Journal of Colloid and Interface Science 364 (2011) 80–84

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Journal of Colloid and Interface Science

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Chitosan and silver nanoparticles as pudding with raisins withantimicrobial properties

M. Carmen Rodríguez-Argüelles a, Carmen Sieiro b, Roberto Cao c,⇑, Lucia Nasi d

a Departamento de Química Inorgánica, Universidade de Vigo, 36310 Vigo, Spainb Departamento de Biología Funcional y Ciencias de la Salud, Area de Microbiología, Universidade de Vigo, 36310 Vigo, Spainc Laboratorio de Bioinorgánica, Facultad de Química, Universidad de La Habana, La Habana 10400, Cubad IMEM-CNR, Parco Area delle Scienze 37/A, I-43124 Parma, Italy

a r t i c l e i n f o a b s t r a c t

Article history:Received 17 May 2011Accepted 3 August 2011Available online 17 August 2011

Keywords:AntimicrobialChitosanNanoparticleNanocompositeSilverTEM

0021-9797/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.jcis.2011.08.006

⇑ Corresponding author.E-mail address: caov@fq.uh.cu (R. Cao).

Chitosan nanoparticles (CS-NP) containing small silver nanoparticles are reported (Ag@CS-NP). CS-NPwas 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(0.93–1.7 nm), with only a discrete dependence from the amount of silver nitrate used (5–200 mg). Theobtained hybrid nanocomposites Ag@CS-NP were characterized by DLS, HRTEM, and HAADF–STEM 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, Staphylococcusaureus, and Bacillus cereus corresponded to MIC values lower than for AgNO3.

� 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, generallypredominating in the former units [1]. At pH < 6, the polymer dis-solves due to protonation of the amine groups, as represented inFig. 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 amongwhich antimicrobial, anti-inflammatory, 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 astrong 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 theinclusion (entrapment) of different types of compounds making itable to efficiently function as bionanocarriers [11–14]. Such prop-erty will permit the development of a wide variety of systems withimportant biomedical and pharmaceutical applications.

ll rights reserved.

Chitosan has served to obtain different types of metal nanopar-ticle-polymer composites. Using chitosan as a polymeric matrixand Na[BH4] as reducing agent, relatively small silver nanoparticles(AgNP) have recently been reported [15,16]. These AgNP presentedsurface plasmon resonance (SPR) maxima within 410–420 nm,assuming diameters lower than 5 nm. The position of the SPR banddepended on the proportions in which the reagents were mixed.

Generally, AgNP are synthesized with diameters of 10 nm or lar-ger, since smaller ones are difficult to obtain [17]. Different ‘‘green’’methods (no use of Na[BH4] nor other contaminating reducingagents) have been reported to synthesize sub10-nm AgNP, but gen-erally of 3 nm or higher diameters [18–21]. For example, 3–5 nmAgNP were obtained using polyphosphonate and H2 as reducingagent [18]. A similar procedure, but using phosphonated calixe-renes, gave 2.1–15 nm AgNP, where the size depended on theamount of AgNO3 and type of calixarene used [19]. H2 is a cleanreducing agent, but must be used with caution. Starch has alsobeen used as a clean reducing agent of AgNO3, but AgNP no smallerthan 5.3 nm have been reported [20,21].

The main goal of the present report consists in obtainingsmall AgNP entrapped in CS-NP (Ag@CS-NP) using a highlyfriendly and inexpensive procedure, since CS is used as bothreducing agent and stabilizer. CS-NP would behave as a bionano-carrier of AgNP. This was because the resulting system couldserve for biological applications combining the interesting prop-erties of both components, including the antimicrobial propertiesof AgNP [22,23].

NH3+

OH

CH2OH

O( (

n

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

M.C. Rodríguez-Argüelles et al. / Journal of Colloid and Interface Science 364 (2011) 80–84 81

2. Materials and methods

2.1. Materials

Low molecular weight chitosan (85% deacetylation) and sodiumtripolyphosphate (TPP) were purchased from Aldrich; AgNO3 wasfrom Scharlau; Mili-X water was used in all cases.

2.2. Spectroscopy

A Cary 50 Conc (Varian) UV–Vis spectrophotomer was used forthe determination of the SPR bands of AgNP.

High resolution transmission electron microscopy (HRTEM), aswell as high angle annular dark field (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 determinedby 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 dropwise to a solution of chitosan (10 mg) dissolved in acetic acid(2%, 10 mL) with constant agitation. A white discrete opalescencewas observed, and the formation of the nanoparticles was con-firmed by the Tyndall effect. This system was maintained underconstant agitation for no less than 1 h.

- - -- - - -

--

-

--- Ag+

- - -- - -

------

Ag+Ag+

Ag+Ag+Ag+

Ag+

Ag+

Ag+Ag+

Ag+

Ag+

- -

---

-

---

- -

---

-

---

2.4. Synthesis of silver nanoparticles inside CS-NP (Ag@CS-NP)

The above solution of ChNP was heated to boiling (under reflux)and then an aqueous solution of AgNO3 (5–200 mg in about 0.6–1 mL) was added drop wise to it. A yellow color appeared after40–80 min of agitation of the boiling solution and was maintainedother 5–6 h under the same conditions.

--

-- -Ag+Ag+

- - -- - - -

--

-- -

---

Δ(100 ºC)

-- --

- - -- - - -

--

-- -

---

- - -- - -- - -- - - -

---

--

-- - -- -

--- ---

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

2.5. Enzymatic treatment of samples

The enzymatic treatment of the Ag@CS-NP samples was carriedout using a chitosanase from Streptomyces griseus (Sigma). Chitosandegradation was evaluated by measuring the reducing power ofthe 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 incubatedin Mueller–Hinton 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 and30 �C, respectively. S. cerevisiae and Salmonella sp. are wild typestrains from our laboratory. CECT: Spanish Type Culture Collection.The visual turbidity of the tubes was noted both before and afterincubation. The media were solidified, when necessary, with 1.5%agar (Cultimed).

2.7. Minimal inhibitory and microbicidal concentration

The antimicrobial properties for the samples were determinedusing the twofold broth dilution technique [26]. All determinationswere performed in duplicate. The samples were used as preparedand tested at final concentrations (of silver) of 24, 12, 6, 3, 1.5,0.75, and 0.37 lg Ag/mL. Inocula of 5 � 104 bacteria/mL and1 � 103 yeast/mL were used. The minimal inhibitory concentration(MIC, lg/mL) was defined 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 readby the visual turbidity of the tubes noted both before and afterincubation. Media and positive growth controls were also runsimultaneously. The minimal bactericidal concentrations (MBC,lg/mL) and the minimal fungicidal concentrations (MFC, lg/mL)were measured by subculturing 100 lL of each sample remainingclear in tubes containing 1 mL of fresh medium.

3. Results and discussion

The reported method for the synthesis of CS-NP based on theuse 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 Tyndalleffect (using a Laser pointer), a more precise procedure.

TPP played three important roles in the synthetic procedureused, all of electrostatic nature. In the formation of CS-NP, thepolyanion TPP served to agglutinate chitosan units as a template,and once CS-NP was formed, it attracted Ag+ ions inside thenanoparticle enhancing the diffusion process. Additionally, theelectrostatic attraction of Ag+ by TPP assisted the regulation ofthe size of the formed AgNP. Once Ag+ diffused inside the CS-

82 M.C. Rodríguez-Argüelles et al. / Journal of Colloid and Interface Science 364 (2011) 80–84

NP matrix and the system was heated up to 100 �C, glucosamineunits of chitosan served as reducing agent to form AgNP insideCS-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 ofAgNP. The formation of Ag@CS-NP is schematically representedin Fig. 2, where the negatively charged semicircular sections cor-respond to TPP.

According to the schematic representation given in Fig. 2, theresulting Ag@CS-NP system could be considered similar to a pud-ding with raisins. Such aspect was confirmed when the systemwas studied by HRTEM. In Fig. 3, two HAADF–STEM images ofAg@CS obtained using 30 mg of AgNO3 are presented. The mea-sured average size of these AgNP was of 2.0 (±0.64) nm accordingto the size distribution histogram (Fig. 4a). The HRTEM images

Fig. 3. HAADF–STEM image of Ag@CS-NP (two different magnifications) obtained from 3structure of AgNP.

Fig. 4. Size distribution diagrams of AgNP (in Ag@CS-NP) formed: (a) with 30 mg of

and the Fast Fourier transform analysis (inset in Fig. 3b) indicatethe formation of AgNP with a fcc structure, which is not the pre-dominant structure expected.

Other amounts of AgNO3 (from 5 to 200 mg) were also used toobtain Ag@CS-NP (TEM images are not given), and in all cases, theaverage 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 ob-tained diameter was of 1.7 (±0.32) nm (Fig. 4c). Therefore, onlysmall variations were observed in the diameters of the obtainedAgNP, with an insignificant dependence on the amount of AgNO3

used. This result was unexpected considering that the concentra-tion of AgNO3 is a determining factor in all the methods reportedon the synthesis of AgNP [27].

0 mg of AgNO3. Inset of (b) is the Fast Fourier transform indicating a crystalline fcc

AgNO3; (b) 5 mg; (c) 200 mg; (d) also AgNP formed in CS with 5 mg of AgNO3.

M.C. Rodríguez-Argüelles et al. / Journal of Colloid and Interface Science 364 (2011) 80–84 83

AgNP was also synthesized in CS (dissolved in acetic acid) usinga similar procedure to that reported here for Ag@CS-NP, but with-out the presence of TPP and, therefore, without the formation ofCS-NP. Under such conditions, larger AgNP were obtained, withan 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 mentionthat 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 thatCS-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 theformation 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 participationof the phosphonate groups. In the case of CS-NP, this tridimensionalsystem should participate as a whole in the complete regulation ofthe size of AgNP with the assistance of negatively charged TPP.

On the other hand, CS-NP behaves as a soft reducing agentthrough the participation of its terminal glucosamine groups. Anexpression of the low reducing capacity of CS-NP is that the reac-tion took place at 100 �C (under reflux). We observed that at lowertemperatures larger AgNP were formed. Generally, strong reducingagents favor the formation of large AgNP, since the reduction oc-curs at a rate higher than the capacity of the capping componentto 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 H2

[17]. In our case, the glucosamine unit constitutes an abundantmild 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 samesynthesis, according to HRTEM determinations. The forms of theobtained Ag@CS-NP varied between oval and spherical, but withno defined regularity. The mean (hydrodynamic) diameter deter-mined by dynamic light scattering (DLS) was of 78 (±19) nm, a bet-ter defined value. Here, it is important to mention that thediameters 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 remainedconstant. Actually, the sizes reported for CS-NP by differentauthors varies in a much wider range and were much larger, from172.6 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 averagesize is about seven times larger than the corresponding value ofAg@CS-NP, as already mention above. The significant difference be-tween the hydrodynamic diameters of Ag@CS-NP and CS-NP can beconsidered as an indication that the silver ions should have played arole in the formation of the nanoparticles of chitosan. Related to thisinterpretation, it is important to mention that in reference [14], theAg@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 flexible structure, strongly dependent on whatit contains and also on the surroundings. We had this considerationin mind from the same beginning and that is the reason for whichwe decided not to wash Ag@CS-NP, once obtained in order to notaffect its initial structure. After the addition of Ag+ to CS-NP, theions should diffuse the membrane electrostatically attracted byTPP. Subsequently, an interaction between the Ag+ ions and theamino groups of glucosamine should have taken place to provokethe observed contraction of CS-NP.

Different forms of chitosan were submitted to the enzymaticcleavage of chitosanase. The amount of free glucosamine formedin each case was used for the comparison. 0.4 UE/mL of chitosanasewas 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 theother 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 speciesas 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), butnot 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 statisticallynot significant and should be attributed to the amount consumedin the reduction of Ag+. On the other hand, the difference betweenthe content of glucosamine in free CS and in CS + AgNP is muchhigher (42 lg/mL), a result that cannot be endorsed to the diffusionof the enzyme. Even so, this difference constitutes only 20% of thecontent of glucosamine in free CS. According to that result it shouldbe assumed that not all of the Ag+ ions added were reduced, andwe 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 AgNO3 added ineach synthesis. When 5 mg of AgNO3 was used, only 18.89% ofAg+ remained unreduced, for 30 mg 16.44%, for 50 mg 13.93%,and for 100 mg 13.33%. These results indicate that the diffusionand steric access of Ag+ to the glucosamine groups constitutedthe main limitation in its reduction, and the reason for which avery low influence of the concentration of AgNO3 used on the sizeof AgNP was observed.

The positively charged surface of CS-NP should favor its dockingon negatively charged biological surfaces and permit the release ofthe species held inside. AgNP could then be able to diffuse outsideCS-NP and interact with the surroundings, as already reported forthe interaction of Ag@CS-NP with human adenocarcinoma cells[14]. In this sense, it is important to mention that in a recent reporton the interaction of CS with a negatively charged liposome (stud-ied by isothermal titration calorimetry), high binding constants ofthe order of 105 M�1 were determined [30].

The results of antimicrobial activity of AgNO3, CS-NP, andAg@CS-NP are presented in Table 1 and expressed as minimalinhibitory 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. aureusand B. cereus. MFC (for the fungi), and MBC values (for the bacteria)are also reported. The amount of AgNP in Ag@CS-NP was correctedaccording to the concentration of Ag+ analytically determined foreach case.

As can be observed from Table 1, Ag@CS-NP presented an anti-microbial activity significantly higher than AgNO3. This result wasnot affected by CS-NP, which presented a low antimicrobial activ-ity, for which no correction was made. Especially significant re-sulted the very low MBC values determined for the five bacteriastudied (1.5–3 lg/mL), except against Salmonella sp (6 lg/mL).The MIC/MBC ratios were of 1–2.

No significant differences between the antimicrobial activity ofAg@CS-NP against the studied Gram positive and Gram negativebacteria were observed, but its antifungal activity was about fourtimes lower. These three biological systems are characterized bypossessing negatively charged surfaces. Therefore, the positivelycharged surface of Ag@CS-NP should favor the interaction withthe 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 thesesystems varies according to the environment and other factors [31].

Table 1Antimicrobial 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 Gram negative bacteria Gram positive bacteria

C. glabrata S. cerevisiae E. coli K. pneumoniae Salmonella sp S. aureus B. cereus

AgNO3 24 >24 6 (12) 12 (24) 24 (>24) 12 (>24) 12 (>4)CS-NP >24 >24 >24 >24 >24 >24 >24Ag@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. Rodríguez-Argüelles et al. / Journal of Colloid and Interface Science 364 (2011) 80–84

The antimicrobial activity of Ag@CS-NP, as a whole, should bestrongly related to the small sizes of both nanoparticles, CS-NPand AgNP, involving a highly positive charge density, able to inter-act with specific areas of the cell surface of the studiedmicroorganisms.

The antimicrobial activity of Ag@CS-NP should be related,among other pathways, to the formation of ROS as a consequenceof the interaction of AgNP with O2, 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 tobehave as a permanent source of the cation [33]. Furthermore,the membrane of CS-NP should regulate the diffusion of Ag+ andAgNP out of the nanoparticle, making Ag@CS-NP behave as a con-trolled release liberation system of Ag+ and AgNP. Therefore, thesecharacteristics should be interpreted as if Ag@CS-NP would be act-ing as a sustained source of antimicrobial agent able to interactwith microorganisms of different nature. Such characteristicshould 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 withthose reported recently. For example, in a paper on AgNP (5–30 nm) dispersed in CS the authors reported MIC and MBC valuesof 10 lg/mL for all the Gram positive and Gram negative bacteriastudied, except P. aeruginosa (2.5 lg/mL) [27]. Then again, ourMIC and MBC values are also better that those reported for AgNO3

included in CS-NP [35].

4. Conclusions

Here, we report a procedure with which it is possible to obtainsmall CS-NP (78 nm) containing very small AgNP (0.93–1.7 nm).The preparation of such small AgNP constitutes a task difficultitself to be achieved. The procedure is environmental friendlyand inexpensive, since chitosan, a biopolymer available as a wasteof fishing industry, behaves as the reducing agent. TPP served as apolyanionic template that favored the formation of the small nano-particles, which also electrostatically attract Ag+ inside CS-NP. Theimportance of the small size of Ag@CS-NP hybrid nanocomposites(and AgNP itself) was expressed in the determined antimicrobial,with significantly small MIC values against two fungi, three Gramnegative bacteria, and two Gram positive bacteria. The Ag@CS-NPsystem should be expected to behave as a biomaterial to be usedin different pharmaceutical applications, mainly in woundtreatments.

Acknowledgments

This work was financed by Xunta de Galicia, Spain, projectPXIB310278PR. The authors are grateful to Prof. Dr. Sabine Schlecht(Giessen, Germany) for the assistance in the characterization of theproducts.

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