Materials Letters 106 (2013) 413416

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Materials Letters
journal homepage: www.elsevier.com/locate/matlet

Bactericide efciency of a combination of chitosan gel

with silver nanoparticles
C. Smano-Valencia a,b, G.A. Martnez-Castan a,b,n, R.E. Martnez-Martnez b,
J.P. Loyola-Rodrguez b, J.F. Reyes-Macas b, G. Ortega-Zarzosa c, N. Nio-Martnez c
a
Autonomous University of San Luis Potos, Salvador Nava Av. Universitary Zone, 78290 San Luis Potos, Mexico
b
Dentistry Faculty, Autonomous University of San Luis Potos, 2 Dr. Manuel Nava Av. University Zone, 78290 San Luis Potos, Mexico
c
Science Faculty, Autonomous University of San Luis Potos, Salvador Nava Av. University Zone, 78290 San Luis Potos, Mexico

art ic l e i nf o a b s t r a c t

Article history: A hybrid hydrogel composed of chitosan and silver nanoparticles was prepared. Silver nanoparticles were
Received 15 January 2013 synthetized prior to their incorporation into the gel and were characterized using DLS and TEM, the size
Accepted 17 May 2013 of silver nanoparticles was 7 nm and present spherical morphology. The chitosan gel was prepared in the
Available online 24 May 2013
presence of silver nanoparticles and was characterized using DSC and ESEM. The ability to release silver
Keywords: ions and the bactericide activity were also evaluated. The thermal properties of the chitosan gel
Nanoparticles containing silver nanoparticles are very similar to those presented by the chitosan gel without silver but
Chitosan with a slight increment on its thermal stability. This hydrogel releases silver ions during at least two
S. mutans weeks and its bactericide properties are comparable to that of chlorhexidine which is the antibacterial
Bactericidal Effect
most commonly used against S. mutans.
Hydrogel
& 2013 Elsevier B.V. All rights reserved.

1. Introduction treatment applications. In this work a hybrid hydrogel composed of

Silver nanoparticles (AgNP) have probed their efcacy as
alternative antimicrobial material over a wide variety of bacteria,
including Streptococcus mutans (S. mutans), a bacterium involved
in the development of dental caries [13]. However a vehicle to
transport nanoparticles to specic sites with a sustained activity is
needed; in this eld, the use of hydrogels in transporting drugs has
showed excellent results and one of the substances used to
prepare these hydrogels is chitosan [47]. Chitosan is a cationic
aminopolysaccharide obtained by the alkaline deacetylation of
chitin, it presents several characteristics such as bioadhesivity,
biodegradablility, biocompatibility, safety, non-toxicity and it pro-
motes drug absorption. All the previously mentioned characteristics
make chitosan a factible option to transport silver nanoparticles [8].
Sanpui et al. and Rhim et al. have reported the preparation of
different chitosanAg composites [9,10], however the way chitosan
and silver nanoparticles are combined is different to ours and what
they prepared are nanoparticles suspensions (where silver nanopar-
ticles are covered with chitosan molecules) or lms which are not as
maneagable as a gel when it comes about possible oral or skin
n
Corresponding author at: Dentistry Faculty, Autonomous University of San Luis
Potos, 2 Dr. Manuel Nava Av. University Zone, 78290 San Luis Potos, Mexico.
Tel./fax: +52 444 8262361.
E-mail addresses: mtzcastanon@fciencias.uaslp.mx, mtzcastanon@gmail.
com (G.A. Martnez-Castan).
chitosan and AgNP was prepared and characterized and its bacter-
icide activity was evaluated.
2. Materials and methods
Preparation and characterization of the solutionsAgNP disper-
sion: 0.169 g of AgNO3 were dissolved in 100 mL of deionized
water, then 0.1 g of gallic acid previously dissolved in 10 mL of
deionized water were added. Immediately after, the pH was
adjusted to 11 using a 0.1 M NaOH solution [2].
Chlorhexidine and gallic acid solutions: 5 mL of a commercial
chlorhexidine solution (2%, Consepsiss, Ultradent) was dissolved
in 95 mL of deionized water (nal concentration 0.1% v/v). On
the other hand, 0.1 g of gallic acid was dissolved in 100 mL of
deionized water. These solutions were prepared in order to obtain
a reference to compare the bactericide activity of the gel
containing AgNP.
AgNP were characterized by Dynamic Light Scattering (DLS) using
a Malvern Zetasizer Nano ZS operating with an HeNe laser at a
wavelength of 633 nm, and a detection angle of 901; all samples were
analyzed for 60 s at 25 1C. To conrm shape, AgNP were analyzed by
a transmission electron microscope (JEOL JEM-1230) at an accelerat-
ing voltage of 100 kV.
Preparation and characterization of gelsChitosan gel with AgNP,
chlorhexidine and gallic acid: 2 mL of acetic acid was dissolved to
48 mL of the previous solutions (or deionized water to prepare a

0167-577X/$ - see front matter & 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.matlet.2013.05.075
414 C. Smano-Valencia et al. / Materials Letters 106 (2013) 413416

chitosan gel as control), after that 1.7 g of chitosan and 0.2 mL of after that the gels were placed in 6 mm diameter wells and the
glycerol were incorporated under magnetic stirring until the gel plate was incubated for 24 h at 37 1C. After the incubation period
was formed. Four different gels were obtained. Chitosan gel and the inhibition zones were measured with a stereomicroscope
chitosan gel with AgNP were characterized using differential (Olympus, Japan) and a digital vernier (Fig. 1).
scanning calorimetry (DSC) in a DSC 500 calorimeter from Waters,
under a nitrogen atmosphere, using a constant heating rate of
20 1C/min from room temperature to 1000 1C. The distribution of
AgNP in the gel was observed using an environmental scanning
electron microscope (ESEM FEI Quanta 200); no special prepara-
tion of the sample was needed for this observation.
Releasing of silver ions: The ability to release silver ions from the
gel with AgNP was evaluated using a methodology reported in
previous references [4,14]. 1 mL of the gel was placed in a glass
test tube and covered with a dialysis tubing cellulose membrane
(12,000 Da). The tube was placed in a plastic container with 5 mL
of PBS. The solution was slightly agitated and after 24, 72 and
96 hours, one week and two weeks an aliquot was removed and
placed in a spectrophotometer (SmartSpec Plus BIO-RAD) to
measure absorbance at 302 nm.
Microbiological analysis: 22 Streptococcus mutans strains were
tested, 21 clinical isolates obtained in a previous study [2] and
1 reference strain. The strains were stored at 40 1C in BHI and
glycerol before used. Each strain was sowed on BHI plates in
triplicate and in each plate the gels were tested (n 66 for
each gel).
50 mL of bacteria were cultured in 5 mL of BHI broth and
incubated for 24 h at 37 1C. From this culture 100 mL (containing Fig. 2. Silver ions release from chitosan gel with AgNP.
2.3  107 UFC/mL) [2] were taken and extended over a BHI plate,

Fig. 1. BHI agar plates with the gels, (a) before and (b) after the incubation period. Fig. 3. DSC analysis of (a) chitosan gel, (b) chitosan gel with AgNP and.
 C. Smano-Valencia et al. / Materials Letters 106 (2013) 413416
3. Results and discussion
Characterization of AgNP and AgNP gel: AgNP size was 7 nm with
spherical shape (results not shown) for more details see references
[2,3]. Results from Ag+ release indicate that silver ion releasing
was continuous and prolonged with no initial burst release which
agrees with other reports where chitosan was used as a vehicle to
transport drugs [47], Fig. 2 shows that the maximum concentra-
tion was reached after one week with 21.0 70.7 mM (which
corresponds to a 70% of the initial silver content).
The TGA analysis for chitosan gel is shown in Fig. 3a, it shows a
rst weight loss of 80.8% between 40 and 180 1C that corresponds
to a water loss; and a second weight loss of 2.2% between 180 and
400 1C which is due to dehydration, depolymerization and decom-
position of the acetylated and deacetylated units of the chitosan.
For the chitosan gel with AgNP (Fig. 3b) the rst weight loss is 93%
and the second one 4.6%. DSC analysis shows two peaks for each
material, the rst one at 108.4 1C for chitosan gel and 116.4 1C for
the chitosan gel with AgNP; the second one at 362.2 1C for
chitosan gel and 366.3 1C for the chitosan gel with AgNP;
Fig. 4. ESEM images of chitosan gel with AgNP. Inset: EDS analysis of chitosan gel
with AgNP.
415
differences could indicate an increase of the thermal stability
associated to the sample containing AgNP [8].
Fig. 4 shows ESEM images (obtained using backscattered
electrons) of chitosan gel with AgNP, from this gure it seeems
that silver nanoparticles (brighter points), although agglomerated,
have a good incorporation into the chitosan matrix and the
distribution is quite uniform. The AgNP presence was corroborated
by the EDS analysis (inset in Fig. 3b).
Microbiological analysis: In the bacterium growth inhibition
test, the chitosan gel with AgNP present an inhibition halo of
10.27 mm 72.129 (n 66) thus conrming the usefulness of chit-
osan gel to provide a vehicle to transport silver nanoparticles with
bactericide activity, this activity is sustained as conrmed by the
release studies (Characterization of AgNP and AgNP gel). A chitosan
gel with chlorhexidine (20.79 mm 72.38) was prepared and used
as a control because this substance is a common oral antimicrobial
agent and the gel presentation is commercially available [11,12],
however the prolonged use of chlorhexidine produces several
secondary effects [13]. When comparing the groups there was no
statistically signicant difference (p4 0.05) between the chitosan
gel with chlorhexidine and the chitosan gel with AgNP, besides the
effect of the AgNP is prolonged. Samples prepared with gallic acid
and neat chitosan gel did not present bactericide activity and their
difference compared with the gel with chlorhexidine and the
chitosan gel with AgNP was statistically signicant (Kolmogorov
Smirnov was used to nd variable distribution, Kruskal Wallis for
analysis of variance and post-hoc Dunn test to nd differences
between the groups).
One major concern in application of nanoparticles and espe-
cially silver nanoparticles is cellular toxicity at high concentrations
of silver ions; we address this issue with a pilot test on wistar rats
were the gels were tested on an injury performed on the rats back
to evaluate the possible absence of gel resorption or any other
secondary reaction. Until now the rats have not showed any
strange reaction to the gels application.
4. Conclusion
A hybrid material composed of chitosan gel and silver nano-
particles was succesfully prepared. This material presents a good
and sustained bactericide activity, this features can be related to
the good distribution of the silver nanoparticles inside the chit-
osan matrix and to the sustained release of silver ions from the
matrix. This material could be a good choice for applications in
oral dessieses.
Acknowledgments
Fondo de Apoyo a la Investigacin (FAI C12-FAI-03-42.42), Pro-
grama de Mejoramiento del Profesorado (PROMEP) and Consejo
Nacional de Ciencia y Tecnologa (CONACYT CB-169020).
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