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International Journal of Biological Macromolecules 48 (2011) 682–687

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules

journa l homepage: www.e lsev ier .com/ locate / i jb iomac

reen synthesis and stabilization of gold nanoparticles in chemically modifiedhitosan matrices

nand D. Tiwaria, Ajay K. Mishrab,∗, Shivani B. Mishraa, Omotayo A. Arotibaa, Bhekie B. Mambaa

Department of Chemical Technology, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South AfricaUJ Nanomaterials Science Research Group, Department of Chemical Technology, University of Johannesburg, P.O. Box 17011, Doornfontein 2028, South Africa

r t i c l e i n f o

rticle history:eceived 28 January 2011eceived in revised form 9 February 2011ccepted 16 February 2011vailable online 22 February 2011

eywords:

a b s t r a c t

Chitosan-N-2-methylhydroxypyridine-6-methylcorboxylate (Ch-PDC) and chitosan-N-2-methylhydroxypyridine-6-methylhydroxy thiocarbohydrazide (Ch-PDC-Th) were synthesized forthe first time using chitosan as precursor. Chitosan, Ch-PDC, Ch-PDC-Th were used in the synthesis ofgold nanoparticles (AuNP) in aqueous medium. Chitosan and Ch-PDC-Th possess reducing propertieswhich enabled the ‘green’ synthesis of AuNPs. The stabilization of the AuNPs was as a result of thethiocarbide (S C) and amine (NH2) groups in the chitosan matrix. The modified chitosan, its deriva-

hitosanreen synthesisold nanoparticle stabilization

tives and the resulting AuNPs were characterized by Fourier transform infrared (FTIR) spectroscopy,Ultraviolet–visible (UV–vis) spectroscopy, Raman scattering measurements, powder X-ray diffraction(PXRD) and thermo gravimetric analysis (TGA). Particle size, morphology, segregation and individualityof the AuNPs were examined by transmission electron microscope (TEM) and energy dispersionspectroscopy (EDS). An average AuNPs size of 20 nm was observed for chitosan and Ch-PDC-Th whileCh-PDC was 50 nm. In comparison, AuNPs resulting from Ch-PDC-Th precursor has the most enhancedRaman and fluorescent intensities and was stable for over 2 months.

. Introduction

Recently, the clinical values of nanometre-scale biomaterialsave become increasingly evident, especially in the context ofanoparticle-mediated drug delivery for cancer therapy [1], tis-ue engineering, device based therapies, and medical imaging [2,3].he Foods and Drugs Association already approved the valida-ion of nanotherapeutic approach in clinical trials with severalanoparticle-based anticancer drugs. Metal nanoparticles withotential applications as biosensors and biolabels are thus under

ntense investigation. In cellular studies, metal nanoparticles haveeen used as enhancers of spectroscopic techniques in biochips forrotein and enzyme analysis. Nanoparticle coordination with bio-

ogical ligands which specifically bind with cellular targets, enableshe recording of diffusion pathways in receptor cells. Vascular com-artments uptake of the nanoparticle around the nucleus of cellsan be used to label the cells so that their pathway and fate can be

nvestigate [4].

Polymers have been applied in controlling the rate of theeduction process in nanomaterial synthesis and thus modu-ate the shapes and sizes of the resultant particles [5]. Small

∗ Corresponding author. Tel.: +27 11 559 6180; fax: +27 11 559 6524.E-mail address: amishra@uj.ac.za (A.K. Mishra).

141-8130/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.ijbiomac.2011.02.008

© 2011 Elsevier B.V. All rights reserved.

bioactive molecules and biomacromolecules are used in the“green” synthesis of nanoparticles [6]. Chitosan is a biopolymerwith biodegradable and biocompatible properties. Chitosan haspotential applications in biofabrication [7], pharmaceutics [8],biomedicine [9–13], food [14] and textiles [15] to mention a few.The polyelectrolyte nature of chitosan is of particular interest inmetal nanoparticle synthesis due to its interaction with metalions and metal nanoparticles. Through chelation, metal ions reg-ularly distributed throughout the polymer matrix. Subsequently,the dispersed metal ions can be reduced to a zero valent stateto form nanoparticles [16–19]. Starting with a chitosan solution,the synthesis of gold nanoparticles (AuNPs) has been extensivelyreported [16,20,21]. Chitosan also possess gel and film makingabilities which when combined with its polyelectrolyte nature,one-dimensional (1-D) chitosan fibres in solution can provide scaf-folds for the adsorption of metal ions with opposite charges. Variousfabrication methods such as wetspinning [22] and electrospinning[10] for 1-D chitosan fibres have been reported. For most appli-cations however, chitosan is usually chemically modified to tailorits properties to its intended use [7]. One of the numerous meth-

ods used in the chemical modification of chitosan is by Schiff basesynthesis through the condensation of amine groups on the chi-tosan backbone with aldehydes or ester group having moieties.Gold nanoparticles with unique chemical and physical proper-ties, have found wide use in fundamental research [23] while in

Biological Macromolecules 48 (2011) 682–687 683

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Scheme 1. Synthesis of chitosan-N-2-methylhydroxypyridine-6-methyl-hycorboxylate (Ch-PDC). Key: acetic acid (AcOH), ethanol (EtOH), Aqueous(aqu), hours (h).

A.D. Tiwari et al. / International Journal of

pplied science the nanoparticle are used in catalytic [24], biolog-cal [25], and sensing applications [26,27] they possess excellentiocompatibility, low toxicity, and relatively low reactivity, thusroviding benefits for use within living systems [28–35]. More-ver, the unique optical properties that result from surface plasmonands (SPBs) in the visible range of the electromagnetic spec-rum make them particularly attractive for optical applications. Thenhancements of absorption, fluorescence emission, and Ramancattering from analytes positioned near the surface of appropriateoble metallic nanoparticles are widely known [36].

We report the stepwise synthesis and characterization ofhitosan-N-2-methylhydroxypyridine-6-methylcorboxylate (Ch-DC) and chitosan-N-2-methylhydroxypyridine-6-methylhydroxyhiocarbohydrazide (Ch-PDC-Th) and their application in theynthesis of gold nanoparticles. Using techniques such as FTIRpectroscopy, UV–vis spectroscopy, Raman spectroscopy, PXRDpectroscopy, TGA, TEM and EDS; the characteristic features ofhese materials were compared.

. Materials and methods

.1. Materials

All compounds were of analytical grade and were used aseceived. Acetic acid (99.7%), sodium borohydride, sodium hydrox-de and pyridine 2,6-dicarboxylic acid were bought from MerckSouth Africa). Gold(III) chloride hydrate (48% gold) was obtainedrom fluka (Switzerland). Thiocarbohydrazide (99.0% syntheticrade) and chitosan (75% deacetylated, low molecular weight)ere obtained from Sigma–Aldrich (South Africa). Pyridine 2,6-imethylcorboxylate was prepared from pyridine 2,6-dicarboxyliccid using methanol and confirmed by NMR. Deionized (DI) wateras used throughout the experiment.

.2. Synthesis ofhitosan-N-2-methylhydroxypyridine-6-methylcorboylateCh-PDC)

The method used in this synthesis was adapted from thateported by Chen and Park [37]. Briefly, 200 mg of chitosanas dissolved in 50 mL of 2% acetic acid. A solution of Pyri-ine 2,6-dimethycarboxylate (1 g in 50 mL ethanol) was added tohe chitosan solution and refluxed for 8 h. The mixture was leftvernight. The gel was formed after neutralization using 1 mMaOH and gel was precipitated in the acetone. Filtered gel was dried

n hot air oven at the 60 ◦C for the 3 h. The yield of the dried productas 300 mg (90%) (Scheme 1).

.3. Synthesis ofhitosan-N-2-methylhydroxypyridine-6-methylhydroxyhiocarbohydrazide (Ch-PDC-Th): functionalization of Ch-PDC byhiocarbohydrazide

Ch-PDC (50 mg) and thiocarbohydrazide (250 mg) were dis-olved in 4% and 2% acetic acid respectively. Both solutions wereixed and refluxed for 24 h. The gel was formed after the neu-

ralization using 1 mM NaOH and was precipitated in acetone. Therecipitated compound was dried in the hot air oven at the 60 ◦Cor 3 h. The yield of the dried product was 50 mg (80%) (Scheme 2).

.4. Synthesis of the gold nanoparticle on the chemically modifiedhitosan biopolymer matrix

Chitosan, modified chitosan Ch-PDC and Ch-PDC-Th solutionsere prepared in 2% aqueous acetic acid (w/v) ratio of 10 mg/mL.

Scheme 2. Synthesis of chitosan-N-2-methylhydroxypyridine-6-methylhydro-xythiocarbohydrazide (Ch-PDC-Th). Key: acetic acid (AcOH), stirring (st), hours (h).

1 mM solution of gold(III) chloride hydrate was added to each of chi-tosan, Ch-PDC and Ch-PDC-Th in 1:1 (v/v) ratio. To the ChPDC andgold(III) chloride solution, excess amount of sodium borohydrate(20 �L of 1 M solution of NaBH4 per mL of the Ch-PDC solution)was added. All solutions were heated at 60 ◦C for 2 h then stirred

for another 1 h at room temp. The acronym CS-AuNPs, Ch-PDC-AuNPs and Ch-PDC-TH-AuNPs were given to the chitosan, Ch-PDCand Ch-PDC-TH stabilized gold nanoparticles respectively.

6 Biological Macromolecules 48 (2011) 682–687

2

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Fig. 1. (a) Comparative FTIR spectra of the biopolymer matrix precursor materialsfrom their scratched film. (b) Comparative FTIR spectra of the nanoparticle biopoly-mer matrix materials from their scratched film.

84 A.D. Tiwari et al. / International Journal of

.5. Characterization

Film samples of the polymers were prepared on glass slide andried in the oven at 60 ◦C then scratched by a razor. FTIR spectraPerkin Elemer spectrum 100) of biopolymer matrix with nanopar-icle and their precursor were recorded on an IR universal ATRampling system accessory transmission mode, accumulation of 16can with resolution of 4 cm−1 in the range of 4000–550 cm−1. TEMeasurements were investigated on a JEOL JEM-3010 instrument

perating at 120 kV coupled with energy-dispersive spectroscopyEDS). Images were collected on a Gatan digital imaging sys-em with Power Mac 8600 computer Digital Micrograph software.he TEM samples were prepared thus: the nanoparticle solu-ions were sonicated for 10 min, and then small drops of theold nanoparticle solution were dropped onto TEM lacey copperrid and allowed to dry. A PHLIPHS XPERT-PRO diffractometerystem (40 kV, 40 mA) equipped with Cu KR radiation with a wave-ength 0.1546 nm and a curved graphite crystal at a scanning

ode continuous rate 1.06◦/min was used. UV–vis absorption spec-roscopy measurements were performed in a Shimadzu UV-2450C dual-beam spectrophotometer using 1 cm path length quartzuvettes. Spectra were collected within a range of 200–800 nmn the aqueous solution. TGA was performed using a Perkin-lmer TGA 4000 thermogravimetric analyzer under a nitrogenas flow rate of 20 mL/min and heating rate 10 ◦C/min at theange of 30–1000 ◦C. Raman spectra were recorded on the Perkinlmer Raman 200 Raman microscope. Raman spectroscopy waserformed on a Raman enhanced surface chip under ambientondition. Confocal Raman microscope equipped with piezo scan-er using 532 nm excitation line directed through microscopebjectives (20×, NA = 0.40, Nikon) was employed. Spectra wereecorded with 1 cm−1 resolution and 10 s accumulation time rangef 200–3200 cm−1. Fluorescence spectroscopy was recorded on aerkin Elmer LS 45 fluorescence spectrometer operated by the FLin lab software.

. Results and discussion

.1. Synthesis and characterization of the chemically modifiedhitosan

.1.1. Fourier transform infrared (FTIR) spectroscopyThe modification on the chitosan was done in a stepwise reac-

ion and synthesized as Ch-PDC and Ch-PDC-Th (Schemes 1 and 2).ig. 1(a) presents the FTIR spectra and characteristic peaks of theodified chitosan respectively. In the Ch-PDC, ester characteristic

eak appeared at 1742 cm−1 due to methyl ester group of the pyri-ine 2,6-dimethycarboxylate. The bending bands of primary amine

n the chitosan at 1647 cm−1 disappeared and the one at 1590 cm−1

hifted to 1571 cm−1 in Ch-PDC due to the ester addition on NH2f chitosan backbone. In the Ch-PDC-Th, new peaks are observed at291 cm−1 and 1406 cm−1 due to amine and N–N groups respec-ively, Fig. 1 (b). Another peak occurring at 922 cm−1 correspondso thiocarbide (C S) group. These FTIR data are indicative of suc-essful synthesis or functionalization of the chitosan [38] and fullyupport the proposed reaction, Schemes 1 and 2.

.1.2. Thermo gravimetric analysis (TGA)TGA (Fig. 2) studies of chitosan have two weight loss steps-one

or the loss of moisture (9.3% weight loss) at the onset temperature

f 105.5 ◦C and the second weight loss (43.5%) at the onset temper-ture of 341.6 ◦C for the thermal degradation of chitosan polymer.n Ch-PDC three weight loss steps were observed: a weight lossf 8.2% at the onset temperature of 133.9 ◦C in the first step dueo the moisture loss. A weight loss of 6.2% was at the tempera-

Fig. 2. Comparative TGA graph of the chitosan, Ch-PDC and Ch-PDC-Th at range of30–1000 ◦C with heating rate of 10 ◦C/min with 20 mL/min flow of nitrogen.

A.D. Tiwari et al. / International Journal of Biological Macromolecules 48 (2011) 682–687 685

Fig. 3. (a) TEM image of Ch-PDC-Th gold nanoparticles in the colloidal aqueous solution for the biopolymer matrix. (b) EDS spectra of Ch-PDC-Th gold nanoparticles. (c) TEMimage of the chitosan-AuNPs in the colloidal aqueous solution for the biopolymer matrix. (d) EDS spectra of chitosan gold nanoparticles. (e) TEM image of the Ch-PDC goldn matr

tdodsPfioWttibTr

anoparticles (Ch-PDC-AuNPs) in the colloidal aqueous solution for the biopolymer

ure range of 201.2 ◦C in the second step, which may be due toegradation of pyridine ester. And in the third step, a weight lossf 44.1% at the onset temperature range of 401.2 ◦C which may beue to the thermal degradation path of the chitosan. The thermaltability of Ch-PDC was lower than the parent chitosan. For Ch-DC-Th, the thermal degradation happened in the four steps. Inrst step, at weight loss of 8.2% (onset temp. 135.2 ◦C) due to lossf residual or physically adsorbed water on membranes surfaces.eight losses of 18.4% (onset temp. 352.5 ◦C) and 22.1% (on set

emp. 496.6 ◦C) were recorded in the second and third steps respec-◦

ively. And finally a weight loss was of 23.9% (onset temp. 913.6 C)

n the fourth step. We however observed an improved thermal sta-ility of Ch-PDC-Th over that of the parent chitosan and Ch-PDC.hese stepwise weight loss observed from TGA corroborates theeactions steps shown in Schemes 1 and 2 [39].

ix. (f) EDS spectra of Ch-PDC gold nanoparticle.

3.2. Synthesis and characterization of the gold nanoparticles onthe chemically modified chitosan biopolymer matrix

The gold nanoparticles synthesized in each of the biopolymersthat is chitosan, Ch-PDC and Ch-PDC-Th showed different prop-erties. It is interesting to note that these differences could also beseen as colour changes. When the gold chloride solution was addedto the chitosan solution, its colour changed to dark brown. We takethis as indication of a reduction reaction of Au as a result of the NH2group present in the chitosan moiety. The pristine chitosan was also

able to stabilize the gold nanoparticle formed as a result of theseamine group. To further establish that the NH2 is responsible for thereduction in chitosan, we added the gold salt solution to Ch-PDCand there was colour change after 12 h because the most of aminehas been substituted by the pyridine 2,6-dimethycarboxylate [7].

6 Biological Macromolecules 48 (2011) 682–687

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to the polymers void of nanoparticles, i.e. precursors. At 488 nm

86 A.D. Tiwari et al. / International Journal of

.2.1. Fourier transform infrared (FTIR) spectroscopyThe FTIR band for the chitosan amine peaks at 1590 and

647 cm−1 shifted in CS-AuNPs to 1555 and 1632 cm−1 (Fig. 1(b)).he shifting of these peaks also confirms that the amine of thehitosan back bone is coordinated to gold nanoparticle for the sta-ilization of the gold nanoparticle. When sodium borohydride wasdded to the Ch-PDC and gold salt mixture, a fast reduction of theold salt to gold nanoparticle occurred as indicated by the rapidolour change. Though the Ch-PDC could not reduce the gold salts fast as CS and Ch-PDC-Th but, it was able to stabilize the goldanoparticle formed after the addition of sodium borohydride aseducing agent and the pyridine act as stabilizer respectively [40].n the case of Ch-PDC-Th, after the addition of the gold salt, theolution changed to brown colour within 2 h due to the formation ofold nanoparticles as a result of the reducing property of NH2 group.e observed that the Ch PDC-Th-AuNPs were the most stable overmonths by the UV–vis spectroscopy. This is attributed to the

H2 and C S groups of thiocarbohydrazide which further stabilizedhe gold nanoparticle as a result of the well known gold–sulphurnteraction. FTIR also confirmed the stabilization effect of the NH2nd C S groups in the Ch-PDC-Th. The peaks associated with themine group in Ch-PDC-Th shifted from 3291, 1571, 1662 cm−1 to275, 1560, 1634 cm−1 respectively. The sharp peak at 922 cm−1

orresponding to the C S bond in the Ch-PDC-Th was absent inhe Ch-PDC-Th-AuNPs composite [38]. This FTIR profiles show thatano-sized gold complex were formed in the biopolymer matrixlm.

.2.2. Transmission electron microscopy (TEM) and powder X-rayiffraction (PXRD)

The TEM in Fig. 3 shows the successful preparation of theold nanoparticles. The Ch-PDC-Th-AuNPs was well dispersed andpherical in shape with an average size less than 20 nm. CS-AuNPslso has an average size of 20 nm but not as well dispersed ash-PDC-Th-AuNP. A larger size of 50 nm was observed for Ch-PDC-uNPs due to the absence of reducing group amine. For all the TEM

mages, EDS was used to confirm that the observed particles wereruly gold.

The crystallinity of the Ch-PDC-Th-AuNPs was investigated withowder X-ray diffraction (Fig. 4) and sharp diffraction peak at

.9, 8.7, 17.9, 26.9 and 36.1 were observed. The peak at 36.1as been reported to be characteristic of gold crystal (1 1 1)41,42].

ig. 4. PXRD spectral pattern for the gold nanoparticles formed on the Ch-PDC-Thlm. (a) Typical TEM image of the gold nanoparticles of the stabilization on theh-PDC-Th biopolymer matrix in the solution.

Fig. 5. UV–vis spectra of chitosan, Ch-PDC and Ch-PDC-Th, gold solution and thegold nanoparticle in the biopolymer matrices from 400 to 650 nm.

3.2.3. Ultraviolet–visible (UV–vis) spectroscopyUV–vis characterization of the nanoparticles and their precur-

sors are presented in Fig. 5. The absorption bands around the500–550 nm are characteristic of the surface plasmon bands (SPBs)for gold nanoparticle [43]. The three gold nanoparticles have thesame SPBs at 525 nm while their precursors have no peak at 525 nm.Ch-PDC-AuNPs has the lowest number of particles as well as largestparticle size (from TEM, Fig. 3) among the three nanoparticles. Thisparticle size can be explained by the very rapid reduction of gold IIIusing sodium borohydride. The reduction process in the other two(i.e. CS-AuNPs and Ch-PDC-Th-AuNPs) were done in the absenceof external reducing agents and thus a slower reduction processwhich made the particle size finer. The lowest absorption observedfor Ch-PDC-AuNPs therefore correlates with the lowest absorptionat SPBs as seen in Fig. 5.

3.2.4. Fluorescence spectraFrom the fluorescence spectra (Fig. 6), the polymers with

embedded nanoparticles showed higher intensities as compared

the magnitude of intensities for the nanoparticles can be writtenas Ch-PDC-Th-AuNPs > Ch-PDC-AuNPs > CS-AuNPs. The increase inintensity at this wavelength (488 nm) is due to the metallic nanos-

Fig. 6. Fluorescence spectra of the chitosan, Ch-PDC, Ch-PDC-Th and gold nanopar-ticle in the biopolymer matrices.

A.D. Tiwari et al. / International Journal of Biolog

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ig. 7. Raman spectra from chitosan, Ch-PDC and Ch-PDC-Th and gold nanoparticlen the biopolymer matrices.

ructures which favorably enhanced the spectral properties ofuorophore and alleviate some of their more classical photophysi-al constraints, such as low quantum yield and poor photo stability44].

.2.5. Raman spectroscopyGenerally, the Raman intensities (Fig. 7) of the gold nanoparti-

les were found to be higher than their corresponding precursors.n consistent with the TEM and UV–vis results, Ch-PDC-Th-AuNPs

hich has the lowest size showed the highest Raman intensity.igh intensity property of the gold nanoparticle can be used as

urface enhanced Raman spectroscopy (SERS) which is applied inhe biological tracing studies [45].

Raman band at the 1399 cm−1 and 1256 cm−1 assigned tohitosan backbone shifts slightly to wave number of 1347 cm−1

nd its intensity gets down after addition of pyridine-2,6-imethylcarboxylate.

. Conclusions

We have successfully prepared novel chitosan derivatives withuitable functional groups such as amine and thiocarbide that cane exploited for various applications. The modified chitosan serveds the matrix within which the gold nanoparticle was synthesizedithout the addition of sodium borohydride which is known to beazardous. This green synthesis is advantageous not only from theoint of solvent elimination but also from the biological applica-ion point of view where substances such as sodium borohydrideave toxic effect. The gold nanoparticles were entirely reduced andtabilized by the biocompatible chitosan derivative. The enhanceduorescence and Raman intensities of the gold nanoparticles overheir precursors are good prospects for surface enhanced Ramanpectroscopy (SERS) which is applied in the biological tracing stud-es. The potentials of chitosan biopolymer and gold nanoparticlen biomedical application is sought to be harnessed in these novelompounds for future biological applications

cknowledgements

Authors are thankful for the financial supports from theintek’s Nanotechnology Innovation Centre (South Africa),

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ical Macromolecules 48 (2011) 682–687 687

National Research Foundation (South Africa), University of Johan-nesburg and UJ commonwealth fellowship to the Author Anand D.Tiwari. Authors are also thankful to Prof. Mike Witkom for TEManalysis and Prof. Rui Krause and Dr. Ajit K Sharma for valuablesuggestions and discussions.

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