Preparation Of Gold Nanoparticle-Quercetin Complexes By Citrate Reduction Method

Rajat Pal and Abhay Sankar Chakraborti #

Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta 92, Acharyya Prafulla Chandra Road, Kolkata 700009, India # ascbmbg@caluniv.ac.in Abstract. Quercetin is an important flavonoid and possesses strong antioxidant property. The aim of the present study is to formulate and characterize quercetin coated gold nanoparticles. Quercetin was conjugated with gold nanoparticle during synthesis of the particle by citrate reduction of chloroauric acid. The conjugates were characterized by different techniques like Atomic Force Microscopy, Dynamic Light Scattering, Transmission Electron Microscopy, Absorption Spectroscopy, Differential Scanning Calorimetry and Thermal Gravimetric Analysis. All these studies suggest formation of stable quercetin-gold nanoparticle complex. Key words: Gold nanoparticles; quercetin; antioxidant. PACS: 81.07.Nb

INTRODUCTION
In recent years, considerable attention has been paid to the synthesis and characterization of gold nanoparticles; because they can be used in different chemical science and technologies.1 Gold nanoparticles are biologically inert and cause no serious side effects in biological systems. In several studies, gold nanoparticles have been used for drug delivery 2-4 and delivery of proteins, peptides, and oligonucleotides etc.5-7 Flavonoids are ubiquitously present in common plant based food items and beverages and they are known to have antioxidant properties that can protect against cardiovascular diseases8, certain kinds of cancers 9, cell apoptosis 10 etc. Since diabetes mellitus is considered as a free radical mediated disease, there has been renewed interest in the use of flavonoids in diabetes research. For example, quercetin, an important flavonol, has been reported to have ameliorative effect in diabetes mellitus.11, 12 To enhance the efficacy of a therapeutic agent, use of nanoparticle-based drug formulation is an important aspect of nanomedicine, for which the first step is the preparation of a stable nanoparticle-drug complex. Wang et al 13 have reported detection of flavonoids namely quercetin, daizeol and puerarin and assay of their antioxidant activities by using gold nanoparticles. The finding is significant but only at a seminal stage. We have, therefore, undertaken the present study to synthesize quercetingold nanoparticle formulation and characterize the composite for further study of its antioxidant and antidiabetic effect.

CREDIT LINE (BELOW) TO BE INSERTED ON THE FIRST PAGE OF EACH PAPER

CP1276, International Conference on Advanced Nanomaterials and Nanotechnology (ICANN-2009) edited by P. K. Giri, D. K. Goswami, A. Perumal, and A. Chattopadhyay 2010 American Institute of Physics 978-0-7354-0825-8/10/$30.00

283

Downloaded 07 Oct 2011 to 210.125.120.79. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions

METHODS Synthesis of Gold Nanoparticles

Gold nanoparticles were synthesized by reducing chloroauric acid by trisodium citrate in the boiling condition according to the method of Turkvitch.14 To synthesize free gold nanoparticles (GNP), 0.1 ml of 1% trisodium citrate was added to 10 ml of 0.01 % chloroauric acid under boiling condition. After few minutes the color changed to wine red. The drug was coated onto the nanoparticles during synthesis of GNP. For preparation of gold nanoparticles-quercetin complexes (GNPQ), different volumes of quercetin (10 mg/ml in DMSO) were added to the chloroauric acid and trisodium citrate mixture (to get desired drug concentration) at the boiling condition prior to the formation of the wine red color.

Characterization of GNP and GNPQ

The sizes of GNP and GNPQ were studied by Atomic Force Microscopy (AFM) using Nanoscope III A system (VECCO Multimedia, USA) and by Transmission Electron Microscopy (TEM) using Techna SE II, Philips Co. Netherlands. The diameters of the particles (GNP and GNPQ) were measured by Dynamic Light Scattering (DLS) method using Zeta Sizer nano ZS and the surface charges were determined from Zeta potential. For spectroscopic study, absorption spectra of GNP and GNPQ were recorded in Hitachi U2800 spectrophotometer. The melting profile of the samples was studied in Perkin Elmer Jade DSC, and Thermal Gravimetric Analysis (TGA) was done by using Mettler SW 9.01.

RESULTS AND DISCUSSION

AFM study shows that the surface of GNP were smooth and had an average diameter 27 4.3 nm (Fig. 1A i), but when the particles were coated with quercetin in GNPQ, the surface of the particles became rough and had larger average diameter of 63 7.89 nm (Fig 1A ii). The finding was also confirmed by TEM study. The TEM images, shown in Fig. 1B, revealed that average diameter of GNP (Fig. 1B i) were 20 2.7 nm and that of GNPQ (Fig. 1B ii) were 42 6.9 nm. Wang et al 13 also reported the enlarged size of flavonoid-coated gold nanoparticles in comparison with free nanoparticles. The diameters of GNP and GNPQ were measured by DLS technique, and the curves of particle distribution are shown in Fig. 2A. The diameter of GNP was found to be 37 nm, while GNPQ made with 0.05 mg/ml and 0.1 mg/ml quercetin exhibited 78 nm and 141 nm diameters, respectively. Increased diameter of GNPQ, as shown by microscopic and DLS methods, suggest that the attached drug has enhanced the diameter of the gold nanoparticle. Gold nanoparticles dispersed in water are negatively charged due to their characteristics of gold micelles 15. The measurements of zeta potential showed (Fig 2B and 2C) that the negative charges present on the surface of free gold nanoparticles were reduced from -28.7 mV to -20.1 mV when quercetin was attached. This further suggests the formation of the complex.

284

Downloaded 07 Oct 2011 to 210.125.120.79. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions

FIGURE 1: A) AFM images of i) Free gold nanoparticles and ii) Gold nanoparticles coated with quercetin (drug concentration 0.1 mg/ml). B) TEM images of i) Free gold nanoparticles and ii) Gold nanoparticles coated with quercetin (drug concentration 0.1 mg/ml).

FIGURE 2: A) Diameter measurement of nanoparticles by DLS i) Free gold nanoparticles, ii) Nanoparticles coated with 0.05 mg/ml quercetin, iii) Nanoparticles coated with 0.1 mg/ml quercetin. B) Zeta potential of free gold nanoparticles, C) Zeta Potential of quercetin coated nanoparticles (drug concentration 0.1 mg/ml).

285

Downloaded 07 Oct 2011 to 210.125.120.79. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions

To confirm the attachment of the drug to the nanoparticles, the spectra of GNP and GNPQ with different concentration of drug were recorded. GNP exhibited only one peak at 530 nm (spectrum a of Fig. 3A), which is in agreement with other reports.16, 17 However, another peak at 292 nm became prominent in the spectra of GNPQ, and its height increased gradually with increased concentration of the flavonoid (spectra b to f), indicating progressive attachment of the drug to the nanoparticles. The absorbance at 292 nm characterized the gold nanoparticlequercetin composite, because free quercetin exhibited a peak at 375 nm only (spectrum g). In TGA analysis (data not shown), quercetin appeared to be completely decomposed around 495OC and no residue was left behind. When GNPQ sample was heated under similar condition, about 15% residue (brittle gold color solid) was left behind. Leff et al 18 have also reported that brittle gold colored solid is left at the end of TGA analysis of gold nanoparticles functionalized with primary amines.

FIGURE 3: UV Vis spectra of a: free gold nanoparticles, b f: nanoparticles with increasing concentrations of drug (0.03, 0.05, 0.07, 0.1 and 0.14 mg/ml respectively), and g: Free quercetin.

FIGURE 4: Thermograms (heat flow vs. temperature) of i) free quercetin, ii) quercetin coated gold nanoparticles (drug concentration 0.1 mg/ml). 3 mg of each sample were heated from 50OC to 400OC at a heating rate of 10OC/min (Data shown from 200 OC to 400OC).

286

Downloaded 07 Oct 2011 to 210.125.120.79. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions

The melting profiles of quercetin and quercetin coated gold nanoparticle were observed by DSC analysis (Fig. 4). Free drug had a melting temperature near 329OC, but the conjugate remained unchanged up to 400OC. All these findings suggest formation of a stable quercetin-gold nanoparticles complex, which will be worth trying to study for therapeutic efficiency in biological systems.

ACKNOWLEDGEMENTS
The work was partly supported by the Centre for Research in Nanoscience & Nanotechnology, CU and DST-FIST program of the Department of Biophysics, Molecular Biology & Bioinformatics, CU. We thank Prof. S. Bhattacharjee, Department of Chemical Engineering, CU, Dr. A. N. Ghosh, NICED, Kolkata, Prof. A. Dasgupta, Department of Biochemistry, CU and Dr. D. Das, Department of Chemistry, CU for allowing us to use AFM, TEM, DLS and TGA facilities, respectively.

REFERENCES
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13 14. 15. 16. 17. 18. T. M. Lee, H. Cai, I. M. Hsing, Analyst 130, 364 369 (2005). M. E. Wieder, D. C. Hone, M. J. Cook, M. M. Handsley, J. Gavrilovic, D. A.Russell, Photochem.. Photobiol. 5, 727 734 (2006). I. H. El-Sayed, X. Huang, M. A. El-Sayed, Cancer Lett. 239, 129 135 (2006). X. Huang, P. K. Jain, I. H. El-Sayed, M. A. El-Saved, Photochem. Photobiol. 82, 412 417 (2006). G. F. Paciotti, D. G. I. Kingston, L. Tamarkin, Drug Dev. Res. 67, 47 54 (2006). G. Han, P. Ghosh, M. De, V. M. Rotello, NanoBiotechnol. 3, 40 45 (2007). G.Han, P.Ghosh, V. M. Rotello, Nanomedicine 2, 113 123 (2007). G. Block Nutr. Rev. 50, 207 213 (1992). G. Block, L. Langseth, Food Technol. 48, 80 84 (1994). M.R. Vijayababu, P. Kanagaraj, A. Arunkumar, R. Ilangovan, A. Dharmarajan, J. Arunkumaran Oncol. Res. 16, 67 74 (2006). M. Vessal, M. Hemmanti, M. Vasei, Comp.Biochem. Physiol. (Part C) 135, 357 364 (2003). F. M. Rouscher, R. A. Sabnders, J.B. Watkins, J. Biochem. Mol. Toxicol. 15, 159 164 (2001). J. Wang, N. Zhou, Z. Zhu, J. Huang and G. Li, Anal. Bioanal Chem. 388, 1199 1205 (2007). J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot, A. Plech. J. Phy. Chem. B 110, 15700 15707 (2006). J. K. Lung, J. C. Huang, D. C. Tien, C. Y. Liao, K. H. Tseng, T. T. Tsung, W. S. Kao, T. H. Tsai, C. S. Jwo, H. M. Lin, L. Stobinski. J. Alloys Compounds 434, 655 658 (2007). A. Henglein, J. Phys. Chem. 97, 5457 5471 (1993). J. J. Storhoff, A. A. Lazarides, R.C. Mucic, C.A. Mirkin, R.L. Letsinger, G.C. Schatz, J. Am. Chem. Soc. 122, 4640 4650 (2000). D. V. Leff, L. Brandt and J. R. Heath, Langmuir 12, 4723 4730 (1996).

287

Downloaded 07 Oct 2011 to 210.125.120.79. Redistribution subject to AIP license or copyright; see http://proceedings.aip.org/about/rights_permissions