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Conjugation of curcumin with PVP capped gold nanoparticles for improving bioavailability
Materials Science and Engineering C 32 (2012) 2659–2663
Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Conjugation of curcumin with PVP capped gold nanoparticles for improving bioavailability Rajesh K. Gangwar a, Vinayak A. Dhumale a, Dimple Kumari b, Umesh T. Nakate a, S.W. Gosavi c, Rishi B. Sharma d, S.N. Kale a,⁎, Suwarna Datar a,⁎ a
Department of Applied Physics, Defence Institute of Advanced Technology (DU), Girinagar, Pune 411025, India Department of Applied Chemistry, Defence Institute of Advanced Technology (DU), Girinagar, Pune 411025, India Department of Physics, University of Pune, Pune 411007, India d DOP, DRDO Bhawan, Rajaji Marg, New Delhi 110105, India b c
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 22 June 2012 Accepted 14 July 2012 Available online 20 July 2012 Keywords: Curcumin Gold nanoparticles Bioavailability
a b s t r a c t Curcumin, a natural polyphenolic compound, has astounding therapeutic applications but lacks in bioavailability mainly due to its poor solubility in water. Polyvinyl pyrrolidone (PVP) which is a proven drug carrier has been used to facilitate the conjugation of curcumin with gold nanoparticles and to improve the solubility of curcumin in water. In this conjugate diaryl heptanoid chromophore group of curcumin which is a much needed group in biomedical applications remains intact as observed from FTIR and UV–vis spectroscopy analysis. The work shows good promise for such conjugates as therapeutic-cum-imaging materials in biomedical field. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Curcumin, a bio-active polyphenol component of curcuma longa (Turmeric), also known as diferuloylmethane (C21H20O6), has become an intense topic of research due to its interesting biological and pharmacological applications [1–3]. It has anti-inflammatory, anti-oxidant, anti-carcinogenic, chemo-preventive, anti-angeogenic, anti-diabetic, anti-viral and antibacterial properties [4,5]. In spite of its wide property range, this polyphenolic compound is less documented for its actual applications, mainly due to its poor bioavailability. Curcumin remains to be less popular as a drug since the basic necessity of any drug to be delivered is its ease in suspension in bio-media (either aqueous solutions or PBS solutions). Curcumin's hydrophobic nature is one of the main reasons for this poor water-solubility/suspension capacity. This leads to its poor activity, low absorption, high rate of metabolism within the living system and rapid elimination from the system [6]. There have been attempts to functionalize the molecule to improve its bioavailability by conjugating it with a hydrophilic molecule. However, this process hampers the therapeutically-active group of the molecule, thereby killing the basic purpose of the conjugation. Numerous approaches have been reported in this context to explore the potent applications of curcumin in very recent times [7–10]. Nanoparticles [11,12], liposomes [13], micelles [14] and phospholipid complexes [15] are being used to improve the bioavailability of curcumin. ⁎ Corresponding authors. Tel.: +91 20 2430 4091; fax: +91 20 2438 9572. E-mail addresses: [email protected] (S. Datar), [email protected] (S.N. Kale). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.022
Bhawana et al. and Wu et al. have prepared curcumin nanoparticles [8] and nanogels [16] for antimicrobial and photo-thermal therapy respectively. Dhule et al. have explored curcumin-loaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma [17]. Ha et al. have synthesized poly(lactide)-vitamin E TPGS (PLA-TPGS) copolymer to formulate a curcumin nanocarrier [18] and Bisht et al. have reported on polymeric nanoparticle-encapsulated curcumin [19] for human cancer therapy. Recently, S. Manju et al. have reported synthesis of water soluble gold nanoparticles in curcumin-polymer conjugate and studied it for blood compatibility and targeted drug delivery onto cancer cells [20]. They have conjugated curcumin with hyaluronic acid (HA) and then synthesized gold nanoparticles within the solution by reducing the chloroauric acid. In this and many other documented protocols, it has been observed that though the bio-availability improves upon conjugation, the main therapeutic group of this molecule gets engaged in the conjugation thereby making itself less-available for its therapeutic activity to any biological system [21]. Therefore, there is a need for an extensive research in this regard which would not only improve the bioavailability of the molecule but also keep the therapeutic activity high and impart multi-functional properties to the conjugated system. On the other side, gold nanoparticles have been immensely explored due to their interesting optical, electrical, physical and chemical properties, which find useful technological applications in the field of opto-electronics [22], chemical and biological sensors [23] and biomedicines [24]. These have been projected to be one of the safest (non-toxic) candidates for DNA-conjugation [25], bio-detection [26], drug-delivery, gene therapy and many other applications [27,28].
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These nanoparticles have also been documented to be good for bio-imaging purposes owing to their interesting surface-plasmon resonance property. In this context, through this manuscript we report a neat protocol which describes the formation of curcumin conjugated with gold nanoparticles using PVP which has been used as drug carrier for various drugs in several reports [29]. This protocol envisages good bio-availability of the curcumin molecule, keeping its therapeutic activity intact. Gold nanoparticles show excellent plasmon resonance characteristic, exhibiting its potential in applications of imaging. 2. Experimental 2.1. Synthesis Chloroauric acid (HAuCl4) has been used as a metal precursor and tri-sodium citrate (Na3C6H5O7) as reducing agent for the synthesis of gold nanoparticles [30]. 200 ml of 1 mM HAuCl4 was boiled and stirred under the reflux condition for 30 min. 20 ml of 38.8 mM aqueous Na3C6H5O7 was added directly into the boiled solution. The color of the solution changed from pale yellow to deep red within 7–10 min after the addition. Further the reaction was continued for additional ~ 20 min after which 20 mg of polyvinyl pyrrolidone (PVP, M.W. = 40,000) in 30 ml of water was added to the above solution and stirred for the next 45 min. The solution was cooled at room temperature. For conjugation of curcumin, 50 mg of crystalline curcumin was dissolved in 25 ml acetone and this solution was added to the 100 ml of PVP capped gold nanoparticles under stirring. Further this mixture was stirred for 3 h at 60 °C and then cooled down to room temperature. The Au–curcumin solution was then centrifuged at 4000 rpm to remove unattached curcumin. This was done three times to ensure that no free curcumin molecules are left in the final conjugate. The final solution was used for spectroscopic study and TEM analysis. Part of this solution was evaporated under vacuum using a rotary evaporator and then the remnant was centrifuged to obtain the powder, which was also used for analysis. 2.2. Characterization The synthesized gold nanoparticles and Au–curcumin samples were characterized by UV–vis Spectroscopy (Ocean Optics, HR4000), Fourier Transform Infrared Spectroscopy (FTIR, Perkin Elmer), Thermogravimetric Analyser (TGA, Perkin Elmer STA 6000) and Transmission Electron Microscopy (TEM, Philips CM 200 and FEI-Tecnai G2 20). For TEM characterization highly diluted specimens were prepared and dilution level was kept constant for all samples. Toxicity analysis was done using MTT assay as described elsewhere [31].
Fig. 1. FTIR spectra of (i) PVP (ii) curcumin (iii) PVP functionalized gold nanoparticles and (iv) PVP functionalized gold nanoparticles with curcumin.
bonding to enolic hydroxyl group which results in shift of O\H stretching from 3514 cm−1 to 3435 cm−1 whereas the basic diaryl heptanoid group, which is the chromophore group of curcumin remains intact [32]. This is also supported by UV–vis spectroscopy explained below. Fig. 2 shows the UV–vis spectra of (i) curcumin in PVP (ii) PVP functionalized gold nanoparticles and (iii) PVP functionalized gold nanoparticles with curcumin in aqueous media. UV–vis spectrum of curcumin in PVP shows an absorption peak at ~ 415 nm which is the signature of basic diaryl heptanoid chromophore group of curcumin [11]. The spectrum of PVP functionalized gold nanoparticles (ii) shows absorption peak at ~520 nm, which is a characteristic peak of gold nanoparticles arising due to Surface Plasmon Resonance (SPR) [33]. The UV–vis spectra of PVP functionalized gold nanoparticles with curcumin (iii) clearly show two distinct peaks at ~418 nm and ~525 nm which confirms that the characteristic peaks for the components in the Au–C complex are intact. The red-shift in the absorption peaks for curcumin (~3 nm) and gold NPs (~5 nm) can be attributed to the conjugation of PVP functionalized gold NPs with curcumin. Thus, the UV–vis data with the support of FTIR results clearly
3. Results and discussion Fig. 1 shows the FTIR spectra of (i) PVP (ii) curcumin (iii) PVP functionalized gold NPs and (iv) PVP functionalized gold nanoparticles with curcumin. Here spectra (i) and (ii) show the typical signatures of both PVP and curcumin. These signatures may be recognized as C_O (1660 cm−1) and C\N (1290 cm−1) in PVP. Signatures of curcumin are free O\H group (3514 cm−1), C_O and C_C (enol) (1450– 1630 cm−1), C\H (methyl) (2845 cm−1), C\H (aryl) (3015 cm−1) and C\O\C (1000–1300 cm−1) typically attributed to symmetric and asymmetric configurations of C\O\C chains [11]. Spectrum (iii) illustrates the functionalization of citrate reduced gold NPs with PVP in the form of shift of C_O stretching from 1660 cm−1 to 1628 cm−1. This may be attributed to the formation of intermolecular hydrogen bonding. Spectrum (iv) shows the conjugation of curcumin with PVP functionalized gold NPs. This figure reveals signatures of both PVP functionalized gold NPs and curcumin molecule. Curcumin molecule may attach to the PVP functionalized gold NPs with intermolecular hydrogen
Fig. 2. UV–vis spectra of (i) curcumin in PVP (ii) PVP functionalized gold nanoparticles and (iii) PVP functionalized gold nanoparticles with curcumin. Inset shows the colorimetric response for each sample (i.e. i, ii and iii).
R.K. Gangwar et al. / Materials Science and Engineering C 32 (2012) 2659–2663
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Fig. 3. Schematic for possible conjugation of curcumin with PVP functionalized gold nanoparticles.
demonstrate that curcumin retains its diaryl heptanoid chromophore group which is much needed in biomedical applications. The suspension stability of these conjugates was checked using aqueous solutions of a) only curcumin in PVP b) gold:PVP and c) gold:PVP:curcumin. The inset of Fig. 2(b) indicates that the conjugates show excellent aqueous-stability, imparted due to the chemistry involved. Fig. 3 shows the schematic for probable conjugation of curcumin molecule with PVP functionalized gold NPs. Citrate reduced gold nanoparticles are capped by PVP as shown in the schematic. This
conjugation takes place by intermolecular hydrogen bonding as shown in the figure which is also supported by the shift of C_O stretching from 1660 cm−1 to 1628 cm−1 in the FTIR spectra of citrate reduced PVP functionalized gold nanoparticles. Curcumin also gets attached to the PVP functionalized gold NPs as shown in the figure by the intermolecular hydrogen bonding to enolic hydroxyl group which results in shift of O\H stretching from 3514 cm−1 to 3435 cm−1. Fig. 4 shows TEM images of (a) PVP functionalized gold NPs (b) curcumin in PVP (c) PVP functionalized gold NPs with curcumin
Fig. 4. TEM images of (a) PVP functionalized gold nanoparticles, (b) curcumin in PVP, (c) PVP functionalized gold nanoparticles with curcumin (Au–C complex) and (d) magnified image of circular region as shown in (c).
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and (d) magnified image of circular region as shown in (c). It is observed from Fig. 4(a) that PVP functionalized gold NPs are spherical in shape having diameter of 13 ± 2 nm. Fig. 4(b) shows the dispersion of curcumin and PVP (2:1) in aqueous medium for reference. PVP functionalized gold NPs with curcumin (Au–C complex) is shown in Fig. 4(c). As observed in the figure, gold NPs (black cores) are surrounded by PVP–curcumin conjugate as a shell. This is supported by the image of PVP–curcumin conjugation (b) as well as the FTIR results showing the hydrogen bonding between the PVP and curcumin. Magnified image of circular region in Fig. 4(c) is shown in Fig. 4(d), reveals that the PVP–curcumin completely surrounds the gold nanoparticles. Here PVP provides the required hydrophilicity to the curcumin molecule making them bioavailable. The TGA curve of gold:PVP:curcumin (Au–C complex) shown in Fig. 5(a) illustrates that the complex begins to lose weight at ~ 235 °C and the loss of weight is about 23% in the temperature range of 50–440 °C. After 440 °C, the rate of weight loss is slower and about 5.5% up to 550 °C. The net weight loss may be attributed to the decomposition of PVP:curcumin coating around the gold nanoparticles. The TGA results show that 26% of curcumin + PVP get loaded on the gold nanoparticles. The cytotoxicity effect of Au:PVP:C conjugate was studied against TZM-bl cell lines, which are modified HeLa derived cell lines. Inset of
Fig. 5(a) shows the cytotoxicity results of the cell lines after incubation with the conjugate for 24 h with increasing concentration. The cell viability of TZM-bl cells after incubation in the medium containing conjugated curcumin demonstrates that the conjugate shows 100% cell viability till the conjugate concentration of ~10 μg/ml. Beyond this concentration the sample exhibits toxicity, which may be useful for the therapeutic applications. Thus by conjugating nanoparticles with curcumin, the cytotoxic dose was found to be much less indicating that not only the anticancer activity of curcumin was intact and with curcumin, the conjugate showed enhanced cytotoxic effect. The results are being evaluated against the primary fibroblasts, which is underway. Fig. 5(b) shows in-vitro release kinetics of curcumin from conjugate. The conjugate was dispersed (1 mg/ml) in the Phosphate Buffer Solution (PBS: 7.4 pH) and the release kinetics was studied using UV vis spectroscopy. The experiment was performed at 35 °C and the data was recorded after every 1 h to monitor the release. As shown in the figure, more than 30% release was observed within 2 h then the rate of the release became slow. From 2 h to 9 h the net release was upto 60%. These results are quite encouraging owing to the applications of curcumin as antimicrobial and antiviral agents. Such gold nanoparticles surrounded by curcumin and PVP providing the bioavailability can be harnessed for excellent applications in targeted drug delivery, antimicrobial activity and bio-imaging. 4. Conclusion In conclusion, we have proposed a simple route to conjugate curcumin with PVP functionalized GNPs which are highly stable in aqueous medium at room temperature. Diaryl heptanoid chromophore group of curcumin and plasmon resonance signature of gold remain intact after conjugation ensuring the bio-active nature of curcumin and gold signature. The conjugate loading percentage was seen to be nearly 26%. The conjugate shows 100% cell viability till the conjugate concentration of ~10 μg/ml. Beyond this concentration the sample exhibits toxicity, which may be useful for the therapeutic applications. More than 30% release was observed within 2 h then the rate of the release became slow. The net release was nearly 60% in 9 h. This work has good potential in antimicrobial studies, bio-imaging and drugdelivery applications. Acknowledgments The authors thank Dr. Prahlada, Vice Chancellor, Defence Institute of Advanced Technology (Deemed University), Pune for providing the laboratory facilities and financial assistance. Thanks are also due to Ms. Ruchira Mukherjee of NCL, Pune for the experimental support. SNK acknowledges funding from DST-Nano Mission (SR/NM/NS-63/ 2009) for this work. SNK and SSD acknowledge funding from “DRDO-DIAT Program on Nanomaterials by ER&IPR, DRDO”. References
Fig. 5. (a) TGA curve for Au:PVP:curcumin (Au–C complex) and (b) shows the graph of in-vitro release kinetics of curcumin at 35 °C in buffer solution (pH = 7.4). Inset of panel (a) shows the cytotoxicity histogram.
[1] R.A. Sharma, A.J. Gescher, W.P. Steward, Eur. J. Cancer 41 (2005) 1955–1968. [2] P. Anand, S.G. Thomas, A.B. Kunnumakkara, C. Sundaram, K.B. Harikumar, B. Sung, S.T. Tharakan, K. Misra, I.K. Priyadarsini, K.N. Rajasekharan, B.B. Aggarwal, Biochem. Pharmacol. 76 (2008) 1590–1611. [3] R.K. Maheshwari, A.K. Singh, J. Gaddipati, R.C. Srimal, Life Sci. 78 (2006) 2081–2087. [4] I. Chattopadhyay, K. Biswas, U. Bandyopadhyay, R.K. Banerjee, Curr. Sci. 87 (1) (2004) 44–53. [5] C. Senft, M. Polacin, M. Priester, V. Seifert, D. Kögel, J. Weissenberger, BMC Cancer 10 (491) (2010) 1–8. [6] P. Anand, A.B. Kunnumakkara, R.A. Newman, B.B. Aggarwal, Mol. Pharm. 4 (6) (2007) 807–818. [7] K. Im, A. Ravi, D. Kumar, R. Kuttan, B. Maliakel, J. Funct. Foods 4 (2012) 348–357. [8] R.K. Bhawana, H.S. Basniwal, V.K. Buttar, N. Jain, J. Agric. Food Chem. 59 (2011) 2056–2061. [9] M.M. Yallapu, M. Jaggi, S.C. Chauhan, Drug Discov. Today 17 (1/2) (2012) 71–80. [10] P. Anand, H.B. Nair, B. Sung, A.B. Kunnumakkara, V.R. Yadav, R.R. Tekmal, B.B. Aggarwal, Biochem. Pharmacol. 79 (3) (2010) 330–338.
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Contents lists available at SciVerse ScienceDirect
Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Short communication
Conjugation of curcumin with PVP capped gold nanoparticles for improving bioavailability Rajesh K. Gangwar a, Vinayak A. Dhumale a, Dimple Kumari b, Umesh T. Nakate a, S.W. Gosavi c, Rishi B. Sharma d, S.N. Kale a,⁎, Suwarna Datar a,⁎ a
Department of Applied Physics, Defence Institute of Advanced Technology (DU), Girinagar, Pune 411025, India Department of Applied Chemistry, Defence Institute of Advanced Technology (DU), Girinagar, Pune 411025, India Department of Physics, University of Pune, Pune 411007, India d DOP, DRDO Bhawan, Rajaji Marg, New Delhi 110105, India b c
a r t i c l e
i n f o
Article history: Received 22 May 2012 Received in revised form 22 June 2012 Accepted 14 July 2012 Available online 20 July 2012 Keywords: Curcumin Gold nanoparticles Bioavailability
a b s t r a c t Curcumin, a natural polyphenolic compound, has astounding therapeutic applications but lacks in bioavailability mainly due to its poor solubility in water. Polyvinyl pyrrolidone (PVP) which is a proven drug carrier has been used to facilitate the conjugation of curcumin with gold nanoparticles and to improve the solubility of curcumin in water. In this conjugate diaryl heptanoid chromophore group of curcumin which is a much needed group in biomedical applications remains intact as observed from FTIR and UV–vis spectroscopy analysis. The work shows good promise for such conjugates as therapeutic-cum-imaging materials in biomedical field. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Curcumin, a bio-active polyphenol component of curcuma longa (Turmeric), also known as diferuloylmethane (C21H20O6), has become an intense topic of research due to its interesting biological and pharmacological applications [1–3]. It has anti-inflammatory, anti-oxidant, anti-carcinogenic, chemo-preventive, anti-angeogenic, anti-diabetic, anti-viral and antibacterial properties [4,5]. In spite of its wide property range, this polyphenolic compound is less documented for its actual applications, mainly due to its poor bioavailability. Curcumin remains to be less popular as a drug since the basic necessity of any drug to be delivered is its ease in suspension in bio-media (either aqueous solutions or PBS solutions). Curcumin's hydrophobic nature is one of the main reasons for this poor water-solubility/suspension capacity. This leads to its poor activity, low absorption, high rate of metabolism within the living system and rapid elimination from the system [6]. There have been attempts to functionalize the molecule to improve its bioavailability by conjugating it with a hydrophilic molecule. However, this process hampers the therapeutically-active group of the molecule, thereby killing the basic purpose of the conjugation. Numerous approaches have been reported in this context to explore the potent applications of curcumin in very recent times [7–10]. Nanoparticles [11,12], liposomes [13], micelles [14] and phospholipid complexes [15] are being used to improve the bioavailability of curcumin. ⁎ Corresponding authors. Tel.: +91 20 2430 4091; fax: +91 20 2438 9572. E-mail addresses: [email protected] (S. Datar), [email protected] (S.N. Kale). 0928-4931/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.msec.2012.07.022
Bhawana et al. and Wu et al. have prepared curcumin nanoparticles [8] and nanogels [16] for antimicrobial and photo-thermal therapy respectively. Dhule et al. have explored curcumin-loaded γ-cyclodextrin liposomal nanoparticles as delivery vehicles for osteosarcoma [17]. Ha et al. have synthesized poly(lactide)-vitamin E TPGS (PLA-TPGS) copolymer to formulate a curcumin nanocarrier [18] and Bisht et al. have reported on polymeric nanoparticle-encapsulated curcumin [19] for human cancer therapy. Recently, S. Manju et al. have reported synthesis of water soluble gold nanoparticles in curcumin-polymer conjugate and studied it for blood compatibility and targeted drug delivery onto cancer cells [20]. They have conjugated curcumin with hyaluronic acid (HA) and then synthesized gold nanoparticles within the solution by reducing the chloroauric acid. In this and many other documented protocols, it has been observed that though the bio-availability improves upon conjugation, the main therapeutic group of this molecule gets engaged in the conjugation thereby making itself less-available for its therapeutic activity to any biological system [21]. Therefore, there is a need for an extensive research in this regard which would not only improve the bioavailability of the molecule but also keep the therapeutic activity high and impart multi-functional properties to the conjugated system. On the other side, gold nanoparticles have been immensely explored due to their interesting optical, electrical, physical and chemical properties, which find useful technological applications in the field of opto-electronics [22], chemical and biological sensors [23] and biomedicines [24]. These have been projected to be one of the safest (non-toxic) candidates for DNA-conjugation [25], bio-detection [26], drug-delivery, gene therapy and many other applications [27,28].
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These nanoparticles have also been documented to be good for bio-imaging purposes owing to their interesting surface-plasmon resonance property. In this context, through this manuscript we report a neat protocol which describes the formation of curcumin conjugated with gold nanoparticles using PVP which has been used as drug carrier for various drugs in several reports [29]. This protocol envisages good bio-availability of the curcumin molecule, keeping its therapeutic activity intact. Gold nanoparticles show excellent plasmon resonance characteristic, exhibiting its potential in applications of imaging. 2. Experimental 2.1. Synthesis Chloroauric acid (HAuCl4) has been used as a metal precursor and tri-sodium citrate (Na3C6H5O7) as reducing agent for the synthesis of gold nanoparticles [30]. 200 ml of 1 mM HAuCl4 was boiled and stirred under the reflux condition for 30 min. 20 ml of 38.8 mM aqueous Na3C6H5O7 was added directly into the boiled solution. The color of the solution changed from pale yellow to deep red within 7–10 min after the addition. Further the reaction was continued for additional ~ 20 min after which 20 mg of polyvinyl pyrrolidone (PVP, M.W. = 40,000) in 30 ml of water was added to the above solution and stirred for the next 45 min. The solution was cooled at room temperature. For conjugation of curcumin, 50 mg of crystalline curcumin was dissolved in 25 ml acetone and this solution was added to the 100 ml of PVP capped gold nanoparticles under stirring. Further this mixture was stirred for 3 h at 60 °C and then cooled down to room temperature. The Au–curcumin solution was then centrifuged at 4000 rpm to remove unattached curcumin. This was done three times to ensure that no free curcumin molecules are left in the final conjugate. The final solution was used for spectroscopic study and TEM analysis. Part of this solution was evaporated under vacuum using a rotary evaporator and then the remnant was centrifuged to obtain the powder, which was also used for analysis. 2.2. Characterization The synthesized gold nanoparticles and Au–curcumin samples were characterized by UV–vis Spectroscopy (Ocean Optics, HR4000), Fourier Transform Infrared Spectroscopy (FTIR, Perkin Elmer), Thermogravimetric Analyser (TGA, Perkin Elmer STA 6000) and Transmission Electron Microscopy (TEM, Philips CM 200 and FEI-Tecnai G2 20). For TEM characterization highly diluted specimens were prepared and dilution level was kept constant for all samples. Toxicity analysis was done using MTT assay as described elsewhere [31].
Fig. 1. FTIR spectra of (i) PVP (ii) curcumin (iii) PVP functionalized gold nanoparticles and (iv) PVP functionalized gold nanoparticles with curcumin.
bonding to enolic hydroxyl group which results in shift of O\H stretching from 3514 cm−1 to 3435 cm−1 whereas the basic diaryl heptanoid group, which is the chromophore group of curcumin remains intact [32]. This is also supported by UV–vis spectroscopy explained below. Fig. 2 shows the UV–vis spectra of (i) curcumin in PVP (ii) PVP functionalized gold nanoparticles and (iii) PVP functionalized gold nanoparticles with curcumin in aqueous media. UV–vis spectrum of curcumin in PVP shows an absorption peak at ~ 415 nm which is the signature of basic diaryl heptanoid chromophore group of curcumin [11]. The spectrum of PVP functionalized gold nanoparticles (ii) shows absorption peak at ~520 nm, which is a characteristic peak of gold nanoparticles arising due to Surface Plasmon Resonance (SPR) [33]. The UV–vis spectra of PVP functionalized gold nanoparticles with curcumin (iii) clearly show two distinct peaks at ~418 nm and ~525 nm which confirms that the characteristic peaks for the components in the Au–C complex are intact. The red-shift in the absorption peaks for curcumin (~3 nm) and gold NPs (~5 nm) can be attributed to the conjugation of PVP functionalized gold NPs with curcumin. Thus, the UV–vis data with the support of FTIR results clearly
3. Results and discussion Fig. 1 shows the FTIR spectra of (i) PVP (ii) curcumin (iii) PVP functionalized gold NPs and (iv) PVP functionalized gold nanoparticles with curcumin. Here spectra (i) and (ii) show the typical signatures of both PVP and curcumin. These signatures may be recognized as C_O (1660 cm−1) and C\N (1290 cm−1) in PVP. Signatures of curcumin are free O\H group (3514 cm−1), C_O and C_C (enol) (1450– 1630 cm−1), C\H (methyl) (2845 cm−1), C\H (aryl) (3015 cm−1) and C\O\C (1000–1300 cm−1) typically attributed to symmetric and asymmetric configurations of C\O\C chains [11]. Spectrum (iii) illustrates the functionalization of citrate reduced gold NPs with PVP in the form of shift of C_O stretching from 1660 cm−1 to 1628 cm−1. This may be attributed to the formation of intermolecular hydrogen bonding. Spectrum (iv) shows the conjugation of curcumin with PVP functionalized gold NPs. This figure reveals signatures of both PVP functionalized gold NPs and curcumin molecule. Curcumin molecule may attach to the PVP functionalized gold NPs with intermolecular hydrogen
Fig. 2. UV–vis spectra of (i) curcumin in PVP (ii) PVP functionalized gold nanoparticles and (iii) PVP functionalized gold nanoparticles with curcumin. Inset shows the colorimetric response for each sample (i.e. i, ii and iii).
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Fig. 3. Schematic for possible conjugation of curcumin with PVP functionalized gold nanoparticles.
demonstrate that curcumin retains its diaryl heptanoid chromophore group which is much needed in biomedical applications. The suspension stability of these conjugates was checked using aqueous solutions of a) only curcumin in PVP b) gold:PVP and c) gold:PVP:curcumin. The inset of Fig. 2(b) indicates that the conjugates show excellent aqueous-stability, imparted due to the chemistry involved. Fig. 3 shows the schematic for probable conjugation of curcumin molecule with PVP functionalized gold NPs. Citrate reduced gold nanoparticles are capped by PVP as shown in the schematic. This
conjugation takes place by intermolecular hydrogen bonding as shown in the figure which is also supported by the shift of C_O stretching from 1660 cm−1 to 1628 cm−1 in the FTIR spectra of citrate reduced PVP functionalized gold nanoparticles. Curcumin also gets attached to the PVP functionalized gold NPs as shown in the figure by the intermolecular hydrogen bonding to enolic hydroxyl group which results in shift of O\H stretching from 3514 cm−1 to 3435 cm−1. Fig. 4 shows TEM images of (a) PVP functionalized gold NPs (b) curcumin in PVP (c) PVP functionalized gold NPs with curcumin
Fig. 4. TEM images of (a) PVP functionalized gold nanoparticles, (b) curcumin in PVP, (c) PVP functionalized gold nanoparticles with curcumin (Au–C complex) and (d) magnified image of circular region as shown in (c).
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and (d) magnified image of circular region as shown in (c). It is observed from Fig. 4(a) that PVP functionalized gold NPs are spherical in shape having diameter of 13 ± 2 nm. Fig. 4(b) shows the dispersion of curcumin and PVP (2:1) in aqueous medium for reference. PVP functionalized gold NPs with curcumin (Au–C complex) is shown in Fig. 4(c). As observed in the figure, gold NPs (black cores) are surrounded by PVP–curcumin conjugate as a shell. This is supported by the image of PVP–curcumin conjugation (b) as well as the FTIR results showing the hydrogen bonding between the PVP and curcumin. Magnified image of circular region in Fig. 4(c) is shown in Fig. 4(d), reveals that the PVP–curcumin completely surrounds the gold nanoparticles. Here PVP provides the required hydrophilicity to the curcumin molecule making them bioavailable. The TGA curve of gold:PVP:curcumin (Au–C complex) shown in Fig. 5(a) illustrates that the complex begins to lose weight at ~ 235 °C and the loss of weight is about 23% in the temperature range of 50–440 °C. After 440 °C, the rate of weight loss is slower and about 5.5% up to 550 °C. The net weight loss may be attributed to the decomposition of PVP:curcumin coating around the gold nanoparticles. The TGA results show that 26% of curcumin + PVP get loaded on the gold nanoparticles. The cytotoxicity effect of Au:PVP:C conjugate was studied against TZM-bl cell lines, which are modified HeLa derived cell lines. Inset of
Fig. 5(a) shows the cytotoxicity results of the cell lines after incubation with the conjugate for 24 h with increasing concentration. The cell viability of TZM-bl cells after incubation in the medium containing conjugated curcumin demonstrates that the conjugate shows 100% cell viability till the conjugate concentration of ~10 μg/ml. Beyond this concentration the sample exhibits toxicity, which may be useful for the therapeutic applications. Thus by conjugating nanoparticles with curcumin, the cytotoxic dose was found to be much less indicating that not only the anticancer activity of curcumin was intact and with curcumin, the conjugate showed enhanced cytotoxic effect. The results are being evaluated against the primary fibroblasts, which is underway. Fig. 5(b) shows in-vitro release kinetics of curcumin from conjugate. The conjugate was dispersed (1 mg/ml) in the Phosphate Buffer Solution (PBS: 7.4 pH) and the release kinetics was studied using UV vis spectroscopy. The experiment was performed at 35 °C and the data was recorded after every 1 h to monitor the release. As shown in the figure, more than 30% release was observed within 2 h then the rate of the release became slow. From 2 h to 9 h the net release was upto 60%. These results are quite encouraging owing to the applications of curcumin as antimicrobial and antiviral agents. Such gold nanoparticles surrounded by curcumin and PVP providing the bioavailability can be harnessed for excellent applications in targeted drug delivery, antimicrobial activity and bio-imaging. 4. Conclusion In conclusion, we have proposed a simple route to conjugate curcumin with PVP functionalized GNPs which are highly stable in aqueous medium at room temperature. Diaryl heptanoid chromophore group of curcumin and plasmon resonance signature of gold remain intact after conjugation ensuring the bio-active nature of curcumin and gold signature. The conjugate loading percentage was seen to be nearly 26%. The conjugate shows 100% cell viability till the conjugate concentration of ~10 μg/ml. Beyond this concentration the sample exhibits toxicity, which may be useful for the therapeutic applications. More than 30% release was observed within 2 h then the rate of the release became slow. The net release was nearly 60% in 9 h. This work has good potential in antimicrobial studies, bio-imaging and drugdelivery applications. Acknowledgments The authors thank Dr. Prahlada, Vice Chancellor, Defence Institute of Advanced Technology (Deemed University), Pune for providing the laboratory facilities and financial assistance. Thanks are also due to Ms. Ruchira Mukherjee of NCL, Pune for the experimental support. SNK acknowledges funding from DST-Nano Mission (SR/NM/NS-63/ 2009) for this work. SNK and SSD acknowledge funding from “DRDO-DIAT Program on Nanomaterials by ER&IPR, DRDO”. References
Fig. 5. (a) TGA curve for Au:PVP:curcumin (Au–C complex) and (b) shows the graph of in-vitro release kinetics of curcumin at 35 °C in buffer solution (pH = 7.4). Inset of panel (a) shows the cytotoxicity histogram.
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