Improving the performance of chitosan in the synthesis and stabilization of gold nanoparticles

European Polymer Journal 68 (2015) 419–431

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European Polymer Journal journal homepage: www.elsevier.com/locate/europolj

Macromolecular Nanotechnology

Improving the performance of chitosan in the synthesis and stabilization of gold nanoparticles Angel Leiva a,⇑, Sebastian Bonardd a, Maximiliano Pino a, Cesar Saldías a, Galder Kortaberria b, Deodato Radic´ a a

Pontificia Universidad Católica de Chile, Facultad de Química, Departamento de Química Física, Santiago 7820436, Chile ‘‘Materials + Technologies’’ Group, Polytechnic School, Dpto. Ingeniería Química y M. Ambiente, Universidad País Vasco/Euskal Herriko Unibertsitatea, Pza. Europa 1, San Sebastian 20018, Spain b

a r t i c l e

i n f o

Article history: Received 11 March 2015 Received in revised form 24 April 2015 Accepted 26 April 2015 Available online 15 May 2015 Keywords: Chitosan Grafted chitosan Gold nanoparticles Nanocomposites

a b s t r a c t This work used chitosan polysaccharide and its graft copolymers with e-caprolactone and N-vinyl-2-pyrrolidone chains to synthesise and stabilise gold nanoparticles. The nanoparticle synthesis was performed directly by reaction of polymers with potassium tetrachloroaurate in an aqueous medium and it was demonstrated that the polymers can act as reducing and stabilizing agents. The modification of chitosan considerably affects the copolymer performance during the gold nanoparticle synthesis. Different synthetic parameters, such as the reaction time, temperature, concentration and polymer and metallic salt feed ratio, were assessed. The gold nanoparticles were characterised via UV–Vis spectroscopy, dynamic light scattering (DLS), transmission electron microscopy (TEM), and zeta potential. In general, using grafted chitosan improves the synthetic performance of gold nanoparticles over unmodified chitosan, which is reflected in the amount, size distribution and suspension stability of the obtained nanoparticles. These results are promising due to the potential technological applications of chitosan derivatives. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction Chitin is one of the most abundant polysaccharides in the earth’s crust and is commonly found in crustacean exoskeletons and the cell walls of several fungi and algae [1,2]. As cellulose, chitin mainly functions structurally in nature, which is easily understood considering the similarity between the chemical structures of both biopolymers. Additionally, the polysaccharide is biodegradable, biocompatible and non-toxic. These features allow for its use in a wide variety of applications [2]. However, despite the desirables properties of chitin, its insolubility in conventional solvents limit both the processing and range of applications for this polysaccharide [1,2]. This fact has prompted the search for chitin derivatives that maintain its ‘‘green’’ properties but simplify the handling and processing. In this context, chitosan, a derivative of chitin, is a biodegradable, biocompatible and non-toxic polymer obtained via the deacetylation of chitin via the hydrolysis of N-acetyl-D-glucosamine to yield D-glucosamine [3]. This modification produces significantly changes the derivative, for example, the solubility, which retains the ‘‘green’’ properties of chitin containing free amino groups. These amino groups enhance chitosan’s solubility in acids and yields a cationic polyelectrolyte; additionally, the amino groups significantly

⇑ Corresponding author. E-mail address: [email protected] (A. Leiva). http://dx.doi.org/10.1016/j.eurpolymj.2015.04.032 0014-3057/Ó 2015 Elsevier Ltd. All rights reserved.

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increases the polymer crystallinity and its ability to interact with certain metals via biosorption [4]. Notably, these functional groups are suitable for chemical modifications by adding reactive sites to the chitosan backbone. Thus, chitosan has been used in several fields and applications such as cosmetics, ophthalmology, medicine and nutrition [1,2]. While chitosan’s solubility in, for example, water is better than chitin, many other conventional solvents have remained a constraint until now. Therefore, scientific work has focused on studying different chemical modifications to increase the functionality and solubility of chitosan and extend its range of applications. Several chemical modifications for chitosan, such as, alkylation, acylation, quaternisation, carboxyalkylation, thiolation, phosphorylation and enzymatic reactions, have been reported [3–6]. Graft copolymerisation is an interesting alternative due to the high versatility of the resultant materials, the reactive sites (hydroxyl and amino groups) along the chitosan polymer chain can be used to covalently bond polymer chains, monomers or small molecules to improve the biomaterial properties. The literature reports the synthetic modification of chitosan by grafting with poly (vinyl alcohol) [3], poly (vinyl acetate) [7] and other compounds. However, these graft copolymers have poor degradability, which affects the ‘‘green’’ properties of chitosan [3] and requires chemical modifications using biodegradable polymers to retain the potential physicochemical and environmentally friendly properties. In contrast, there has been great interest in both the scientific and technologic aspects of metal nanoparticles, especially gold nanoparticles, over the last two decades. Because gold nanoparticles exhibit special physical, chemical and biocompatibility properties, they have been the focus for a wide range of applications, e.g., optics, photonics, catalysis, and pharmaceutics. Additionally, the size, shape, specific surface area and plasmon absorbance of metal nanoparticles are highly relevant to their applications [8]. Metal nanoparticle syntheses using coordination agents that stabilise and control the morphological evolution of the metal nanostructures in solution or suspension are useful tools [9,10]. The coordination of a stabilizing agent during nanoparticle synthesis directly affects their size and shape because it is involved in, for example, the nucleation and growth stages due to the selective adsorption to and desorption from specific metal nanoparticle faces [11]. This behaviour could be attributed to the selectivity between the metal surfaces and coordination agent functional groups. For instance, the importance of oxygen- or nitrogen-metal coordination bonds to forming metal nanostructures with different dendrimers has been reported [12,13]. Polymers with the appropriate functional groups have been demonstrated as alternative coordinating and stabilizing agents to control the metal nanoparticle characteristics and surface properties [14–17]. Numerous reports address using polymers in nanoparticle synthesis [18,19]. These studies use copolymers containing sites and functional groups capable of stabilizing several types of metal nanoparticles in solution. For example, amphiphilic copolymers with a hydrophobic region capable of interacting with the metal surface and hydrophilic side groups linked to the polymeric chain have been suggested to stabilise nanoparticles in a polar dispersion medium [20]. Some water-soluble homopolymers such as poly (vinylpyrrolidone) and poly (ethylene glycol) have been used extensively as steric stabilizers in metal nanoparticle syntheses [21,22]. Furthermore, biodegradable materials [23] and polyelectrolytes have been used to synthesise and stabilise metal nanoparticles; polyelectrolytes are a good option because they exhibit special features due to their combination of steric and electrostatic stabilisation capabilities [24]. Chitosan and its derivatives have also been used to synthesise gold nanoparticles. Spano and coworkers reported the in situ formation of gold nanoparticles in hydrogel-type chitosan films loaded with chloroauric acid (HAuCl4) [25]. The preparation of colloidal gold nanoparticles (AuNPs) stabilized in a chitosan matrix by reducing Au III to Au 0 in aqueous chitosan and different organic acids (i.e., acetic, malonic, or oxalic acid) has also been reported [26]. The authors demonstrated that varying the acid nature tuned the gold precursor (HAuCl4) reduction rate and modified the resultant metal nanoparticle morphology. Due to the excellent film-forming capabilities of chitosan, AuNPs–chitosan solutions were used to obtain hybrid nanocomposite films combining highly conductive AuNPs with a large number of organic functional groups. Au–chitosan nanocomposites were thus successfully proposed as sensitive and selective electrochemical sensors for detecting and quantifying caffeic acid. Additionally a clean, nontoxic and environmentally friendly synthesis to generate a large variety of gold nanoparticles (GNPs) using chitosan polysaccharide as the reducing and stabilizing agent was reported by Potara and coll [27]. The results indicate the reaction temperature plays a crucial role in controlling the size, shape and crystalline structure of GNPs. The authors demonstrated chitosan can perform as a scaffold for assembling GNPs, which were successfully used as a substrate for surface-enhanced Raman scattering (SERS). This synthesis procedure was developed between 4 and 100 °C by reacting chitosan in acetic acid and HAuCl4 solutions. Furthermore, chitosan and derivatives have also been used to coat gold nanoparticles, obtaining nanocomposites applicable to cancer therapy under physiological conditions [28]. Other polycarbohydrates, such as Xanthan gum and starch, have been used to synthesise gold nanoparticles [29,30]. In this work, we improve the chitosan performance during gold nanoparticle syntheses. The best performance was achieved using N-vinyl-2-pyrrolidone and e-caprolactone grafted chitosan copolymers. The results indicate more nanoparticles with better stabilities in aqueous suspension were obtained in a shorter time using grafted chitosan. 2. Experimental 2.1. Materials Chitin was obtained from Chilean shrimp shells provided by Antartic Seafood S.A., Coquimbo, Chile. Chitosan (MW = 70,000 g/mol, deacetylation degree 75%) was obtained from chitin. N,N-Dimethyl Formamide (DMF), calcium

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chloride (CaCl2), calcium hydride (CaH2), N-vinyl-2-pyrrolidone (VP), e-caprolactone (e-CL), tin 2-ethylhexanoate (II), potassium tetrachloroaurate (KAuCl4), hydrogen peroxide (H2O2), iron sulphate heptahydrate (FeSO47H2O), phthalic anhydride, and hydrazine monohydrate were obtained from Aldrich. Glacial acetic acid, toluene, acetone, hydrochloric acid and methanol were obtained from Merck Chile S.A. e-Caprolactone (e-CL) was purified via a reduced pressure distillation; all other chemical reagents were of analytical grade and used without further purification. Deionised water (specific resistance 18.25 MX) was used in all experiments. 2.2. Characterisation of chitosan (Q) 2.2.1. Deacetylation degree The degree of chitosan deacetylation was determined via an acid–base titration, where 0.3 g of chitosan were dissolved in a 0.1 M HCl solution to obtain a 100 mL solution with an ionic strength of 0.1 (controlled using KCl). The titrant was 0.05 M NaOH, and the solution pH was continuously measured during the titration until pH = 12. 2.2.2. Viscosimetric molecular weight The molecular weight of the chitosan was determined via viscosity measurements in aqueous solution 5% v/v acetic acid at an ionic strength of 0.1 (adjusted with KCl) using an Ostwald viscometer immersed in a 25 ± 0.1 °C water bath. Values of 0.85 and 1.38  104 were determined for a and K, respectively, for chitosan in 5% v/v acetic acid in 0.1 M aqueous HCl via the Mark–Houwink relationship–Sakurada [31]. 2.3. Synthesis of the graft chitosan-g-poly (e-caprolactone) copolymer This copolymer was synthesised via the following three successive steps starting from chitosan with a molecular weight of 70,000 g/mol: first, protect the amino groups, second, graft poly (e-caprolactone), and finally, deprotect the amino groups. 2.3.1. Protection of amino groups A reported method [32] was used. In a glass reaction flask connected to a reflux system, 30 mmol of phthalic anhydride was dissolved in a 95/5% v/v DMF/H2O mixture. Once the phthalic anhydride had dissolved, 2 g of chitosan were added, and the reaction occurred at 120 °C for 8 h. The resultant reaction mixture was poured into 200 mL of cold water and decanted for 14 h. The precipitate was then filtered using a vacuum trap before washing the precipitate with 300 mL of methanol for 2 h and filtering again. Finally, the obtained product was dried under vacuum to a constant mass. 2.3.2. Grafting of poly (e-caprolactone) The synthetic route described by Liu et al. [33] was used. The system containing 0.5 g of chitosan with protected amino groups in a two-neck glass reaction flask was purged with dry nitrogen gas, and a mixture containing 3 ml of toluene, 1 mL of caprolactone and 20 lL of tin octanoate was added using a syringe. The reaction mixture was stirred vigorously at 100 °C for 20 h. Finally, the reaction mixture was filtered, and the obtained precipitate was washed with acetone for 24 h in a Soxhlet apparatus and dried under vacuum to a constant mass. 2.3.3. Deprotection of amino groups A previously reported methodology [34–37] was used. In a two-neck glass reaction flask connected to a reflux system, 0.5 g of the graft copolymer with protected amino groups were added in 10 mL of DMF with constant stirring, and the system was heated to 100 °C. Once this temperature was reached, 1 mL of hydrazine monohydrate was added, and the reaction system was held for 2 h. The obtained yellow heterogeneous mixture was filtered under vacuum, and the obtained precipitate was washed with methanol and dried under vacuum to a constant mass. 2.4. Synthesis of graft copolymer chitosan-g-poly (N-vinyl-2-pyrrolidone) The graft copolymer was synthesised directly from 70,000 g/mol chitosan solubilised in a 0.1 M HCl solution in the presence of the N-vinyl-2-pyrrolidone monomer and Fenton reagent as a radical initiator. In a glass reaction flask, 0.5 g of chitosan was dissolved in 25 mL of 0.1 M HCl. Once the chitosan dissolved, 10.4 mL of N-vinyl-2-pyrrolidone, 12.6 ml of distilled water and 50 mg of FeSO47H2O were added [6]; the reaction mixture was stirred vigorously until the slat completely dissolved, and the system was then placed in an ultrasonic bath under a nitrogen atmosphere. Finally, 2 mL of H2O2 were added, and the reaction was held for 2 h. The reaction mixture was diluted using 250 ml of water and an excess of 1 M NaOH was added, the precipitate was filtered under a reduced pressure and washed first with 400 mL of water for 24 h and then with 100 mL of ethanol. Finally, the product was dried under a reduced pressure to constant mass.

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2.5. Synthesis of gold nanoparticles The gold nanoparticles were synthesised in solution in a glass reaction flask coupled with a reflux system and containing 10 mL of a 3% polymer solution with constant stirring until the desired temperature was reached. Then, 5 mL of aqueous KAuCl4 solution was added, and the reaction was maintained for 210 min. The syntheses were performed using chitosan, chitosan-g-PCL or chitosan-g-PVP at 40, 60 and 80 °C and three KAuCl4 concentrations (0.035, 0.07 and 0.14 mM).

3. Results and discussion 3.1. Characterisation of chitosan The degree of deacetylation was determined to be of 75% via the acid–base titration [38], which indicates the product was chitosan and corresponded to a random copolymer because the N-acetyl-D-glucosamine and D-glucosamine units were distributed along the polymer backbone without a defined pattern or order [1,2]. A viscosimetric molecular weight of 70,000 g/mol was obtained for chitosan via the intrinsic viscosity measurements using a and Ka values of 0.85 and 1.38  104, respectively, as reported for chitosan solubilised in 5% v/v acetic acid and 0.1 M HCl [31] based on the Mark–Houwink–Sakurada relation. Both unmodified and modified chitosan polymers were characterised via 1H NMR and FTIR. Fig. 1a shows the IR spectrum for chitosan. The high-intensity signal at 3449 cm1 corresponded to the amine and hydroxyl groups distributed along the polymer backbone stretching. The band centred at 2880 cm1 was assigned to the C–H bond stretching from the pyranose rings. Moreover, the high-intensity signal between 1685 and 1600 cm1 corresponded to the characteristics carbonyl vibration in the N-acetyl-D-glucosamine units and NH2 bending vibration from the D-glucosamine units, respectively. The signal between 1160 and 1010 cm1 was assigned to the C–C and C–O bond vibrations from the pyranose ring. The IR spectrum for chitosan-g-PCL contained the described chitosan signals with the main differences being the enhanced signal at 2939 cm1, which corresponded to C–H bond stretching from the methylene carbons in the PCL chain and a new signal at 1725 cm1 corresponding to carbonyl groups in the grafted e-caprolactone [34,35]. Additionally, the IR spectrum for chitosan-g-PVP yielded a signal at 2990 cm1 attributed to the C–H bonds in the methylene groups from both the pyranose rings and N-vinyl-2-pyrrolidone blocks. The carbonyl signal at 1655 cm1 increased in intensity due to the PVP blocks. Finally, a characteristic band of amides appear peak at <1385 cm1, which confirms the presence of N-vinyl-2-pyrrolidone. Fig. 2 shows the 1H NMR spectra for chitosan and the chitosan-g-PCL and chitosan-g-PVP derivatives. The H1, H2 and H3– H6 signals typically appear at 5.4, 3.7 and 4.2–4.4 ppm, respectively, for chitosan [39]. The 1H NMR spectrum for chitosan-g-PCL exhibited these proton signals from the chitosan backbone in addition to those for the e-caprolactone units for a (2.2 ppm), b and d (1.4 ppm), c (1.2 ppm) and e (3.4 ppm) [34]. Similarly, the chitosan-g-PVP spectrum mainly exhibited signals a, b and c from the pyrrolidonic ring at 1.8, 2.1 and 2.3 ppm, respectively. The PVP and PCL contents in the graft copolymers could then be estimated from the integrated values of 2 and a signals for chitosan-g-PCL and 2 and c for chitosan-g-PVP [40]. The PVP and PCL content grafted to the chitosan backbone were 8% and 18% w/w, respectively.

3.2. Synthesis of gold nanoparticles using chitosan and its graft copolymers Fig. 3 shows the UV–Vis spectra for gold nanoparticles obtained via the direct reaction of chitosan and its respective graft copolymers with the gold metal precursor. A polymer concentration 3% m/v was constantly maintained, and different KAuCl4 concentrations (0.035, 0.070 and 0.140 mM) were assayed for each case. All reactions occurred at 60 °C for 3.5 h and were monitored via UV–Visible spectrophotometry using the typical absorbance between 500 and 550 nm from the surface plasmon resonance of the gold nanoparticles [41]. As expected, the plasmon absorbance band for all copolymers increased with increasing gold (III). However, the absorbance increment was considerably higher using chitosan-g-PCL and chitosan-g-PVP in the synthesis relative to the unmodified chitosan. Additionally, significantly lower plasmon bands were obtained for chitosan at all KAuCl4 concentrations compared to the corresponding copolymers; these results indicate the gold nanoparticles are more efficient when the copolymers are used due to the presence of e-caprolactone and N-2-vinyl pyrrolidone. The highest absorbance was obtained with the grafted con e-caprolactone copolymer due to the higher e-caprolactone content of 18% relative to the 8% N-vinyl-2-pyrrolidone in the corresponding copolymers. As mentioned above, chitosan has been used as a stabilizer [42,43] and reductant [44–46,27] to synthesise gold nanoparticles. Huang and coll. [45] were most likely the first to report the dual stabilizer and reductant role for chitosan in gold and silver nanoparticle syntheses. They obtained nanoparticles with high suspension stabilities and different size distributions using different chitosan concentrations and molecular weights during the synthesis; the Au III reduction was attributed to either the hydrolysis of chitosan to generate a reductant glucose derivative or the –CH2–OH groups in the chitosan structure.

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Fig. 1. Infrared spectra for chitosan and its graft copolymers.

Previously, our group demonstrated both experimentally and theoretically that terminal hydroxyl groups in either the polymer backbone or grafted chains were responsible for the reduction of Au (III) to Au (0) during gold nanoparticle syntheses [47–50]. Therefore, we postulated the reduction occurred via the –CH2–OH groups for chitosan and terminal hydroxyl groups in the side chains for the grafted copolymers. This last result would explain the higher nanoparticle synthesis

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Fig. 2. 1H NMR spectra for chitosan and its grafted copolymers.

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Absorbance (a.u.)

0.3

Chitosan

0.2 0,14mM 0,07mM 0,035mM

0.1

0.0

300

400

500

600

700

800

wavelength (nm) 0.3 0,035mM 0,07mM 0,14mM

0.2

0.0

Absrorbance (a.u.)

Absorbance (a.u.)

Chitosan-g-PCL

Chitosan-g-PVP

0.2 0,14mM 0,07mM 0,035mM

0.1

0.0

-0.1

300

400

500

600

700

wavelength (nm)

800

300

400

500

600

700

800

wavelength (nm)

Fig. 3. Absorption bands of plasmon, for gold nanoparticles synthesised at different concentrations of KAuCl4, with chitosan and their graft copolymers.

efficiency for the grafted copolymers because the hydroxyl end groups in the grafts have a higher reducing power. To clarify, a simple computational chemistry exercise focused on the reactivity of the hydroxyl groups in chitosan and its graft copolymers was performed and is discussed below. To complement the previously discussed results, the nanoparticle synthesis was monitored at different reaction times. Fig. 4 shows the evolution of the UV–Vis spectra for the gold nanoparticle syntheses using chitosan and its grafted copolymers, chitosan-g-PCL and chitosan-g-PVP, at 60 °C for a constant KAuCl4 concentration of 0.07 mM. All of the polymers exhibited a similar behaviour; the plasmon band absorbance increased as time increase, which indicates the reaction progress. Additionally, the maximum absorbance wavelength shifted slight to higher values due to the nanoparticle growth [47,51]. The spectra were recorded until the absorbance and wavelength stopped changing; at this point, the nanoparticle syntheses had completed [52]. Based on these results, two facts deserve emphasis: (i) the maximum obtained absorbances were higher for the graft copolymers than for chitosan and (ii) the required reaction time was at least one order of magnitude shorter for the grafted chitosan. These facts confirm both the higher efficiency and improved performance of these copolymers in the gold nanoparticle synthesis. The effect the reaction temperature had on the gold nanoparticle syntheses was also monitored via UV–Vis spectroscopy. The results for different temperatures are shown in Fig. 5. As expected, the general results indicate the synthetic efficiency was higher when the temperature increased, which agrees with other reports on gold nanoparticle syntheses using polymers where the reaction temperature significantly affected the structure, size distribution and optical properties of the final product [53]. Fig. 6 shows the TEM images for gold nanoparticles obtained by reacting chitosan and graft copolymers at 60 °C with 0.14 mM KAuCl4. The nanoparticles obtained using chitosan were approximately 25.8 nm in diameter with a predominantly spherical shape. Using chitosan-g-PVP nanoparticles of 34.8 nm and with less well-defined shapes including triangular nanoparticles were obtained. Finally, nanoparticles obtained using chitosan-g-PCL were spherical with the larger diameter of approximately 40.4 nm. These results are coincident with the bibliography since, generally gold nanoparticles synthesised using chitosan are reported spherical in shape [45,46,54], although some triangular shaped nanoparticles have also been observed [55]. The nanoparticle sizes determined by TEM and DLS are summarised in Table 1. The values obtained via both technics are consistent, i.e., both show larger nanoparticles formed using chitosan-g-PCL, whereas smaller nanoparticles formed using chitosan. These observations suggest the copolymers perform differently during the nucleation, growth or stabilisation stages of the synthesis.

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Fig. 4. Evolution of the absorption band of plasmon in function of time, for gold nanoparticles synthesised with chitosan and their graft copolymers.

Fig. 5. Absorption bands of plasmon, for gold nanoparticles synthesised at different temperatures, with chitosan and their graft copolymers.

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Fig. 6. TEM images of gold nanoparticles synthesised using, (a) chitosan, and its grafted copolymers, (b) chitosan-g-PVP and c chitosan-g-PCL.

Table 1 Distribution of gold nanoparticle sizes obtained by DLS and TEM for systems synthesised with chitosan and graft copolymers.

a b

Polymer

[KAuCl4] (mM)

Sizea (nm)

Sizeb (nm)

Chitosan-g-PCL Chitosan-g-PVP Chitosan

0.14 0.14 0.14

42.1 ± 5.5 32.2 ± 4.8 28.8 ± 5.8

40.4 ± 3.7 34.8 ± 7.5 25.8 ± 8.4

Obtained by dynamic light scattering. Obtained by transmission electron microscopy.

Zeta potential measurements were performed to evaluate the nanoparticle stability in aqueous suspension. Table 2 summarises the zeta potentials for nanoparticles synthesised at different temperatures using chitosan and its grafted copolymers. A large negative or positive zeta potential for suspended particles indicates the system is stable against flocculation or coagulation [56]. In general, the zeta potentials for gold nanoparticles prepared using grafted copolymers show a tendency

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A. Leiva et al. / European Polymer Journal 68 (2015) 419–431 Table 2 Zeta potential of gold nanoparticles synthesised with chitosan, chitosan-g-PCL and chitosan-g-PVP, at different temperatures. Polymer

Temperature (°C)

Zeta potential (mV)

Chitosan-g-PCL

40 60 80

58.3 ± 11.3 94.5 ± 7.3 91.0 ± 4.2

Chitosan-g-PVP

40 60 80

90.1 ± 11.0 73.5 ± 8.5 98.4 ± 14.0

Chitosan

40 60 80

60.0 ± 4.8 46.2 ± 5.2 81.9 ± 12.0

R

O

Poly (vinylpyrrolidone)

Poly (ε-caprolactone)

C3 chitosan

C6 chitosan

Fig. 7. Reduced (R) and oxidised (O) optimised conformers for chitosan, poly (vinylpyrrolidone) and poly (e-caprolactone). The circles indicate the hydroxyl groups analysed.

to be higher compared to those synthesised with chitosan, this could indicate a better stability of nanoparticles synthesised with chitosan grafted copolymers. This trend was confirmed by the experimental observation that, while nanoparticles synthesised with chitosan are decanted in few weeks, the nanoparticles synthesised and stabilized by the grafted copolymers remain in solution at least two months at room temperature. As previously stated, to gain insight about the reactivity for the studied nanoparticles syntheses, monomeric units of chitosan, PVP and PCL were modelled and optimised (Fig. 7). The hybrid exchange–correlation functional, B3LYP (Becke’s three-parameter and Lee–Yang–Parr), level of approximation implemented in the Gaussian 09 package was used for all of the electronic structure calculations. The 6-311 G(d, p) basis set for O, C, N, and H was assigned, and conformational analyses were used to determine the most stable conformers for all structures. A selective analysis of the hydroxyl groups was performed using these tools to determine the probability in energetic terms they act as reducing agents assuming they can be oxidised to carboxylic acid groups. Thus, the oxidised chitosan, PVP and PCL monomer units were also modelled and optimised (Fig. 7). Table 3 summarises the energy values for the oxidised (Eox) and reduced (Ered) conformers and the conversion energy, DEconversion, for each monomer based on the following equation:

DEconversion ¼ Eox  Ered

ð1Þ

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Table 3 Oxidation (Eox), reduction (Ered) and conversion (DE) energy values for chitosan, poly (vinylpyrrolidone) and poly (e-caprolactone) monomer units. All values are in kcal mol1. Conformer

Eox

Ered

DE

Chitosan (C3) Chitosan (C6) Poly (vinyl pyrrolidone) (PVP) Poly (e-caprolactone) (PCL)

467435.70013 514669.30067 347629.6009 360858.8989

468196.1008 468196.1008 301152.1994 314380.3996

17535.2504 46473.1999 46477.4016 46478.4992

Scheme 1. Schematic representation for non-oxidable (red circle) and oxidable (green circle) hydroxyl groups of A chitosan and B grafted chitosan polymers. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

The conformer analyses focused on the hydroxyl end groups for both the PVP and PCL monomers while chitosan centred around the hydroxyls bonded to both C3 and C6. The results indicate the oxidised monomer formation is energetically favoured except for hydroxyl groups bonded to C3 in chitosan, which prevents any significant contribution for the latter to the reduction process during gold nanoparticles synthesis. Hence, the ability of chitosan to reduce Au III and produce gold nanoparticles would mainly be determined by the reactivity of the hydroxyl group bonded to C-6 (Scheme 1). The hydroxyl groups in both the PVP and PCL conformers are also primary alcohols, which explains the increased reducing power of grafted chitosan relative to unmodified chitosan based on the previously discussed experimental results. Since, the DE values are the same for the hydroxyl groups of PVP and PCL and these are slightly lower than of the hydroxyl groups of C6 of chitosan, it should be expected that the oxidation of the first ones would be lightly favored. Additionally the interactions between the grafted PVP and PCL blocks and gold nanoparticles during either, the nucleation and growth stages would be more probable than between nanoparticles, and chitosan backbone due to steric considerations. Thus, the better ability of grafted chitosan to reduce Au III and produce gold nanoparticles would mainly be determined by a higher number of oxidisable hydroxyl groups provided by the grafted chains and to a lesser extent to a lower steric hindrance of the same ones. However, more information on the role and nature of interactions during the nanoparticles synthesis is required.

4. Conclusions Although chitosan is a biopolymer, which has already been used in the synthesis and stabilisation of gold nanoparticles, this work shows a remarkable improvement on the performance of the polysaccharide in this application by grafting poly e-caprolactone and poly N-vinyl-2-pyrrolidone chains onto chitosan backbone. The graft copolymers exhibit higher yields, shorter reaction times and improved stability of nanoparticles in aqueous suspensions. The grafted side chains and its hydroxyl end groups would be responsible for the substantial enhanced performance, this due to its capabilities to interact with the surface of the nanoparticles and act as reductants respectively. The scope of this study is to contribute to the extensive applications of chitosan in the field of nanostructured materials, which is a field of great technological importance.

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