Journal of Colloid and Interface Science 364 (2011) 8084

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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 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
b

a r t i c l e

i n f o

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 electrostatically attract Ag+ inside CS-NP, where it was reduced by the terminal glucosamine units of the biopolymer. 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 presenting a mean hydrodynamic diameter of 78 nm. The antimicrobial activity of Ag@CS-NP against Candida glabrata, Sacharomyces cerevisiae, Escherichia coli, Klebsiella pneumoniae, Salmonella, Staphylococcus aureus, and Bacillus cereus corresponded to MIC values lower than for AgNO3. 2011 Elsevier Inc. All rights reserved.

Article history: Received 17 May 2011 Accepted 3 August 2011 Available online 17 August 2011 Keywords: Antimicrobial Chitosan Nanoparticle Nanocomposite Silver TEM

1. Introduction Chitosan is a biopolymer of (1 ? 4)-2-amino-2-deoxy-b-D-glucan and (1 ? 4)-2-acetamido-2-deoxy-b-D-glucan units, generally predominating in the former units [1]. At pH < 6, the polymer dissolves due to protonation of the amine groups, as represented in Fig. 1. Chitosan is a non-toxic, inexpensive, and biocompatible polymer, biodegradable by different hydrolytic enzymes [2]. This biopolymer presents very important biological properties among which antimicrobial, anti-inammatory, antioxidant, and antitumoral can be cited [3]. Chitosan has been widely used in the regeneration 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 tripolyphosphate (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 property will permit the development of a wide variety of systems with important biomedical and pharmaceutical applications.
Corresponding author.
E-mail address: caov@fq.uh.cu (R. Cao). 0021-9797/$ - see front matter 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.jcis.2011.08.006

Chitosan has served to obtain different types of metal nanoparticle-polymer composites. Using chitosan as a polymeric matrix and Na[BH4] 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 nm or larger, since smaller ones are difcult to obtain [17]. Different green methods (no use of Na[BH4] nor other contaminating reducing agents) have been reported to synthesize sub10-nm AgNP, but generally of 3 nm or higher diameters [1821]. For example, 35 nm AgNP were obtained using polyphosphonate and H2 as reducing agent [18]. A similar procedure, but using phosphonated calixerenes, gave 2.115 nm AgNP, where the size depended on the amount of AgNO3 and type of calixarene used [19]. H2 is a clean reducing agent, but must be used with caution. Starch has also been used as a clean reducing agent of AgNO3, 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 bionanocarrier of AgNP. This was because the resulting system could serve for biological applications combining the interesting properties of both components, including the antimicrobial properties of AgNP [22,23].

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CH2OH

2.6. Strains and culture conditions Escherichia coli (E. coli) CECT 101, Klebsiella pneumoniae (K. pneumoniae) 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. cerevisiae) 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 5 104 bacteria/mL and 1 103 yeast/mL were used. The minimal inhibitory concentration (MIC, lg/mL) was dened as the lowest concentration of compound inhibiting the growth of each strain. The tubes were incubated 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 optimum amounts of each reagent. The formation of CS-NP was controlled 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-

OH NH3
+

Fig. 1. Schematic representation of chitosan through its predominating protonated (1 ? 4)-2-amino-2-deoxy-b-D-glucan units.

2. Materials and methods 2.1. Materials Low molecular weight chitosan (85% deacetylation) and sodium tripolyphosphate (TPP) were purchased from Aldrich; AgNO3 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 working at 200 kV. Dynamic light scattering (DLS) measurements of obtained CSNP and Ag@CS-NP aqueous solutions were performed on a Nanotrac 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 previous 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 conrmed 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 AgNO3 (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.
- --

---

Ag+ (100 C)

2.5. Enzymatic treatment of samples The enzymatic treatment of the Ag@CS-NP samples was carried out using a chitosanase from Streptomyces 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 samples was adjusted to 5.5 using 1 M Tris buffer pH 7. Reactions containing 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.
---

- --

Fig. 2. Schematic representation of the formation of AgNP inside CS-NP, where the negatively charged semicircular sections correspond to TPP.

-- -

-- -

Ag+

- -

- -

- -

---

- - - Ag+ - - -+ Ag+ Ag+

Ag+ Ag Ag+ Ag+ Ag+ Ag+ Ag+

- -

Ag+

- -

Ag+

- -

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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 synthesis 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 correspond to TPP. According to the schematic representation given in Fig. 2, the resulting Ag@CS-NP system could be considered similar to a pudding 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 AgNO3 are presented. The measured 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 predominant structure expected. Other amounts of AgNO3 (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 AgNO3, the average diameter of AgNP was of 0.95 (0.30) nm (Fig. 4b), while when using 200 mg of AgNO3, the obtained 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 AgNO3 used. This result was unexpected considering that the concentration of AgNO3 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 AgNO3. 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 AgNO3; (b) 5 mg; (c) 200 mg; (d) also AgNP formed in CS with 5 mg of AgNO3.

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AgNP was also synthesized in CS (dissolved in acetic acid) using a similar procedure to that reported here for Ag@CS-NP, but without 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 AgNO3 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 nanoparticles (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 attributed 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 modulate the size of AgNP. As mentioned in the Introduction, with phosphonated calixarenes, relatively small AgNP have been reported [19], where the former 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 reaction 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 occurs at a rate higher than the capacity of the capping component to cover the formed nanoparticle. An opposite tendency is observed when a mild reducing agent is used, especially when it participates at low and sustained concentrations, as is the case of H2 [17]. In our case, the glucosamine unit constitutes an abundant mild reducing agent, which is accessed by Ag+ by a constrained diffusion 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 determined by dynamic light scattering (DLS) was of 78 (19) nm, a better dened value. Here, it is important to mention that the diameters that are obtained by DLS are larger than those determined 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 distribution of our CS-NP (without the presence of silver) was of 576 (145) nm according to DLS determinations. This average size is about seven times larger than the corresponding value of Ag@CS-NP, as already mention above. The signicant difference between 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 formation 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 produced 173 (0.75) lg/mL, while for Ag@CS-NP only 134 (9.6) lg/mL of glucosamine was formed. Evidently, CS-NP is permeable to small species (as Ag+ cations), but not to large species as enzymes. The enzyme used was of 39 kDa, with a mean diameter 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 between 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 concentration of unreduced Ag+ that remained in the Ag@CS-NP solution presented a discrete dependence on the amount of AgNO3 added in each synthesis. When 5 mg of AgNO3 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 AgNO3 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 (studied by isothermal titration calorimetry), high binding constants of the order of 105 M1 were determined [30]. The results of antimicrobial activity of AgNO3, 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. cerevisiae, 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 antimicrobial activity signicantly higher than AgNO3. This result was not affected by CS-NP, which presented a low antimicrobial activity, for which no correction was made. Especially signicant resulted 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 electrostatically. Nevertheless, such type of interaction is dynamic in nature, since the density of the negatively charged surfaces of these systems varies according to the environment and other factors [31].

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Table 1 Antimicrobial activity of AgNO3, 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 C. glabrata AgNO3 CS-NP Ag@CS-NP 24 >24 5 (5) S. cerevisiae >24 >24 5 (20) Gram negative bacteria E. coli 6 (12) >24 1.3 (1.3) K. pneumoniae 12 (24) >24 2.5 (2.5) Salmonella sp 24 (>24) >24 2.5 (5) Gram positive bacteria S. aureus 12 (>24) >24 2.5 (2.5) B. cereus 12 (>4) >24 1.3 (2.5)

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 interact 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 O2, a process that provokes apoptosis [14,23,32]. In this sense, it is important to take into consideration 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 controlled release liberation system of Ag+ and AgNP. Therefore, these characteristics should be interpreted as if Ag@CS-NP would be acting 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 activity 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 AgNO3 included in CS-NP [35].

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. References
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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 nanoparticles, 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.