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Materials Letters
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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.
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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 415
3. Results and discussion differences could indicate an increase of the thermal stability
associated to the sample containing AgNP [8].
Characterization of AgNP and AgNP gel: AgNP size was 7 nm with Fig. 4 shows ESEM images (obtained using backscattered
spherical shape (results not shown) for more details see references electrons) of chitosan gel with AgNP, from this gure it seeems
[2,3]. Results from Ag+ release indicate that silver ion releasing that silver nanoparticles (brighter points), although agglomerated,
was continuous and prolonged with no initial burst release which have a good incorporation into the chitosan matrix and the
agrees with other reports where chitosan was used as a vehicle to distribution is quite uniform. The AgNP presence was corroborated
transport drugs [47], Fig. 2 shows that the maximum concentra- by the EDS analysis (inset in Fig. 3b).
tion was reached after one week with 21.0 70.7 mM (which Microbiological analysis: In the bacterium growth inhibition
corresponds to a 70% of the initial silver content). test, the chitosan gel with AgNP present an inhibition halo of
The TGA analysis for chitosan gel is shown in Fig. 3a, it shows a 10.27 mm 72.129 (n 66) thus conrming the usefulness of chit-
rst weight loss of 80.8% between 40 and 180 1C that corresponds osan gel to provide a vehicle to transport silver nanoparticles with
to a water loss; and a second weight loss of 2.2% between 180 and bactericide activity, this activity is sustained as conrmed by the
400 1C which is due to dehydration, depolymerization and decom- release studies (Characterization of AgNP and AgNP gel). A chitosan
position of the acetylated and deacetylated units of the chitosan. gel with chlorhexidine (20.79 mm 72.38) was prepared and used
For the chitosan gel with AgNP (Fig. 3b) the rst weight loss is 93% as a control because this substance is a common oral antimicrobial
and the second one 4.6%. DSC analysis shows two peaks for each agent and the gel presentation is commercially available [11,12],
material, the rst one at 108.4 1C for chitosan gel and 116.4 1C for however the prolonged use of chlorhexidine produces several
the chitosan gel with AgNP; the second one at 362.2 1C for secondary effects [13]. When comparing the groups there was no
chitosan gel and 366.3 1C for the chitosan gel with AgNP; 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
Acknowledgments
References
[1] Morones JR, Elechiguerra JL, Camacho A, Holt K, Kouri JB, Tapia Ramrez J, et al.
Nanotechnology 2005;16:234653.
[2] Espinosa Cristbal LF, Martnez Castan GA, Martnez Martnez RE,
Loyola Rodrguez JP, Patio Marn N, Reyes Macas JF, et al. Mater Lett
2009;63:26036.
[3] Martnez Castan GA, Nio Martnez N, Martnez Gutirrez F, Martnez
Mendoza JR, Ruiz F. J Nanopart Res 2008;10:13438.
[4] Gomes do Nascimento E, Montenegro Sampaio TB, Cunha Madeiros A,
Pereira de Azevedo E. Acta Cir Bras 2009;24:4605.
[5] Wang Y, Chen M, Li X, Huang Y, Liang W. J Biomater Sci Polym Ed
2008;19:123947.
Fig. 4. ESEM images of chitosan gel with AgNP. Inset: EDS analysis of chitosan gel [6] Fontana CR, Dos Santos Jr DS, Bosco JM, Spolidorio DM, Marcantonio RAC. Drug
with AgNP. Delivery 2008;15:41722.
416 C. Smano-Valencia et al. / Materials Letters 106 (2013) 413416
[7] Smelcerovic A, Knezevic Jugovic Z, Petronijevic Z. Curr Pharm Des [11] Xia JQ, Sheng ZQ, Deng J, Lu R. J Mater Sci: Mater Med 2010;21:243542.
2008;14:316895. [12] Slot DE, Rosema NAM, Hennequin-Hoenderdos NL, Versteeg PA, Van der
[8] Hernndez R, Zamora Mora V, Sibaja Ballesteros M, Vega Baudrit J, Lpez D, Velden U, Van der Weijden GA. Int J Dent Hyg 2010;8:294300.
Mijangos C. J Colloid Interface Sci 2009;339:539. [13] Seymour RA, Meechan JG, Yates MS. Pharmacology and dental therapeutics.
[9] Sanpui P, Murugadoss A, Prasad PVD, Ghosh SS, Chattopadhyay A. Int J Food 3th ed. New York: Oxford University Press; 2001.
Microbiol 2008;124:1426. [14] Mallik M, Mandal RK. Indian J Eng Mater Sci 2008;15:4258.
[10] Rhim JW, Hong SI, Park HM, Perry KWN. J Agric Food Chem 2006;54:581422.