reducing capacity of CS-NP is that the reac-
tion took place at 100 C (under reux). We observed that at lower
temperatures larger AgNP were formed. Generally, strong reducing
agents favor the formation of large AgNP, since the reduction oc-
curs at a rate higher than the capacity of the capping component
to cover the formed nanoparticle. An opposite tendency is ob-
served when a mild reducing agent is used, especially when it par-
ticipates at low and sustained concentrations, as is the case of H
2
[17]. In our case, the glucosamine unit constitutes an abundant
mild reducing agent, which is accessed by Ag
+
by a constrained dif-
fusion process.
The sizes of CS-NP varied from 20 to 70 nm even within a same
synthesis, according to HRTEM determinations. The forms of the
obtained Ag@CS-NP varied between oval and spherical, but with
no dened regularity. The mean (hydrodynamic) diameter deter-
mined by dynamic light scattering (DLS) was of 78 (19) nm, a bet-
ter dened value. Here, it is important to mention that the
diameters that are obtained by DLS are larger than those deter-
mined by HRTEM.
Therefore, the Ag@CS-NP nanoparticles varied in size and form,
while the size of the spherical AgNP cores practically remained
constant. Actually, the sizes reported for CS-NP by different
authors varies in a much wider range and were much larger, from
172.6 nm [14] up to 3.1 lm [29].
The size distributionof our CS-NP(without the presence of silver)
was of 576(145) nmaccordingtoDLS determinations. This average
size is about seven times larger than the corresponding value of
Ag@CS-NP, as already mention above. The signicant difference be-
tween the hydrodynamic diameters of Ag@CS-NP and CS-NP can be
considered as an indication that the silver ions should have played a
role in the formation of the nanoparticles of chitosan. Related to this
interpretation, it is important to mention that in reference [14], the
Ag@CS-NP reported (of 172 nm) were obtained by a previous forma-
tion of CS-NP, with no direct participation of silver.
CS-NP presents a exible structure, strongly dependent on what
it contains and also on the surroundings. We had this consideration
in mind from the same beginning and that is the reason for which
we decided not to wash Ag@CS-NP, once obtained in order to not
affect its initial structure. After the addition of Ag
+
to CS-NP, the
ions should diffuse the membrane electrostatically attracted by
TPP. Subsequently, an interaction between the Ag
+
ions and the
amino groups of glucosamine should have taken place to provoke
the observed contraction of CS-NP.
Different forms of chitosan were submitted to the enzymatic
cleavage of chitosanase. The amount of free glucosamine formed
in each case was used for the comparison. 0.4 UE/mL of chitosanase
was used. Free CS produced 215 (0.51) lg/mL of glucosamine,
while the cleavage of CS-NP only gave 142 (4.6) lg/mL. On the
other hand, the cleavage of free CS in the presence of AgNP pro-
duced 173 (0.75) lg/mL, while for Ag@CS-NP only 134
(9.6) lg/mL of glucosamine was formed. Evidently, CS-NP is per-
meable to small species (as Ag
+
cations), but not to large species
as enzymes. The enzyme used was of 39 kDa, with a mean diame-
ter of about 8 nm, which makes it smaller than CS-NP (78 nm), but
not small enough to favor its diffusion inside the latter.
The difference in the enzymatic production of glucosamine be-
tween chitosan with and without AgNP (8 lg/mL) is statistically
not signicant and should be attributed to the amount consumed
in the reduction of Ag
+
. On the other hand, the difference between
the content of glucosamine in free CS and in CS + AgNP is much
higher (42 lg/mL), a result that cannot be endorsed to the diffusion
of the enzyme. Even so, this difference constitutes only 20% of the
content of glucosamine in free CS. According to that result it should
be assumed that not all of the Ag
+
ions added were reduced, and
we decided to determine that value quantitatively. The concentra-
tion of unreduced Ag
+
that remained in the Ag@CS-NP solution pre-
sented a discrete dependence on the amount of AgNO
3
added in
each synthesis. When 5 mg of AgNO
3
was used, only 18.89% of
Ag
+
remained unreduced, for 30 mg 16.44%, for 50 mg 13.93%,
and for 100 mg 13.33%. These results indicate that the diffusion
and steric access of Ag
+
to the glucosamine groups constituted
the main limitation in its reduction, and the reason for which a
very low inuence of the concentration of AgNO
3
used on the size
of AgNP was observed.
The positively charged surface of CS-NP should favor its docking
on negatively charged biological surfaces and permit the release of
the species held inside. AgNP could then be able to diffuse outside
CS-NP and interact with the surroundings, as already reported for
the interaction of Ag@CS-NP with human adenocarcinoma cells
[14]. In this sense, it is important to mention that in a recent report
on the interaction of CS with a negatively charged liposome (stud-
ied by isothermal titration calorimetry), high binding constants of
the order of 10
5
M
1
were determined [30].
The results of antimicrobial activity of AgNO
3
, CS-NP, and
Ag@CS-NP are presented in Table 1 and expressed as minimal
inhibitory concentrations (MIC, lg/mL) for C. glabrata and S. cerevi-
siae, which are fungi; for E. coli, K. pneumoniae and Salmonella,
Gram negative bacteria; and also Gram positive bacteria S. aureus
and B. cereus. MFC (for the fungi), and MBC values (for the bacteria)
are also reported. The amount of AgNP in Ag@CS-NP was corrected
according to the concentration of Ag
+
analytically determined for
each case.
As can be observed from Table 1, Ag@CS-NP presented an anti-
microbial activity signicantly higher than AgNO
3
. This result was
not affected by CS-NP, which presented a low antimicrobial activ-
ity, for which no correction was made. Especially signicant re-
sulted the very low MBC values determined for the ve bacteria
studied (1.53 lg/mL), except against Salmonella sp (6 lg/mL).
The MIC/MBC ratios were of 12.
No signicant differences between the antimicrobial activity of
Ag@CS-NP against the studied Gram positive and Gram negative
bacteria were observed, but its antifungal activity was about four
times lower. These three biological systems are characterized by
possessing negatively charged surfaces. Therefore, the positively
charged surface of Ag@CS-NP should favor the interaction with
the three studied systems by a docking process governed electro-
statically. Nevertheless, such type of interaction is dynamic in nat-
ure, since the density of the negatively charged surfaces of these
systems varies according to the environment and other factors [31].
M.C. Rodrguez-Argelles et al. / Journal of Colloid and Interface Science 364 (2011) 8084 83
The antimicrobial activity of Ag@CS-NP, as a whole, should be
strongly related to the small sizes of both nanoparticles, CS-NP
and AgNP, involving a highly positive charge density, able to inter-
act with specic areas of the cell surface of the studied
microorganisms.
The antimicrobial activity of Ag@CS-NP should be related,
among other pathways, to the formation of ROS as a consequence
of the interaction of AgNP with O
2
, a process that provokes apopto-
sis [14,23,32]. In this sense, it is important to take into consider-
ation that AgNP have the property of releasing Ag
+
in solution to
behave as a permanent source of the cation [33]. Furthermore,
the membrane of CS-NP should regulate the diffusion of Ag
+
and
AgNP out of the nanoparticle, making Ag@CS-NP behave as a con-
trolled release liberation system of Ag
+
and AgNP. Therefore, these
characteristics should be interpreted as if Ag@CS-NP would be act-
ing as a sustained source of antimicrobial agent able to interact
with microorganisms of different nature. Such characteristic
should reduce the known toxicity of small AgNP [34].
The sizes of both nanoparticle components (AgNP and CS-NP)
could be considered as important factors in the antimicrobial activ-
ity of Ag@CS-NP, if one compares our MIC and MBC values with
those reported recently. For example, in a paper on AgNP (5
30 nm) dispersed in CS the authors reported MIC and MBC values
of 10 lg/mL for all the Gram positive and Gram negative bacteria
studied, except P. aeruginosa (2.5 lg/mL) [27]. Then again, our
MIC and MBC values are also better that those reported for AgNO
3
included in CS-NP [35].
4. Conclusions
Here, we report a procedure with which it is possible to obtain
small CS-NP (78 nm) containing very small AgNP (0.931.7 nm).
The preparation of such small AgNP constitutes a task difcult
itself to be achieved. The procedure is environmental friendly
and inexpensive, since chitosan, a biopolymer available as a waste
of shing industry, behaves as the reducing agent. TPP served as a
polyanionic template that favored the formation of the small nano-
particles, which also electrostatically attract Ag
+
inside CS-NP. The
importance of the small size of Ag@CS-NP hybrid nanocomposites
(and AgNP itself) was expressed in the determined antimicrobial,
with signicantly small MIC values against two fungi, three Gram
negative bacteria, and two Gram positive bacteria. The Ag@CS-NP
system should be expected to behave as a biomaterial to be used
in different pharmaceutical applications, mainly in wound
treatments.
Acknowledgments
This work was nanced by Xunta de Galicia, Spain, project
PXIB310278PR. The authors are grateful to Prof. Dr. Sabine Schlecht
(Giessen, Germany) for the assistance in the characterization of the
products.
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Table 1
Antimicrobial activity of AgNO
3
, CS-NP, and Ag@CS-NP (obtained as described and discounting the amount of unreduced Ag
+
) expressed as MIC (MFC/MBC) in lg of Ag/mL.
System Fungi Gram negative bacteria Gram positive bacteria
C. glabrata S. cerevisiae E. coli K. pneumoniae Salmonella sp S. aureus B. cereus
AgNO
3
24 >24 6 (12) 12 (24) 24 (>24) 12 (>24) 12 (>4)
CS-NP >24 >24 >24 >24 >24 >24 >24
Ag@CS-NP 5 (5) 5 (20) 1.3 (1.3) 2.5 (2.5) 2.5 (5) 2.5 (2.5) 1.3 (2.5)
84 M.C. Rodrguez-Argelles et al. / Journal of Colloid and Interface Science 364 (2011) 8084