The aim of this work is to synthesize chitosan-gold nanoparticles films by direct chemical reduction of HAuCl4 in a chitosan solution and to investigate the influence of gold nanoparticles concentration on the structure of films, conductivity and healing effect on mice skin after surgery. Results obtained have shown that new chitosan-gold nanoparticle-collagen bionananocomposites demonstrated better healing effect on the mice skin after surgery than control performed on commercial TheraFormTM material.
When you are citing the document, use the following link http://essuir.sumdu.edu.ua/handle/123456789/3540

Franco Molina, M.A.

Luna-Bárcenas, G.

Prokhorov, E.

Rodríguez Padilla, C.

Valenzuela Acosta, E.M.

Zarate-Triviño, D.G.

Electronic Sumy State University Institutional Repository

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  NANOMATERIALS: APPLICATIONS AND PROPERTIES Vol. 2 No 4, 04NABM01(4pp) (2013)   2304-1862/2013/2(4)04NABM01(4) 04NABM01-1  2013 Sumy State University Chitosan-Gold Nanoparticle Composites for Biomedical Application  D.G. Zarate-Triviño1, E.M. Valenzuela Acosta1, E. Prokhorov1*, G. Luna-Bárcenas1,  C. Rodríguez Padilla2, M.A. Franco Molina2  1 CINVESTAV del IPN, Juriquilla 2000, Querétaro, México 2 Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, México  (Received 09 May 2013; published online 30 August 2013)  The aim of this work is to synthesize chitosan-gold nanoparticles films by direct chemical reduction of HAuCl4 in a chitosan solution and to investigate the influence of gold nanoparticles concentration on the structure of films, conductivity and healing effect on mice skin after surgery. Results obtained have shown that new chitosan-gold nanoparticle-collagen bionananocomposites demonstrated better healing effect on the mice skin after surgery than control performed on commercial TheraFormTM material.   Keywords: Chitosan, Gold nanoparticles, Dielectric spectroscopy, Healing effect.    PACS numbers: 77.84.Jd, 87.85.Qr                                                           * prokhorov@qro.cinvestav.mx 1. INTRODUCTION  Metal nanoparticles such as gold (AuNP’s) are ex-cellent candidates for biomedical and tissue engineer-ing applications. AuNP’s have unique properties, such as size- and shape-dependent optical and electronic features, a high surface-to-volume ratio, and surfaces that can be readily modified with ligands containing functional groups such as thiols, phosphines, amines [1, 2]. Different methods for synthesis of gold nanopar-ticles have been proposed. The most common method for AuNP’s synthesis is based on the reduction of HAuCl4 by sodium citrate in solution at high tempera-ture (Turkevitch method) [3]. The Brust-Schiffrin method usually involves use of organic solvents [4]. However, the use of organic solvents render AuNP’s unsuitable for application in biomedicine. In ref. [1] to improve the stability and uptake of nanoparticles has been proposed a novel method for synthesis and stabili-zation of gold nanoparticles using a natural, biocompat-ible polymer, chitosan. Chitosan (CS) is well known for its hydrophilic, biocompatible, biodegradable and non-toxic properties. Furthermore, the NH2 and/or OH groups of chitosan may act as reducing agents for the formation of gold nanoparticles [5]. Additional, chi-tosan promotes adsorption onto the surfaces of gold nanoparticles via electrostatic interactions between the amino groups and gold [6]. It is noteworthy that AuNP’s embedded in the three-dimensional chitosan matrix according to the effective media theory [7] must sufficiently change con-ductivity mechanism of CS / AuNP’s nanocomposites. But the majority of recent publications have been fo-cusing on the fabrication, microstructural characteriza-tion and sensibility of CS / AunP’s as biosensors, how-ever, electrical properties of such CS / AuNP’s films have not been reported which are important for optimi-zation material for different applications. Additional, in spite of fact that many studies have been reported that chitosan  is  a  substitute material in the  skin tissue engineering [8, 9], but from our knowledge in the liter-ature there are no any reports about application of CS / AuNP’s nanocomposites for treatment of skin damaged. Based upon the above discussion on the potential of AuNP’s, the aim of this work is to synthesize CS / AuNP’s films by direct reduction of HAuCl4 in chi-tosan solution and to investigate the influence of gold nanoparticles concentration on the structure, conduc-tivity and healing effect on the mice skin after surgery.  2. MATERIALS AND METHODS  The nanocomposites were synthesized by dissolving 2 wt. % of chitosan (with medium molecular weight 300,000 g/mol and 85 % of degree of deacetilation) in 1 wt. % acetic acid solution, afterward the HAuCl4 so-lution ranging from 0.06 mM to 1.7 mM were added and the mixture. The solutions were mixed and heated at 75 ºC under magnetic stirring until the solution changed its color from slight yellow to red. CS / AuNP’s thin films (thickness ca. 30 nm) were prepared by the solvent cast method by pouring the final solution into a plastic Petri dish and allowing the solvent to evaporate in oven for 24 h at 60 °C. CS / AuNP’s films morphology was analyzed by JEOM JSM-7401F Field emission scanning electron microscope. Crystal structure analysis was performed using a Rigaku diffractometer ULTIMA IV, equipped with the CuKα radiation (  1.5406 Å). The amount of free water was determined by ther-mogravimetric analysis (TGA) (Metler Toledo 851e model). The measurements were made with a dry air flow from 25 to 300 ºC with rate 10 ºC/min. Dielectric measurements in the frequency range from 40 Hz to 110 MHz were carried out Agilent Preci-sion Impedance Analyzer 4249A. The amplitude of measuring signal was 100 mV.  3. RESULTS AND DISCUSSION   3.1 Structure and Properties of CS/AuNP’s films  SEM micrographs show a homogeneous distribution  D.G. ZARATE-TRIVIÑO, E.M. VALENZUELA ACOSTA, ET AL. PROC. NAP 2, 04NABM01 (2013)   04NABM01-2 of the nanoparticles with dimension between 8 and 11 nm embedded in the chitosan matrix (not shown). XRD measurements (Fig. 1) show the peak of Au (111) appeared at around 38.1  of 2 which is in agreement with previous reports [10]. It is clear that volume fraction of AuNP’s increases with increasing with HAuCl4 concentration (increasing of intensity of Au diffraction line on Fig. 1). The crystalline size of nanoparticles d was estimated from XRD measure-ments using the Scherrer formula [11]:   0.9cosdFWHM,   where,  is wavelength, FWHM is full-width at half-maximum and  is the peak position of Au. The value of d was calculated about of 9 nm and is close to SEM measurements.    Fig. 1 – XRD pattern of films obtained from solution with dif-ferent HAuCl4 concentration  Free water content was determined by thermograv-imetric analysis (TGA). The amount of free water may be evaluated by the decrease of sample weight during the heating scan. The loss in weight at the temperature 120 C was taken to be the result of water evaporation. Fig. 2 shows the dependence of moisture content on the HAuCl4 concentration: increasing of HAuCl4 leads to the lower moisture content in CS / AunP’s composites.     Fig. 2 – Dependence of water content on HAuCl4 concentration  This effect can be related to the interaction of AuNP’s with the reactive NH2 and OH groups and the formation of a polymeric composite structure with na-noparticles which impeded the formation of H-bonding with water and decrease the water absorption ability with increasing of AuNP’s concentration in composites. This lower weight loss by increasing of the nanoparti-cles concentration has been previously observed in chi-tosan-Ag nanocomposites [12]. Fig. 3 shows the plots of the DC electrical conductivi-ty as a function of the HAuCl4 concentration obtained from impedance measurements at room temperature. Insert in Fig. 3 shows the typical complex imped-ance spectra measured in all films (open circles). This plot exhibits characteristic depressed semicircles at high frequencies and a quasi-linear response at low frequencies. According to previously reported studies [13], a depressed semicircle describes properties of bulk material and a quasi-linear response at low frequencies is associated with interfacial polarization and/or sur-face and contact effects.     Fig. 3 – DC conductivity of CS / AuNP’s films as a function of the of the HAuCl4 concentration obtained from impedance measurements at room temperature. Insert shows complex impedance spectra  The values of DC resistance Rdc, and the corre-sponding DC conductivity dc (dc  d/(Rdc  S), where d  film thickness and S  contact area) have been ob-tained from the intersection of the high frequency sem-icircle and the real-part axis on the impedance plane as it is shown in insert of Fig. 3 (red line).    Fig. 4 – Surgery process. The lesion nearby to head was implanted with material synthetized,  the lesion ubicated in the inferior part of animal was used like control  CHITOSAN-GOLD NANOPARTICLE COMPOSITES… PROC. NAP 2, 04NABM01 (2013)   04NABM01-3 According to effective media theory DC conductivi-ty of system with conductivity inclusion in polymer dielectric matrix must increasing [7]. But, in the case of CS / AuNP’s systems, DC conductivity at the low nano-particles concentration decreases. This conductivity decreasing can be explained by interaction of AuNP’s with citosan matrix. Such interaction is specific for chitosan composites and related with decreasing of moisture content (Fig. 2) and number of free H + and OH – ions which responsible for conductivity of pure chitosan [14]. At the higher AuNP’s concentration con-ductivity of CS / AunP’s nanocomposites increases due to the formation of conductivity paths in the composite formed by nanoparticles. That is why DC conductivity demonstrate minimum. This minimum appear due to competition of two mechanisms: decreasing number of free H + and OH – ions and the formation of conductivi-ty paths in the composite formed by nanoparticles. As a rule, interaction of AuNP’s with chitosan  matrix can be confirm by FTIR measurements by changing the bands shapes and frequencies. But these changes can be observed at relative high AuNP’s con-centration [15]. In our case the formation of a polymer-ic composite structure with nanoparticles has been con-firmed by TGA and conductivity measurements as have been discussed above.  3.2 Healing effect of CS/AuNP’s nanocomposites  To check the possibility application of CS / AuNP’s nanocomposites in the tissue engineering healing effect on the mice skin have been investigated. For applica-tion in tissue engineering, the material should meet the following requirements:  biocompatibility with the tissues; biodegradability; nontoxicity and has good me-chanical property. CS / AuNP’s nanocomposite re-spondents to all these requirements but has relative bad mechanical strength. To improve mechanical prop-erties 20 % of Glydidyl methacrylate (GMA) has been add to CS / AunP’s solution. GMA is a synthetic mono-mer which belongs to epoxy group. GMA can be func-tionalized on aqueous medium and can react with amines, carboxylic acids and hydroxides groups. This monomer has been used in dentistry like diluents of epoxy resins and is biodegradable. This new hybrid natural-synthetic hydrogel (CTS-g-GMA) change the mechanical properties, viscosity and swelling index of CS and can be used like cellular support for biomedical applications [16].  Additional to GMA 0.3 % of collagen (Col) dissolved in the acetic acid solution has been added to CS / AuNP’s solution. Collagen is a major protein com-ponent of bone, cartilage, skin, and connective tissue and also an important part of extracellular matrixes in animals [17]. More, chitosan-collagen membranes have great potential in skin tissue engineering [18]. CS-g-GMA-AuNP´s-Col thin films were prepared by the sol-vent cast method by pouring the final solution into a plastic Petri dish and allowing the solvent to evaporate in oven for 24 h at environment temperature.  Four CS-g-GMA-AuNP´s-Col films obtained from solution with different HAuCl4 concentration (0.15, 0.3, 0.6 and 1,07 mM) have been evaluated for investigation healing effect. Additional, commercial material Thera-FormTM and CS-g-GMA film were used like control. TheraFormTM is a commercial absorbable biomaterial based on collagen fabricated by Regenerative Medical System industry. It is biocompatible implant and using in the tissue regeneration [19]. The materials were cut into squares of 8  8 mm; afterwards cut materials were washed with buffer phosphate solution (PBS) 600 L by 5 minutes two times and 600 L of Dulbec-co's Modified Eagle Medium (DMEM) by 5 minutes three times. Finally the materials were sterilized by ultraviolet light during 10 minutes.  In vivo tests were carried out on the female mice stain BALB / C at 6 weeks old, with an average weight of 21 g. Each mouse was anesthetized with ketamine hydrochloride 100 mg/kg and xylazine 5 mg/kg. Subse-quently the back of animal was shaved carefully and the surgery consisted on removed two skins square of 5  5 mm (dermis and epidermis) on the back. The square nearby to head of mice was implanted with the synthesized materials and the other square was used like a control, without any materials (Fig. 4).    Fig. 5 – Cicatrization process of mices after 18 days.  Numbers on Figure show HAuCl4 concentration in solution from which films have been prepared  Fig. 5 shows cicatrization process of mices after 18 days after surgery. All materials have shown that new skin was growing without infection and inflamma-tory sings during healing process. Fig. 6 shows percent of recuperation of skin (esti-mated from dimension of cicatrize) using different ma-terials: TheraFormTM, CTS-g-GMA and CS-g-GMA-AuNP´s-Col films obtained from solution with different HAuCl4 concentration (0.15, 0.3, 0.6 and 1,07 mM). TheraFormTM, CTS-g-GMA and CS-g-GMA-AuNP´s-Col with low AuNP´s (below 0.6 mM) demonstrated appro-ximately the same recuperation ability. But films with high higher AuNP´s concentration have been demon-strated better recuperation ability than control com-mercial TheraFormTM and CTS-g-GMA materials. The healing of a skin wound is complicated courses,  D.G. ZARATE-TRIVIÑO, E.M. VALENZUELA ACOSTA, ET AL. PROC. NAP 2, 04NABM01 (2013)   04NABM01-4 including a wide range of cellular, molecular, physio-logical, and biological processes. Many studies and dif-ferent explanations have been reported on the use of chitosan as a skin substitute material in skin tissue engineering due to its many advantages such as hemo-stasis, accelerating the tissue regeneration and stimu-lating the fibroblast synthesis of collagen [8]. One of explanation proposed that the cationic nature of chi-tosan allows the electrostatic interactions with anionic glycosaminoglycans and proteoglycans distributed widely throughout the body and other negatively charged species [8].    Fig. 6 – Estimation of healing effect on mices.  Numbers on Figure show HAuCl4 concentration in solution from which films have been prepared  From another point of view AuNP´s wide used in fundamental cell biology applications [1]. As is the case with all nanomaterials, little is known about the inter-actions of nanoparticle with cells at the molecular level [1]. But it was been reported that AuNP´s can be modi-fied with chitosan reactive groups [20, 21]. So, we can propose that high surface area of AuNP´s and the cati-onic nature of chitosan increase the electrostatic inter-actions with anionic group and other negatively charged species that accelerate the tissue regeneration.   4. CONCLUSION  Chitosan-AuNP´s (with nanoparticle dimension about of 9 nm) films have been synthesized by direct reduction of HAuCl4 in chitosan solution. It was shown that DC conductivity dependent on AuNP’s concentra-tion and demonstrate minimum. This minimum appear due to competition of two mechanisms:  decreasing number of free H + and OH – ions and the formation of conductivity paths in the composite formed by nano-particles. Possible application of CS / AunP’s nanocomposites in the tissue engineering has been demonstrated on the healing effect on the mice skin. New CS / AuNP´s-Glydidyl methacrylate-collagen nanocomposites have been synthesized. It has been shown that new nanocomposite demonstrated better recuperation ability of skin than control commercial TheraFormTM material. This study indicated that a small amount of AuNP´s could induce significant changes in the physicochemical properties of chitosan, which may increase its biocompatibility and can be used in the tissue engineering.  AKNOWLEDGEMENTS  This work was partially supported by CONACYT of Mexico. The authors are grateful to M.A. Hernandez-Landaverde and J.A. Munoz-Salas for technical assis-tance.   REFERENCES  1. D.A. Giljohann, D.S. Seferos, W.L. Daniel, M.D. Massich, P.C. Patel, C.A. Mirkin, Angew. Chem. Int. Ed. 49, 3280 (2010). 2. D.R. Bhumkar, H.M Joshi, M. Sastry, V.B. Pokhar, Phar-maceutical Research 24, 1415 (2007). 3. J. Turkevich, P.C. Stevenson, J. Hillier, Discuss. Faraday Soc. 11, 55 (1951). 4. M. Brust, M. Walker, D. Bethell, D.J. Schiffrin, R. Whyman, Chem. Commun. 7, 801 (1994). 5. J. Shan, H. Tenhu, Chem. Commun. 44, 4580 (2007). 6. M.J. Hortiguela, I.A. Marıa, C. Gutierrez, M.L. Ferrer, F. del Monte, Biomacromolecules 12, 179 (2011). 7. D.S. McLachlan, J.H. Hwang, T.O. Mason, J. Elec-troceramics 5, 37 (2000).  8. I.Y. Kim, S.J. Seo, H.S. Moon, M.K. Yoo, I.Y. Park, B.C. Kim, C.S. Cho, Biotechnology Adv. 26, 1 (2008). 9. W. Gao, J. Lai, S.W. Leung, Frontiers in Physiology 3, 321 (2012). 10. A.N. Ananth, S. Umapathy, G. Ghosh, T. Ramprasath, M.A.J. Rajan, Adv. Nat. Sci.: Nanosci. Nanotechnol. 3, 045010 (2012). 11. Kratos Analytical Applications Note "Percent Crystallinity Determination by X-Ray Diffraction" (1999). 12. J.B. González-Campos, E. Prokhorov, J.G. Luna-Bárcenas, J. Lara-Romero, M.E. Mendoza Duarte, J. Polym. Sci. B. 48, 739 (2010). 13. J.B. González-Campos, E. Prokhorov, G. Luna-Bárcenas, A. Fonseca-García, I.C. Sanchez, J. Polym. Sci. B 47, 2259 (2009). 14. R.S. Radhakrishnan, A.A. Begum, S.S. Sri, Int. J. Cur. Res. 11, 085 (2010). 15. S.H. Hsu, Y.B. Chang, C.L. Tsai, K.Y. Fu, S.H. Wang, H.J. Tseng, Colloids Surf. B, 85, 198 (2011). 16. E.A. Elizalde Peña, D.G. Zarate Triviño, S.M. Nuño Donlucas, L. Medina Torres, J.E. Gough, I.C. Sanchez, F. Villaseñor, G. Luna Barcenas, J. Biomat. Sci. Polymer Ed. (2013). 17. R. Parenteau-Bareil, R. Gauvin, F. Berthod, Materials  3, 1863 (2010). 18. K.Y. Chen, W.J. Liao, S.M. Kuo, F.J. Tsai, Y.S. Chen, C.Y. Huang, C.H. Yao, Biomacromolecules 10, 1642 (2009). 19. Regenerative Bio-matrix. http://RMSbio.net. 20.  H. Huang, Q. Yuan, X. Yang, J. Colloid Interf. Sci. 282, 26 (2005). 21. D.R. Bhumkar, H.M. Joshi, M. Sastry, V.B. Pokharkar, Pharmaceutical Res. 24, 8, 1415 (2007).  

Chitosan-Gold Nanoparticle Composites for Biomedical Application

The aim of this work is to synthesize chitosan-gold nanoparticles films by direct chemical reduction of HAuCl4 in a chitosan solution and to investigate the influence of gold nanoparticles concentration on the structure of films, conductivity and healing effect on mice skin after surgery. Results obtained have shown that new chitosan-gold nanoparticle-collagen bionananocomposites demonstrated better healing effect on the mice skin after surgery than control performed on commercial TheraFormTM material

D. G. Zarate-triviño

E. M. Valenzuela Acosta

E. Prokhorov

G. Luna-bárcenas

C. Rodríguez Padilla

M. A. Franco Molina

CiteSeerX

SocioEconomic Challenges Journal (Sumy State University)

PROCEEDINGS OF THE INTERNATIONAL CONFERENCE  
NANOMATERIALS: APPLICATIONS AND PROPERTIES 
Vol. 2 No 4, 04NABM01(4pp) (2013) 
 
 
2304-1862/2013/2(4)04NABM01(4) 04NABM01-1  2013 Sumy State University 
Chitosan-Gold Nanoparticle Composites for Biomedical Application 
 
D.G. Zarate-Triviño1, E.M. Valenzuela Acosta1, E. Prokhorov1*, G. Luna-Bárcenas1,  
C. Rodríguez Padilla2, M.A. Franco Molina2 
 
1 CINVESTAV del IPN, Juriquilla 2000, Querétaro, México 
2 Facultad de Ciencias Biológicas, Universidad Autónoma de Nuevo León, México 
 
(Received 09 May 2013; published online 30 August 2013) 
 
The aim of this work is to synthesize chitosan-gold nanoparticles films by direct chemical reduction of 
HAuCl4 in a chitosan solution and to investigate the influence of gold nanoparticles concentration on the 
structure of films, conductivity and healing effect on mice skin after surgery. Results obtained have shown 
that new chitosan-gold nanoparticle-collagen bionananocomposites demonstrated better healing effect on 
the mice skin after surgery than control performed on commercial TheraFormTM material.  
 
Keywords: Chitosan, Gold nanoparticles, Dielectric spectroscopy, Healing effect.  
 
 PACS numbers: 77.84.Jd, 87.85.Qr 
 
 
                                                        
* prokhorov@qro.cinvestav.mx 
1. INTRODUCTION 
 
Metal nanoparticles such as gold (AuNP’s) are ex-
cellent candidates for biomedical and tissue engineer-
ing applications. AuNP’s have unique properties, such 
as size- and shape-dependent optical and electronic 
features, a high surface-to-volume ratio, and surfaces 
that can be readily modified with ligands containing 
functional groups such as thiols, phosphines, amines 
[1, 2]. Different methods for synthesis of gold nanopar-
ticles have been proposed. The most common method 
for AuNP’s synthesis is based on the reduction of 
HAuCl4 by sodium citrate in solution at high tempera-
ture (Turkevitch method) [3]. The Brust-Schiffrin 
method usually involves use of organic solvents [4]. 
However, the use of organic solvents render AuNP’s 
unsuitable for application in biomedicine. In ref. [1] to 
improve the stability and uptake of nanoparticles has 
been proposed a novel method for synthesis and stabili-
zation of gold nanoparticles using a natural, biocompat-
ible polymer, chitosan. Chitosan (CS) is well known for 
its hydrophilic, biocompatible, biodegradable and non-
toxic properties. Furthermore, the NH2 and/or OH 
groups of chitosan may act as reducing agents for the 
formation of gold nanoparticles [5]. Additional, chi-
tosan promotes adsorption onto the surfaces of gold 
nanoparticles via electrostatic interactions between the 
amino groups and gold [6]. 
It is noteworthy that AuNP’s embedded in the 
three-dimensional chitosan matrix according to the 
effective media theory [7] must sufficiently change con-
ductivity mechanism of CS / AuNP’s nanocomposites. 
But the majority of recent publications have been fo-
cusing on the fabrication, microstructural characteriza-
tion and sensibility of CS / AunP’s as biosensors, how-
ever, electrical properties of such CS / AuNP’s films 
have not been reported which are important for optimi-
zation material for different applications. Additional, in 
spite of fact that many studies have been reported that 
chitosan  is  a  substitute material in the  skin tissue 
engineering [8, 9], but from our knowledge in the liter-
ature there are no any reports about application of 
CS / AuNP’s nanocomposites for treatment of skin 
damaged. 
Based upon the above discussion on the potential of 
AuNP’s, the aim of this work is to synthesize 
CS / AuNP’s films by direct reduction of HAuCl4 in chi-
tosan solution and to investigate the influence of gold 
nanoparticles concentration on the structure, conduc-
tivity and healing effect on the mice skin after surgery. 
 
2. MATERIALS AND METHODS 
 
The nanocomposites were synthesized by dissolving 
2 wt. % of chitosan (with medium molecular weight 
300,000 g/mol and 85 % of degree of deacetilation) in 
1 wt. % acetic acid solution, afterward the HAuCl4 so-
lution ranging from 0.06 mM to 1.7 mM were added 
and the mixture. The solutions were mixed and heated 
at 75 ºC under magnetic stirring until the solution 
changed its color from slight yellow to red. CS / AuNP’s 
thin films (thickness ca. 30 nm) were prepared by the 
solvent cast method by pouring the final solution into a 
plastic Petri dish and allowing the solvent to evaporate 
in oven for 24 h at 60 °C. 
CS / AuNP’s films morphology was analyzed by 
JEOM JSM-7401F Field emission scanning electron 
microscope. Crystal structure analysis was performed 
using a Rigaku diffractometer ULTIMA IV, equipped 
with the CuKα radiation (  1.5406 Å). 
The amount of free water was determined by ther-
mogravimetric analysis (TGA) (Metler Toledo 851e 
model). The measurements were made with a dry air 
flow from 25 to 300 ºC with rate 10 ºC/min. 
Dielectric measurements in the frequency range 
from 40 Hz to 110 MHz were carried out Agilent Preci-
sion Impedance Analyzer 4249A. The amplitude of 
measuring signal was 100 mV. 
 
3. RESULTS AND DISCUSSION  
 
3.1 Structure and Properties of CS/AuNP’s films 
 
SEM micrographs show a homogeneous distribution 
 D.G. ZARATE-TRIVIÑO, E.M. VALENZUELA ACOSTA, ET AL. PROC. NAP 2, 04NABM01 (2013) 
 
 
04NABM01-2 
of the nanoparticles with dimension between 8 and 
11 nm embedded in the chitosan matrix (not shown). 
XRD measurements (Fig. 1) show the peak of Au 
(111) appeared at around 38.1  of 2 which is in 
agreement with previous reports [10]. It is clear that 
volume fraction of AuNP’s increases with increasing 
with HAuCl4 concentration (increasing of intensity of 
Au diffraction line on Fig. 1). The crystalline size of 
nanoparticles d was estimated from XRD measure-
ments using the Scherrer formula [11]: 
 
 
0.9
cos
d
FWHM




,  
 
where,  is wavelength, FWHM is full-width at half-
maximum and  is the peak position of Au. The value of 
d was calculated about of 9 nm and is close to SEM 
measurements. 
 
 
 
Fig. 1 – XRD pattern of films obtained from solution with dif-
ferent HAuCl4 concentration 
 
Free water content was determined by thermograv-
imetric analysis (TGA). The amount of free water may 
be evaluated by the decrease of sample weight during 
the heating scan. The loss in weight at the temperature 
120 C was taken to be the result of water evaporation. 
Fig. 2 shows the dependence of moisture content on the 
HAuCl4 concentration: increasing of HAuCl4 leads to 
the lower moisture content in CS / AunP’s composites.  
 
 
 
Fig. 2 – Dependence of water content on HAuCl4 concentration 
 
This effect can be related to the interaction of 
AuNP’s with the reactive NH2 and OH groups and the 
formation of a polymeric composite structure with na-
noparticles which impeded the formation of H-bonding 
with water and decrease the water absorption ability 
with increasing of AuNP’s concentration in composites. 
This lower weight loss by increasing of the nanoparti-
cles concentration has been previously observed in chi-
tosan-Ag nanocomposites [12]. 
Fig. 3 shows the plots of the DC electrical conductivi-
ty as a function of the HAuCl4 concentration obtained 
from impedance measurements at room temperature. 
Insert in Fig. 3 shows the typical complex imped-
ance spectra measured in all films (open circles). This 
plot exhibits characteristic depressed semicircles at 
high frequencies and a quasi-linear response at low 
frequencies. According to previously reported studies 
[13], a depressed semicircle describes properties of bulk 
material and a quasi-linear response at low frequencies 
is associated with interfacial polarization and/or sur-
face and contact effects.  
 
 
 
Fig. 3 – DC conductivity of CS / AuNP’s films as a function of 
the of the HAuCl4 concentration obtained from impedance 
measurements at room temperature. Insert shows complex 
impedance spectra 
 
The values of DC resistance Rdc, and the corre-
sponding DC conductivity dc (dc  d/(Rdc  S), where 
d  film thickness and S  contact area) have been ob-
tained from the intersection of the high frequency sem-
icircle and the real-part axis on the impedance plane as 
it is shown in insert of Fig. 3 (red line). 
 
 
 
Fig. 4 – Surgery process. The lesion nearby to head was 
implanted with material synthetized,  the lesion ubicated in 
the inferior part of animal was used like control 
 CHITOSAN-GOLD NANOPARTICLE COMPOSITES… PROC. NAP 2, 04NABM01 (2013) 
 
 
04NABM01-3 
According to effective media theory DC conductivi-
ty of system with conductivity inclusion in polymer 
dielectric matrix must increasing [7]. But, in the case of 
CS / AuNP’s systems, DC conductivity at the low nano-
particles concentration decreases. This conductivity 
decreasing can be explained by interaction of AuNP’s 
with citosan matrix. Such interaction is specific for 
chitosan composites and related with decreasing of 
moisture content (Fig. 2) and number of free H + and 
OH – ions which responsible for conductivity of pure 
chitosan [14]. At the higher AuNP’s concentration con-
ductivity of CS / AunP’s nanocomposites increases due 
to the formation of conductivity paths in the composite 
formed by nanoparticles. That is why DC conductivity 
demonstrate minimum. This minimum appear due to 
competition of two mechanisms: decreasing number of 
free H + and OH – ions and the formation of conductivi-
ty paths in the composite formed by nanoparticles. 
As a rule, interaction of AuNP’s with chitosan  
matrix can be confirm by FTIR measurements by 
changing the bands shapes and frequencies. But these 
changes can be observed at relative high AuNP’s con-
centration [15]. In our case the formation of a polymer-
ic composite structure with nanoparticles has been con-
firmed by TGA and conductivity measurements as have 
been discussed above. 
 
3.2 Healing effect of CS/AuNP’s nanocomposites 
 
To check the possibility application of CS / AuNP’s 
nanocomposites in the tissue engineering healing effect 
on the mice skin have been investigated. For applica-
tion in tissue engineering, the material should meet 
the following requirements:  biocompatibility with the 
tissues; biodegradability; nontoxicity and has good me-
chanical property. CS / AuNP’s nanocomposite re-
spondents to all these requirements but has relative 
bad mechanical strength. To improve mechanical prop-
erties 20 % of Glydidyl methacrylate (GMA) has been 
add to CS / AunP’s solution. GMA is a synthetic mono-
mer which belongs to epoxy group. GMA can be func-
tionalized on aqueous medium and can react with 
amines, carboxylic acids and hydroxides groups. This 
monomer has been used in dentistry like diluents of 
epoxy resins and is biodegradable. This new hybrid 
natural-synthetic hydrogel (CTS-g-GMA) change the 
mechanical properties, viscosity and swelling index of 
CS and can be used like cellular support for biomedical 
applications [16].  
Additional to GMA 0.3 % of collagen (Col) dissolved 
in the acetic acid solution has been added to 
CS / AuNP’s solution. Collagen is a major protein com-
ponent of bone, cartilage, skin, and connective tissue 
and also an important part of extracellular matrixes in 
animals [17]. More, chitosan-collagen membranes have 
great potential in skin tissue engineering [18]. CS-g-
GMA-AuNP´s-Col thin films were prepared by the sol-
vent cast method by pouring the final solution into a 
plastic Petri dish and allowing the solvent to evaporate 
in oven for 24 h at environment temperature.  
Four CS-g-GMA-AuNP´s-Col films obtained from 
solution with different HAuCl4 concentration (0.15, 0.3, 
0.6 and 1,07 mM) have been evaluated for investigation 
healing effect. Additional, commercial material Thera-
FormTM and CS-g-GMA film were used like control. 
TheraFormTM is a commercial absorbable biomaterial 
based on collagen fabricated by Regenerative Medical 
System industry. It is biocompatible implant and using 
in the tissue regeneration [19]. The materials were cut 
into squares of 8  8 mm; afterwards cut materials 
were washed with buffer phosphate solution (PBS) 
600 L by 5 minutes two times and 600 L of Dulbec-
co's Modified Eagle Medium (DMEM) by 5 minutes 
three times. Finally the materials were sterilized by 
ultraviolet light during 10 minutes.  
In vivo tests were carried out on the female mice 
stain BALB / C at 6 weeks old, with an average weight 
of 21 g. Each mouse was anesthetized with ketamine 
hydrochloride 100 mg/kg and xylazine 5 mg/kg. Subse-
quently the back of animal was shaved carefully and 
the surgery consisted on removed two skins square of 
5  5 mm (dermis and epidermis) on the back. The 
square nearby to head of mice was implanted with the 
synthesized materials and the other square was used 
like a control, without any materials (Fig. 4). 
 
 
 
Fig. 5 – Cicatrization process of mices after 18 days.  Numbers 
on Figure show HAuCl4 concentration in solution from which 
films have been prepared 
 
Fig. 5 shows cicatrization process of mices after 
18 days after surgery. All materials have shown that 
new skin was growing without infection and inflamma-
tory sings during healing process. 
Fig. 6 shows percent of recuperation of skin (esti-
mated from dimension of cicatrize) using different ma-
terials: TheraFormTM, CTS-g-GMA and CS-g-GMA-
AuNP´s-Col films obtained from solution with different 
HAuCl4 concentration (0.15, 0.3, 0.6 and 1,07 mM). 
TheraFormTM, CTS-g-GMA and CS-g-GMA-AuNP´s-Col 
with low AuNP´s (below 0.6 mM) demonstrated appro-
ximately the same recuperation ability. But films with 
high higher AuNP´s concentration have been demon-
strated better recuperation ability than control com-
mercial TheraFormTM and CTS-g-GMA materials. 
The healing of a skin wound is complicated courses, 
 D.G. ZARATE-TRIVIÑO, E.M. VALENZUELA ACOSTA, ET AL. PROC. NAP 2, 04NABM01 (2013) 
 
 
04NABM01-4 
including a wide range of cellular, molecular, physio-
logical, and biological processes. Many studies and dif-
ferent explanations have been reported on the use of 
chitosan as a skin substitute material in skin tissue 
engineering due to its many advantages such as hemo-
stasis, accelerating the tissue regeneration and stimu-
lating the fibroblast synthesis of collagen [8]. One of 
explanation proposed that the cationic nature of chi-
tosan allows the electrostatic interactions with anionic 
glycosaminoglycans and proteoglycans distributed 
widely throughout the body and other negatively 
charged species [8]. 
 
 
 
Fig. 6 – Estimation of healing effect on mices.  Numbers on 
Figure show HAuCl4 concentration in solution from which 
films have been prepared 
 
From another point of view AuNP´s wide used in 
fundamental cell biology applications [1]. As is the case 
with all nanomaterials, little is known about the inter-
actions of nanoparticle with cells at the molecular level 
[1]. But it was been reported that AuNP´s can be modi-
fied with chitosan reactive groups [20, 21]. So, we can 
propose that high surface area of AuNP´s and the cati-
onic nature of chitosan increase the electrostatic inter-
actions with anionic group and other negatively 
charged species that accelerate the tissue regeneration.  
 
4. CONCLUSION 
 
Chitosan-AuNP´s (with nanoparticle dimension 
about of 9 nm) films have been synthesized by direct 
reduction of HAuCl4 in chitosan solution. It was shown 
that DC conductivity dependent on AuNP’s concentra-
tion and demonstrate minimum. This minimum appear 
due to competition of two mechanisms:  decreasing 
number of free H + and OH – ions and the formation of 
conductivity paths in the composite formed by nano-
particles. 
Possible application of CS / AunP’s nanocomposites 
in the tissue engineering has been demonstrated on the 
healing effect on the mice skin. 
New CS / AuNP´s-Glydidyl methacrylate-collagen 
nanocomposites have been synthesized. It has been 
shown that new nanocomposite demonstrated better 
recuperation ability of skin than control commercial 
TheraFormTM material. This study indicated that a 
small amount of AuNP´s could induce significant 
changes in the physicochemical properties of chitosan, 
which may increase its biocompatibility and can be 
used in the tissue engineering. 
 
AKNOWLEDGEMENTS 
 
This work was partially supported by CONACYT of 
Mexico. The authors are grateful to M.A. Hernandez-
Landaverde and J.A. Munoz-Salas for technical assis-
tance. 
 
 
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