Curcumin conjugated gold nanoparticle synthesis and its biocompatibility

Abstract

Gold nanoparticles have gained much attention due to their widespread biological and technological applications, and consequently their simpler synthesis via green chemistry has also become of foremost importance. We report the room temperature synthesis of spherical gold nanoparticles using curcumin alone as the reducing and stabilizing agent. The pH is found to have an important role in curcumin solubilisation and subsequent formation of curcumin conjugated gold nanoparticles (cAuNPs). UV-visible studies show that the cAuNPs formed are of uniform size and HRTEM studies confirm spheres of average size 18 nm. The DLS measurements show a particle size of 58 nm. The crystallinity has been determined by HRTEM and XRD. The conjugation of stable curcumin on the cAuNPs is indicated by FTIR spectra which also suggest that the phenolic and enolic groups of curcumin bring about the reduction. The zeta potential value of cAuNPs is −23 mV which is stable for up to 6 months at room temperature. The mechanism of cAuNP formation is inferred to be through temporal evolution. This is the first demonstration where curcumin is solubilized at alkaline pH without using any external agent and is used for reducing HAuCl₄ to form cAuNPs. The non toxic nature of the cAuNPs is evidenced through biocompatibility studies using human blood cells.

---

Introduction

Gold nanoparticles (AuNPs) can find applications in diverse fields like medical diagnosis, drug delivery, biosensors, cancer treatment etc.^1–5 AuNPs synthesized by the conventional Turkevich method of citrate reduction are well defined spherical particles, but their use in biomedicine is limited due to the cytotoxicity elicited by citrate.^6,7 The surfactant, CTAB used for the preparation also results in toxic responses in cellular systems.⁸ Another extensively used surfactant, PEG increases the stability of AuNPs and also inhibits the direct interaction of biological fluids thus decreasing toxicity.⁹ However, concerns on the size specific toxicity of PEG coated AuNPs have been raised.¹⁰ As a consequence, AuNPs made by employing green chemistry principles based on extracts or compounds from plants, microbes, algae etc. are receiving considerable attention.^11,12

The secondary metabolites of plants such as tannins, flavones and polyphenols have antioxidant, antimicrobial and anti-carcinogenic properties. The ability to release H atom enables them to reduce metal precursors and form the corresponding nanoparticles.¹³ Recent reports using the extracts of soy bean (Glycine max), cumin (Cuminum cyminum) seeds, onion (Allium cepa), red cabbage (Brassica oleracea capitata), Calotropis procera and Dalbergia sissoo for synthesizing AuNPs indicate the continued interest in the field.^14–19 These AuNPs are proposed to have suitability in biomedical applications as mentioned before. Very recently a few reports suggest that the AuNPs prepared using phytochemicals as reductants show anti-cancer activity while being nontoxic to normal cell lines.^20,21 Polyphenols such as tannic acid, quercetin, rutin and luteolin are also able to form AuNPs.^22,23 However, the potential of these nanoparticles in nanomedicine has not yet been investigated.

Curcumin, derived from the rhizome of turmeric is one of the cash crops extensively grown in Asia and has been used to treat various illnesses from ancient times. It is a flavonoid (1,7-bis-(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione) well known for its antioxidant, anti-inflammatory, antimicrobial, anti-carcinogenic and immunomodulatory activities.^24–26 The bio protective properties stem from its distinct chemical structure.²⁷ A number of reports show the ability of curcumin to inhibit free radical species generation which is attributed to the abstraction of H atom from the C12 methylene group or from phenolic groups.^25,28–30 The unsaturated heptadiene chain participates in inhibiting the transcription factor of NFκB and TNFα and hence acts as an anti-inflammatory and anti-carcinogenic agent.^31,32 The chromophore has a strong absorption maximum at 428 nm and a weaker absorption of light above 300 nm.³³ Due to the latter reason, curcumin can prevent damage induced by UV radiation.³⁴ Further, Phase I clinical trials indicate that humans can tolerate a dose as high as 12 g per day without any toxic side effects.²⁴

Utility of such a promising, multi-targeting molecule with an exemplary safe profile is limited by the lack of solubility, bioavailability and target specificity.³⁵ At least 8000 mg of free curcumin should be administered for its detection in systemic levels as it is found to undergo intestinal metabolism.³⁶ Consequently conjugation of curcumin with β-cyclodextrin, cellulose acetate or encapsulation in liposomes have been attempted to increase its solubility.^36–39

Manju and Sreenivasan solubilized curcumin using hyaluronic acid and prepared curcumin conjugated AuNPs where hyaluronic acid and not curcumin functions as the reductant.²⁰ Very recently, Singh et al. also prepared curcumin reduced AuNPs by solubilising curcumin at very high temperature.⁴⁰ Gangwar et al. synthesized curcumin conjugated, (poly(vinyl pyrrolidone)) PVP capped, trisodium citrate reduced AuNPs to increase the bioavailability of curcumin.⁴¹ The presence of PVP acts as a capping agent over the citrate reduced AuNPs and also as a carrier of curcumin. Here we report a one step synthesis of AuNPs where curcumin acts both as the reducing and stabilizing agent. The distinction in our study is that we have not only prepared cAuNPs at room temperature but also solubilized curcumin without using any external agent. The synthesis process has also been optimized and the cAuNPs have been characterized by employing various spectroscopic and electron microscopic techniques. The mechanism of evolution of crystalline and spherical cAuNPs is elucidated using HRTEM and absorbance spectroscopy. The results indicate that spherical cAuNPs are obtained with curcumin present in its stable conformation on the nanoparticle and that they are not toxic to human blood cells at micromolar concentrations. We believe that cAuNP will emerge as an attractive therapeutic agent as they are nontoxic and cost effective. Also, this synthesis process represents an added advantage in that the limitation of insolubility of curcumin is overcome (thus increasing its bioavailability) and its conjugation with AuNPs ensures its bioactivity.

Results

Synthesis of cAuNPs

The synthesis of cAuNPs has been carried out by reacting curcumin with HAuCl₄ at different molar ratios (MRs) and pH values (8–11) and the colour change visually monitored and spectroscopically recorded (Fig. 1). No particle formation is observed by mixing both the solutions without changing their pH. Increasing the pH of curcumin to 9 also does not bring about cAuNP formation. This might be due to the insolubility of curcumin at this pH as the spectra in Fig. 1a illustrates and hence it is not available for reducing Au³⁺. When the pH of curcumin is adjusted to a range of 9.2–9.6 followed by dropwise addition of HAuCl₄, a rapid and progressive change in colour from pale yellow to colourless, black and burgundy red is observed. The narrow SPR peak formed by the colloid at 528 nm, confirms the formation of cAuNP. On the other hand, increasing the pH of curcumin above 9.6 lowers the intensity and broadens the SPR peak. Based on the SPR spectra obtained, pH 9.3 is selected for further experiments. On increasing the pH of curcumin to 10.6 a black coloured colloid is formed. The corresponding broad plasmon resonance band with a peak at 544 nm indicates a broad size distribution of the cAuNPs formed (Fig. 1a). Further reacting curcumin at a pH of 11.0 with HAuCl₄ results in the formation of a black coloured colloid that precipitates immediately.

Fig. 1 UV-visible spectra of cAuNP at different synthesis conditions. Concentration of curcumin is 0.25 mM and HAuCl₄ is 1 mM (a) pH dependent formation of cAuNP is observed where a high SPR intensity is exhibited by the burgundy red coloured colloid obtained when the pH of curcumin is increased to 9.3. SPR intensities shown by the black coloured colloid on increasing the pH of curcumin to 10.6 are low. (b) A concentration dependent increase in SPR intensity for cAuNPs (pH of curcumin at 9.3), prepared at different curcumin to HAuCl₄ (Cur/Au) MRs is observed. The highest yield is given by the MR 1 : 4 with high SPR and low plasmon wavelength width.

The SPR spectra of cAuNPs synthesized at different MRs when pH of curcumin is at 9.3 is given in Fig. 1b. A concentration dependent increase in SPR intensity without any significant shift in SPR is obtained at different MRs. A MR of 1 : 4 (curcumin – 0.25 mM and HAuCl₄ – 1 mM) is optimum for the formation of cAuNPs as obtained from the maximum SPR peak at 528 nm with a correspondingly low plasmon wavelength width. Therefore, for further characterization, cAuNPs obtained at a MR of 1 : 4 with the pH of curcumin adjusted to 9.3 have been used. The yield of cAuNPs as observed from the extinction value of 2 for 1 mM of HAuCl₄ is found to be comparable with that of AuNPs produced by the standard citrate reduction method where 0.91 mM of HAuCl₄ was used.⁴²

It has been reported that at alkaline pH, curcumin degrades to condensed products like ferulic acid and vanillin.⁴³ In order to find out the time interval at which this reaction is initiated and completed, the absorption spectral changes of curcumin at alkaline pH has been monitored (Fig. S1a and S1b, ESI†). It is observed that the absorption intensity at 438 nm is not altered up to 15 min indicating that no degradation occurs within this time period (The shift in absorbance maxima of curcumin from 428 nm to 438 nm is discussed later in the text). On adding HAuCl₄ the peak at 438 nm disappears completely with the simultaneous appearance of a broad spectrum indicating the formation of cAuNPs (Fig. 2). The broad SPR spectra formed initially narrows off with time and the intensity also increases. This indicates that the particles formed initially may be of different sizes which gradually convert to a set of particles with similar size. Hence in our further experiments, care has been taken to add HAuCl₄ within two minutes of adjusting the pH of curcumin to 9.3.

Fig. 2 Kinetics of cAuNP formation. Concentration of curcumin is 0.25 mM and HAuCl₄ is 1 mM. The absorbance maximum of curcumin at 438 nm disappears initially and a serial increase in the SPR at 528 nm is observed which narrows off with time.

Structural analysis by FTIR spectroscopy

To determine the specific site of interaction of Au³⁺ on curcumin, FTIR has been performed. Fig. 3 shows FTIR spectra of both curcumin and cAuNP. The bands at 1383 cm⁻¹, 1233 cm⁻¹ and 962 cm⁻¹ represent the in-plane bending of the hydroxyl groups of the two phenolic and an enolic group respectively. All the three bands are completely absent in cAuNPs which suggests the interaction of HAuCl₄ at these sites. The bands at 767 cm⁻¹ and 1425 cm⁻¹ corresponding to the olefinic in-plane bending vibrations of the heptadiene chain of curcumin are observed in cAuNP (though broadening was observed for the vibrations at 1425 cm⁻¹) indicating the presence of intact curucmin moiety in the cAuNP. Similarly the bands 2920 cm⁻¹, 1461 cm⁻¹, 1024 cm⁻¹ and 989 cm⁻¹ that appear in cAuNP are due to vibrations of aliphatic C–H stretches and mixed vibrations of CH₃, aromatic CCC and CCH of curcumin confirming the action of curcumin as a capping agent.

Fig. 3 FTIR spectra of curcumin and cAuNPs.

Electron microscopy studies

The cAuNPs are found to be uniformly spherical in shape with an average diameter of 47 nm by SEM analysis (Fig. 4a). HR-TEM image also indicates that the particles are homogenous and spherical in shape (Fig. 4b). The particles are predominantly 18 ± 3.3 nm in size as indicated by the size distribution histogram (Fig. 4b, inset). An RSD of 18% is observed which is comparable to the RSD of 10–16% obtained for AuNPs of size 12–20 nm, synthesized by Frens method.⁴⁴Fig. 4c shows the lattice planes over a single nanoparticle. The average distance between the planes is calculated to be 0.23 nm and 0.204 nm, which is typical of the (111) and (200) lattice planes respectively of face centered cubic gold. The corresponding Fast Fourier Transform (FFT) (Fig. 4c, inset) image shows the diffraction pattern of the sample, used to study the interplanar distance and also gives information on the type of lattice planes and symmetry of the particle. The split in the FFT points out that the particles formed are multiply twinned.^45–47Fig. 4d shows two aggregated particles and other individual spherical particles. The TEM image of the black coloured colloid of cAuNPs formed on increasing the pH of curcumin above 10.6 is found to be formed of a mixture of particles with size ranging from 70–200 nm (Fig. 4e). The HRTEM image of the same colloid shows an aggregation of three nanoparticles with each particle having an average diameter of 18 nm. From the corresponding FFT image, D spacing of 0.23 nm and 0.12 nm is obtained which are the interplanar distances for (111) and (220) planes of gold respectively (Fig. S2, ESI†).

Fig. 4 EM images of cAuNP. (a), (b) (c) and (d) show cAuNPs prepared with pH of curcumin at 9.3 (a) SEM image of cAuNPs show a large number of spherical shaped particles. (b) Lower magnification HR-TEM image of spherical cAuNPs and the inset showing particle size distribution, (c) high resolution TEM image of a single particle showing lattice fringes and the inset shows the FFT (d) two conjoined cAuNPs that have not dissociated during the course of formation along with independent spherical particles. (e) TEM image of larger spherical cAuNPs of varied size when prepared with pH of curcumin at 10.6 (particle diameter marked in figure is in nm).

X-ray crystallographic studies

The crystallinity of the cAuNPs has been further confirmed using XRD (Fig. 5). The Braggs reflections at 2θ angles of 38.15°, 44.29°, and 64.37° correspond to the (111), (200) and (220) planes respectively of cubic gold and are clearly visible. The presence of higher intensity peak of (111) plane of gold is in accord with the HRTEM data which confirms that majority of the formed nanocrystals are oriented along the (111) lattice plane.⁴⁸ The crystallite size of cAuNPs as calculated from Scherrer equation is 34.94 nm.

Fig. 5 XRD of cAuNPs. The peaks at 2 theta angles 38.15°, 44.29° and 64.37° corresponding to (111), (200), and (220) planes of gold respectively are shown.

DLS and zeta potential measurements

The majority of the formed cAuNPs are found to be 58 nm (92.8%) in size, while a small percentage of particles with size 4 nm (7%) is also observed (intensity average diameter). This difference in size observed between HRTEM and DLS data may be due to the curcumin acting as a capping agent and forming a covering on the nanoparticles. A zeta potential value of −23 mV shows that cAuNP is highly stable (Fig. S3, ESI†).

Stability in different media and dilutions

The cAuNPs have been suspended in cell culture medium, different buffers and at different pH conditions at a ratio of 1 : 4 (Fig. S4a and b, ESI†). There is no change in the SPR peak wavelength or intensity in different buffers and medium while a small red shift of 5 nm is observed in the presence of PBS. The zeta potential value for the cAuNPs in PBS is −20 mV, which is similar to that obtained in Milli Q water, confirming its stability. No significant change in the size is observed. A concentration dependent increase in SPR intensity is also observed (Fig. S4c, ESI†). This indicates that the cAuNPs are stable in these buffers, at a wide pH range, cell culture medium and also at working concentrations at cellular levels. The SPR spectra of cAuNPs in Milli Q water are monitored for over 6 months and no change is observed which clearly proves its stability at RT (Fig. S4d, ESI†). The cAuNPs, after isolation by centrifugation and filtering in a 0.22 μm filter is resuspended in Milli Q water at higher concentration for cell culture experiments. The absence of any change in the SPR spectra of the prepared and resuspended cAuNPs further confirms its high redispersibility (Fig. S4e, ESI†). The particle size of the resuspended cAuNPs is 56 nm from DLS intensity measurements.

Biocompatibility of cAuNPs

Hemocompatibility. In vitro hemocompatibility assay is a simple and reliable method to find out the toxicity of materials. In order to compare the hemolytic activity of cAuNPs with a standard, AuNPs of 30 nm size are synthesized using citric acid and ascorbic acid (ctAuNPs) according to the method of Zeigler and Eychmuller.⁴⁹ The hemolysis is 0.66 ± 0.10% in the presence of cAuNPs which compared well with that of ctAuNPs at similar concentration (Table 1).

Table 1 Hemocompatibility of cAuNPs

Concentration of AuNPs	% hemolysis by cAuNPs	% hemolysis by ctAuNPs
43 μM	0.06 ± 0.01	0.046 ± 0.015
86 μM	0.315 ± 0.07	0.405 ± 0.093
172 μM	0.664 ± 0.11	1.375 ± 0.10

---

Effect on PBMCs. The ability of cAuNPs to induce cytotoxicity in normal PBMCs is investigated by exposing them to a range of cAuNP concentrations (43 μM, 86 μM and 172 μM). Three different parameters are tested. Untreated cells are taken as control.

Trypan blue dye exclusion method. PBMCs treated with various concentrations of cAuNPs indicate that almost all the cells are live. In fact, the percentage of healthy cells is found to increase slightly with concentration and time (Fig. 6a).

Fig. 6 Cytotoxicity of cAuNPs towards PBMCs. (a) Trypan blue assay shows the absence of any cytotoxicity of cAuNPs at all concentrations (43 μM, 86 μM and 172 μM) even for 72 h. (b) A safe profile is also detected in Alamar blue assay. Each point is represented as mean ± SEM of triplicate experiments. Representative EtBr/AO pictures of three independent experiments show the morphology of control cells (a) and treated cells (b) to be similar. The percentage of viable cells is quantitated and the mean ± SEM for control is 97.43 ± 1.1 and treated is 95.38 ± 1.19.

Alamar blue assay. Spectroscopic analysis has been performed to confirm the non-toxicity of cAuNPs using Alamar blue assay by quantifying the viable cells. There is no considerable change in the fluorescence intensity for both treated and untreated cells. Concentrations of cAuNPs as high as 172 μM on treating for 72 h show more than 90.5 percentage viability (Fig. 6b).

Ethidium bromide/acridine orange (EtBr/AO) staining. Both the control and treated cells show similar morphology with nearly all the cells fluorescing green indicating that they are viable. Only few cells show apoptotic features in treated cells (Fig. 6c and d).

Discussion

The preliminary studies on synthesis and cytotoxicity of cAuNPs were reported by us recently.⁵⁰ The results obtained in the present study indicate that the –OH groups of curcumin reduce HAuCl₄ to Au⁰ and also stabilize the nanoparticle by acting as a capping agent. That the pH dependent solubility and stability of curcumin, are the factors which drive the formation of cAuNP is demonstrated. The pK_a of the enolic and the two phenolic groups of curcumin ranges from 7–10.6.⁵¹ When the pH is increased to a range of 9.2–9.6 using K₂CO₃, curcumin becomes soluble and the shift in wavelength from 428 nm to 438 nm is related to deprotonation of the enolic and phenolic groups and it exists as Cur³⁻. These observations corroborate those of Zsila et al.⁵¹ These phenolate and enolate anions seem to bring about the reduction of Au³⁺ to Au⁰. Thus curcumin in the pH range of 9.2–9.6 (Cur³⁻) is in its most favorable state to reduce Au³⁺. Influence of pH of the reductant solution on AuNP formation has been observed earlier. In the case of tannic acid, only the tannin ions formed above its pK_a values can reduce Au³⁺ effectively.²² Optimal formation of citrate reduced AuNPs is also observed when pH of the solution is above the pK_a of sodium citrate.⁵² The pH dependent solubility of curcumin also explains the observation that no reduction takes place when pH of curcumin is lower than 9.2 due to its low solubility. When the pH is increased to 11 curcumin may not be stable and hence the formed cAuNPs precipitates. When HAuCl₄ is added to Cur³⁻, the disappearance of the characteristic peak at 438 nm of curcumin observed may indicate binding and interaction of the acid with curcumin (Fig. 2). Similar result has been obtained when human serum albumin (HSA) is reacted with curcumin which has been attributed to cancelling of the π conjugation when the two vinyl guaiacol parts rotate due to the binding of ionized curcumin to HSA.⁵¹ It is also observed that increasing the pH of HAuCl₄ and adding curcumin results in very low SPR values indicating poor AuNP formation. This may be due to the weak reactivity of the acid at highly alkaline pH conditions.^22,52

The increase in SPR intensity without any significant change in the plasmon wavelength, as the MR increases indicates a concentration dependent formation of cAuNPs. Even though the formation and changes in SPR spectra is dependent on many physical factors, shift in plasmon wavelength has been correlated to changes in size or shape and the narrow SPB likewise to narrow size distribution or homogeneity.^{15,18,53–56} Hence we conclude that the cAuNPs formed at different MRs may be of similar size.

The extinction value, 2 obtained for cAuNPs (curcumin – 0.25 mM, HAuCl₄ – 1 mM) is high when compared with the absorbance values of AuNPs synthesised by other bioreductants such as rutin and tannic acid, indicating curcumin to be a more potent reductant.^22,23 Synthesis in the presence of plant extracts such as soy bean, red cabbage etc. shows much lower extinction values since concentration of the reducing biomolecules may be low in the extract.^14,18 Even though, the hyaluronic acid–curcumin conjugate reduced AuNPs synthesized by Manju and Sreenivasan gives a higher yield, in this case, the reduction to AuNPs is brought about by hyaluronic acid and not by curcumin.²⁰ Thus the synthesis methodology reported here confirms that making charged molecules of curcumin by increasing the pH is essential for enhancing its reducing and stabilizing ability.

The FTIR data of cAuNPs also confirm the reduction of Au³⁺ by enolic and phenolic –OH groups of curcumin (Fig. 3). This is in contrast to the AuNPs synthesized using hyaluronic acid–curcumin conjugate in which the carboxyl group of hyaluronic acid is the reacting moiety.²⁰ The presence of peaks corresponding to the out-of-plane bending vibrations of aromatic rings and the CCH bonds of the heptadiene chain in cAuNP spectra proves the presence of the reductant curcumin on the formed particle. That curcumin is present in its stable conformation is confirmed by the presence of vibrations corresponding to the intact olefinic bonds of the heptadiene chain in cAuNP.^57,58 Further the peak at 1425 cm⁻¹ also corresponds to the olefinic C–H in-plane vibrations which broaden in cAuNP spectra and might be due to the influence of Au interaction with the aromatic rings of curcumin. Similar change in the olefinic double bond vibrations due to the interaction of curcumin with β-cyclodextrin at the aromatic rings has been observed by Tang et al.⁵⁹ The important role played by the ionization of –OH groups of the flavanoids tannic acid and rutin in forming AuNPs has already been reported by Sivaraman et al. and Levchenko et al.^22,23 According to their reports, the dissociation of the H⁺ groups from the reductants transforms them to their ionized or charged state. These charged molecules not only aid in reduction but also act as capping agents leading to the formation of stable AuNPs. Scheme 1 represents the interaction sites of Au with curcumin as concluded from our observations.

Scheme 1 Proposed sites for the reduction of Au³⁺ by curcumin. The predominant enol form of curcumin, at alkaline pH forms Cur³⁻ and the O⁻ moieties formed is assumed to reduce Au³⁺ to Au⁰.

Interpretation of the mechanism of formation of AuNPs by reduction has been attempted by many.^42,52,60,61 Correlation between colour of AuNP colloid to absorption spectra and size has been reported. Chow and Zukoski suggested temporal evolution of AuNPs from larger spherical intermediates (black coloured colloid) which disperses to smaller spherical particles (burgundy red colloid) at the final stage.⁶⁰ By following the kinetics of cAuNP formation, a broad absorption spectra is observed at the initial time periods. As the reaction progresses the plasmon bandwidth decreases which also shifts towards the blue region and after 4 h, maximum SPR intensity is obtained. This is accompanied by a rapid and progressive change in colour from yellow to black and finally to burgundy red. Even though the burgundy red colour of cAuNPs is attained in 10 min of the reaction, the SPR narrows down and the intensity increases up to a time period of 4 h. This may be attributed to processes like Ostwald maturation or intra-particle ripening. Citrate reduction of AuNPs is also found to progress through such a process of maturation for 15–20 min in which conjoined spherical gold particles dissociate into individual nanoparticles as observed through TEM analysis.^42,52 Similar observation has been reported by Zeigler and Eychmuller during seed mediated AuNP synthesis where blackberry shaped AuNPs are formed upon addition of the reactants which on heating transform to smooth spheres.⁴⁹ The HRTEM of cAuNPs shows most of the particles to be uniformly dispersed and spherical. However, evidence for a few particles remaining together without separation (Fig. 4d) probably indicate the occurrence of intermediate sized particles which then mature to spherical shaped and dispersed cAuNPs.

From these results it is deduced that the mechanism of formation of cAuNPs starts and progresses through the following steps: (1) deprotonation – the hydrogen atoms are dissociated from the –OH groups of curcumin to form Cur³⁻. (2) Reduction – the electrons on the O⁻ aids in the reduction of Au³⁺ to form Au⁰. (3) Nucleation – the Au⁰ atoms form clusters and nucleates. (4) Growth – further growth occur on these nanoclusters as more Au⁰ is added on the surface. Random growth occur leading to the formation of particles with wide ranging size as evidenced from the black colour and broad absorption spectra of the colloid. (5) Cleavage – as more Au⁰ are adsorbed onto the surface, the size of the cluster increases leading to instability and consequently cleaves to smaller fragments. (6) Maturation – the final spherical, solid, dense and crystalline cAuNPs are formed over a period of maturation for nearly 4 h through processes such as Ostwald ripening or intraparticle ripening. They are stabilized by the ionised curcumin molecules on the surface of cAuNPs. Scheme 2 represents the possible mechanism of cAuNP formation as substantiated from our data.

Scheme 2 Mechanism of cAuNP formation. Curcumin, insoluble in water at neutral pH, gets solubilized at alkaline pH and reduces Au³⁺ to Au⁰. The Au⁰ nucleates and grows forming larger sized intermediates that cleaves and matures to form individual, spherical cAuNPs.

When the pH of curcumin is above 10.6 the SPR peak obtained is broad even after 4 h and the formed colloid is black in colour indicating the inability of curcumin to completely reduce Au³⁺ at this pH. At high alkaline pH, interaction of protons in water with the β-diketone group of curcumin occurs.⁶² A hydrogen bond might form with the anions making it inefficient to completely reduce Au³⁺. This result is further confirmed by the TEM images of the black coloured colloid (Fig. 4e) showing particles of varied size. It is possible that the black coloured colloids are larger intermediates not completely cleaved off to smaller particles since all the curcumin molecules are not in Cur³⁻ form which is the favorable state for reducing Au³⁺. Aggregation of unstable AuNPs to form nanowire like structures has been reported by Pei et al.⁶¹

An average size of 18 ± 3.3 nm and an RSD of 18% obtained through HRTEM analysis confirm the uniformity of the synthesized cAuNPs. Seed mediated method has been adopted by many to obtain highly monodisperse AuNPs with relatively narrow size distribution to be used in electronic and plasmonic applications, as catalysts etc.^44,49,63,64 On the other hand, AuNPs synthesized by phytochemicals show a variation of 2.7 nm to 10 nm from the average size of the particle.^14,17,20,22 Thus a standard deviation of 3.3 nm obtained for cAuNPs is superior to the AuNPs obtained using other plant extracts indicating the synthesis of spherical AuNPs of narrow size distribution.

The stability of AuNPs as a colloid is critical since the unique optical properties depend on its ability to stay dispersed in solution. Polymers and biomolecules act as good stabilizing agents.^4,9 AuNPs synthesized by phytochemicals such as tannic acid, rutin etc. show no significant change in SPR spectra up to almost 1 year at RT indicating the ability of these flavanoids to act as stabilizers.^22,23 The cAuNP spectrum also shows no change for more than 6 months when kept at room temperature which indicates that curcumin, similar to other flavanoids can stabilize the AuNPs. Hyaluronic acid–curcumin conjugate reduced AuNPs are reported to be stable for 3 months at 20 °C.²⁰ The high redispersibility of cAuNPs even in concentrated solutions without any aggregation as indicated by the particle size shows its efficacy to be used as stable nanoparticles in biological environments. Further, its stability in different media and buffers, in the presence of salt concentrations and at a wide range of pH values also proves its suitability to be used in vitro and in vivo.

For the application of any material therapeutically it is essential to evaluate its biocompatibility particularly its non-toxicity. Studies show that presence of excess sodium citrate on AuNPs reduces proliferation of epithelial (HDMEC) and endothelial cell lines (hCMEC/D3).⁷ Similar observations were made in lung endothelial carcinoma cell lines A549 and NCIH441.⁶ The presence of CTAB on AuNPs is also demonstrated to be toxic to cells.⁸ Other commonly used surfactants like PEG are also found to express varied levels of toxicity in vitro and in vivo depending on their size.¹⁰ Chompoosor et al. report that as the hydrophobicity of the alkyl tail in an ammonium functionalized surfactant increases, the AuNPs' ability to generate ROS also increases, which decreases the viability of cells.⁶⁵ AuNPs stabilized by trisulphonated triphenyl phosphines also induced toxicity in different cell lines.⁶⁶ These studies underline the role of surfactants in causing cytotoxicity and consequently emphasize the need for synthesizing biocompatible AuNPs.

Curcumin is a phytochemical, well known for its safe profile in the human cell. This is further confirmed by the very low hemolytic activity of cAuNPs suggesting good hemocompatibility. This is also important as NaBH₄ reduced AgNPs are found to show high hemolytic activity at a concentration of 30 mg l⁻¹ and thereby unsuitable for in vivo experiments.⁶⁷ The presence of the biomolecule BSA, renders the BSA reduced AuNPs non-toxic towards RBC's.⁶⁸ This indicates that the nature of surface coating influences the compatibility of nanoparticles and the hemocompatible nature of cAuNPs can be due to the presence of curcumin on the Au core. Hyaluronic acid curcumin conjugated AuNPs also show less hemolysis percentage confirming that curcumin is compatible towards human RBC's.²⁰

The cytotoxicity assays using PBMCs indicate a safe profile for cAuNP. Only 9.5 percentage toxicity is observed in PBMCs when exposed to a cAuNP concentration of 172 μM for a treatment period of 72 h. On the other hand, citrate reduced AuNPs at a concentration of 50 μM show cyto- and genotoxicity in PBMCs.⁶⁹ They are also found to activate the NF-κB signaling pathway adversely affecting B-lymphocyte cell lines.⁷⁰ AuNPs prepared using NaBH₄ as reductant are non toxic to PBMCs only upto a concentration of 101.5 μM.⁷¹ Report also suggests the high toxicity caused by gold salt.⁷²

Generally, AuNPs prepared through plant extract or phytochemicals are reported to be non-toxic towards normal human cells.^14,21,73 Some reports suggests that even cancer cells ((PC-3) and (MCF-7)) show good compatibility when treated with AuNPs prepared by extracts of onion, tea, cumin and cinnamon.^16,74–76 These AuNPs are envisaged to have applications in diagnostics and imaging. Cinnamon reduced AuNPs are found to be internalized in cancer cells, and hence the possibility of using it as a molecular imaging agent was suggested.⁷⁶ Non-toxicity to normal cells and cytotoxicity to cancer cells due to any phytoreductant may be advantageous in treatment procedures. Kumar et al. report that AuNPs prepared by banana stem powder are non-toxic towards normal human kidney cell lines but decrease viability in cancer cells.²¹ Such nanoparticles can be applied in diverse fields of medicine such as diagnostics or imaging, drug delivery and also in therapy. Therefore, it is obvious that those AuNPs which are biocompatible as well as toxic to cancer cells can combine diagnosis and therapy and can contribute broadly to biomedicine.

Numerous reports cite the ability of curcumin to kill cancerous cells while being harmless to normal cells at the same concentration.^25,35,77 In addition, curcumin loaded fibrinogen nanoparticles revealed a cell selective response with increased apoptosis in tumor cell lines (MCF 7) when compared to normal mouse fibroblast (L929) cells.⁷⁸ Curcumin loaded magnetic nanoparticles are also reported to cause a loss of mitochondrial membrane potential and increased ROS production in breast cancer cells (MDA MB 231).⁷⁹ Hyaluronic acid–curcumin conjugate reduced AuNPs and curcumin loaded PVP capped AuNPs also showed efficacy to kill cancer cells suggesting the ability of curcumin when conjugated with AuNPs in expressing its therapeutic properties.^20,41

Our observations in the present study suggest the non-toxic nature of cAuNPs towards normal human blood cells. The possible applications of these cAuNPs can be in biomedicine e.g. imaging, diagnostic and drug delivery. The presence of the curcumin, a phytochemical with proven medicinal properties in its stable form also suggests its use as an anti-carcinogenic agent. Consequently the promising applications of cAuNPs in diagnostic and nanopharmaceutics are being explored.

Conclusions

We have shown that curcumin is a potent reducing agent of Au³⁺ resulting in the formation of stable, crystalline and spherical nanoparticles with an RSD of 18%. While the O⁻ moiety of Cur³⁻ reduces Au³⁺ to Au⁰, the aromatic rings and the heptadiene chain of curcumin remains unaltered forming a capping around Au⁰ and stabilizing the formed nanoparticle. Even though the interaction of Au⁰ at the –OH group of curcumin might reduce its antioxidant activity, all other functional groups such as methoxy, diketo and unsaturated chain are present unaltered, and hence we expect that the activation of several biochemical pathways that are implicated in the anti-inflammatory, anti-carcinogenic and anti-angiogenic potential of curcumin may be retained on the cAuNPs in a biological environment. The cAuNPs are also biocompatible as shown by our studies on its treatment with normal human blood cells. Thus cAuNPs show promising features for its applications in biomedicine which is under investigation in our laboratory.

Experimental

Chemicals

Gold(III) chloride hydrate (HAuCl₄) 99.99% pure, Phosphate Buffered Saline (PBS), RPMI 1640 medium, Hanks balanced salt solution (HBSS), Ficoll-Hypaque (Histopaque 1077), heparin, sodium bicarbonate, pencillin, streptomycin, gentamycin, amphotericin B, dimethylsulphoxide (DMSO), trypan blue dye, resazurin (alamar blue), ethidium bromide (EtBr) and acridine orange (AO) were purchased from Sigma-Aldrich, USA. Curcumin (1,7-bis(4-hydroxy-3-methoxy phenyl)hepta-1,6-diene-3,5-dione) 98.5% pure was obtained from Alexis Biochemicals, USA. Purity was checked by NMR spectra (Fig. S6, ESI†).⁸⁰ Potassium carbonate (K₂CO₃) was purchased from Merck, USA.

Synthesis and isolation of cAuNPs

A stock solution of 20 mM curcumin was prepared in 100% DMSO. Stock solutions of HAuCl₄ and K₂CO₃ of concentrations 4 mM and 150 mM respectively were prepared in Milli Q water. The pHs were adjusted separately for both the solutions using K₂CO₃. In a typical experiment 0.25 mM of curcumin was taken in Milli Q water and its pH adjusted using K₂CO₃. To this solution 1 mM of HAuCl₄ was added drop wise with simultaneous shaking. cAuNP formation was investigated using different concentrations of both the reactants (curcumin 0.125 mM to 1 mM and HAuCl₄ 0.25 mM to 2 mM) at different pHs (8–11). The final reaction mixture was kept at room temperature for 3 days and subjected to centrifugation at a speed of 10 000 rpm for 15 min in Hitachi Himac CR22 model centrifuge at a temperature of 14 °C. The pellet obtained was resuspended in Milli Q water and centrifuged at the same speed to remove any unreacted curcumin or HAuCl₄ present. The pellet was then resuspended in Milli Q water and used for further characterizations. For XRD and FTIR measurements the isolated cAuNPs were freeze dried to obtain dry powder.

Characterization of cAuNPs

The prepared cAuNPs were monitored spectroscopically at a wavelength range of 300–700 nm. All the absorbance and fluorescence readings were measured in multi mode plate reader (Tecan Infinite M200). Potassium bromide pellets of curcumin and cAuNPs were prepared and FT-IR spectra were collected in a Nicolet spectrometer over a range of 4000 cm⁻¹ to 400 cm⁻¹. Morphological features of cAuNPs were analyzed by electron microscopy studies. A drop of the sample was allowed to dry on carbon coated copper grids in air. For SEM studies the dried samples were gold coated and observed through JEOL Cold Field Emission scanning electron microscope operated at 3 kV. For TEM analysis the images were acquired through FEI Tecnai 200 TEM operated at 80 kV and high resolution images in Tecnai transmission electron microscope at 300 kV. The crystalline nature of cAuNPs was studied by XRD in a Seifert ISO Bebyeflex 2002 instrument at a voltage of 40 kV and a current of 30 mA. The core size of cAuNPs was estimated by Scherrer formula

D = 0.9λ/β cos θ,

β is the full width at half maximum of the peak, θ is the angle of diffraction, and λ is the wavelength of the X-ray radiation.

Malvern Zetasizer, Nano ZS was used to measure particle size and zeta potential of cAuNPs at a wavelength of 632.8 nm using a He–Ne laser beam. Stability of the cAuNPs was measured by adding cAuNPs to the buffers (HBSS, PBS), medium (RPMI), 5% NaCl and BSA-1 mg ml⁻¹ at 1 : 4 ratio and absorbance spectra obtained after 6 h. The stability of cAuNPs was also investigated at a pH range from 4–9. The Au content in the cAuNPs was determined by ICP using a Perkin Elmer Optima 5300 DV instrument.

Biocompatibility studies

Isolation of human RBC's and peripheral blood mononuclear cells. Blood was drawn from healthy donors, heparinized, layered onto Histopaque-1077 solution and centrifuged at 400 × g for 30 min at room temperature. This gave an upper layer of plasma, followed by a buffy coat consisting of mononuclear cells and a pellet of RBC's. The pellet was separated, washed thrice, diluted 10 times with PBS and used for hemolysis assay. The buffy coat was separated and washed twice with HBSS for 10 min. The supernatant was removed and to the pellet, RPMI 1640 medium containing 10% autologous plasma, sodium bicarbonate (2 mg ml⁻¹), pencillin (100 IU per ml), streptomycin (100 μg ml⁻¹), gentamicin (30 μg ml⁻¹), and amphotericin B (2.5 μg ml⁻¹) were added. The cells were maintained at 37 °C temperature, 5% CO₂ and 95% air in humidified atmosphere provided by Binder CO₂ incubator, Germany. Both RBCs and PBMCs were treated with isolated cAuNPs at 43 μM, 86 μM and 172 μM concentrations and toxicity analysed through the following studies.

Hemolysis assay. To 100 μl of the RBC suspension, 400 μl of the desired concentration of AuNPs suspended in PBS was added. 400 μl of H₂O and PBS served as the positive and negative controls respectively. After incubation for 4 h, the samples were vortexed, centrifuged and the supernatant was collected. The absorbance of supernatant at 577 nm was recorded and the percentage hemolysis calculated by the formula:

((sampleabsorbance − negative control absorbance)/(positive control absorbance − negative control absorbance)) × 100.

Trypan blue dye exclusion method. The viability of PBMCs was examined microscopically by Trypan blue dye exclusion method using Nikon Eclipse E600, Japan. 10 μl of the treated cells were stained with the dye (0.4%) and loaded onto a haemocytometer. Cells that excluded the dye were counted as viable cells and the percentage was calculated with reference to control (taken as 100% viable).

Alamar blue assay. PBMC's were seeded at a density of 2 × 10⁵ cells per well in a 96 well micro plate and treated. 10 μg of resazurin in PBS was added to each well before 18 h of incubation period and fluorescence measured (Ex 530 nm and Em 590 nm). The percentage of cell viability was calculated with reference to control. Blanks were set with RPMI medium, cAuNPs and Alamar blue without cells for each cAuNP concentration. Control cells were considered to be 100% viable.

Ethidium bromide (EtBr) acridine orange (AO) staining. The treated PBMCs were washed with PBS. EtBr and AO (0.25 μg of each) in PBS were added to 10 μl of cells and the morphological changes observed by Nikon Eclipse E600 confocal laser scanning microscope C1 (Ex 490 nm) and images captured using EZ-C1 software. An average of 4 scans (512 × 512 pixel array) was taken at 40× magnification. Three independent experiments were performed and a total of 100 cells counted in each experiment. Cells fluorescing green were considered as viable and those with red fluorescence as dead cells. The percentage of viable cells is calculated by the formula

(Number of viable cells counted/Number of total cells counted) × 100.

Statistical analysis. Statistical analyses were performed using GraphPad Prism 5 (GraphPad Software). The results of AuNP size distribution is expressed as mean ± SD and the results for cytotoxicity assays expressed as mean ± SEM.

Conflict of interest

The authors do not have any conflict of interest.

Acknowledgements

The authors thank the Director, CLRI, Chennai, India for the support provided and CSIR NWP-0035 (Nanomaterials and Nanodevices for application in health and diseases) for financial assistance. One of the authors (K. Sindhu) wishes to thank the CSIR, New Delhi, India, for the Junior Research Fellowship. The HR-TEM assistance by National Institute of Interdisciplinary Science and Technology (NIIST), Trivandrum, ICP-OES facility by IIT-Madras, DLS facility in Anna University, Chennai and SEM facility at Sastra University, Tanjore are gratefully acknowledged.

References

1. R. A. Sperling, P. R. Gil, F. Zhang, M. Zanella and W. J. Parak, Chem. Soc. Rev., 2008, 37, 1896.
2. E. C. Dreaden, M. A. Mackey, X. Huang, B. Kang and M. A. El-Sayed, Chem. Soc. Rev., 2011, 40, 3391.
3. P. M. Tiwari, K. Vig, V. A. Dennis and S. R. Singh, Nanomaterials, 2011, 1, 31.
4. A. Llevot and D. Astruc, Chem. Soc. Rev., 2012, 41, 242.
5. Z. Zhang and D. Fu, J. Nanosci. Nanotechnol., 2012, 12, 7265.
6. C. Uboldi, D. Bonacchi, G. Lorenzi, M. I. Hermanns, C. Pohl, G. Baldi, R. E. Unger and C. J. Kirkpatrick, Part. Fibre Toxicol., 2009, 6, 18.
7. C. Freese, C. Uboldi, M. I. Gibson, R. E. Unger, B. B. Weksler, I. A. Romero, P. O. Couraud and C. J. Kirkpatrick, Part. Fibre Toxicol., 2012, 9, 23.
8. E. E. Connor, J. Mwamuka, A. Gole, C. J. Murphy and M. D. Wyatt, Small, 2005, 1, 325.
9. J. Manson, D. Kumar, B. J. Meenan and D. Dixon, Gold Bull., 2011, 44, 99.
10. W. Cho, M. Cho, J. Jeong, M. Choi, H. Cho, B. S. Han, S. H. Kim, H. O. Kim, Y. T. Lim, B. H. Chung and J. Jeong, Toxicol. Appl. Pharmacol., 2009, 236, 16.
11. P. Mohanpuria, N. K. Rana and S. K. Yadav, J. Nanopart. Res., 2008, 10, 507.
12. S. Iravani, Green Chem., 2011, 13, 2638.
13. N. Ahmed, S. Sharma, M. K. Alam, V. N. Singh, S. F. Shamsi, B. R. Mehta and A. Fatma, Colloids Surf., B, 2010, 81, 81.
14. R. Shukla, S. K. Nune, N. Chanda, K. Katti, S. Mekapothula, R. R. Kulkarni, W. V. Welshons, R. Kannan and K. V. Katti, Small, 2008, 4, 1425.
15. K. Sneha, M. Sathishkumar, S. Y. Lee, M. A. Bae and Y. S. Yun, J. Nanosci. Nanotechnol., 2011, 11, 1811.
16. U. K. Parida, B. K. Bindhani and P. Nayak, World J. Nano Sci. Eng., 2011, 1, 93.
17. R. K. Das, P. Sharma, P. Nahar and U. Bora, Mater. Lett., 2011, 65, 610.
18. D. D. Lekeufack and A. Brioude, Dalton Trans., 2012, 41, 1461.
19. C. Singh, R. K. Baboota, C. K. Naik and H. Singh, Adv. Mater. Lett., 2012, 3, 279.
20. S. Manju and K. Sreenivasan, J. Colloid Interface Sci., 2012, 368, 144.
21. P. Arunkumar, H. Vedagiri and K. Premkumar, J. Nanopart. Res., 2013, 15, 1481.
22. S. K. Sivaraman, S. Kumar and V. Santhanam, Gold Bull., 2010, 43, 275.
23. L. A. Levchenko, S. A. Golovanova, N. V. Lariontseva, A. P. Sadkov, D. N. Voilov, Y. M. Shuĺga, N. G. Nikitenko and A. F. Shestakov, Russ. Chem. Bull., Int. Ed., 2011, 60, 426.
24. P. Anand, A. B. Kunnumakkara, R. A. Newman and B. B. Aggarwal, Mol. Pharm., 2007, 4, 807.
25. H. Hatcher, R. Planalp, J. Cho, F. M. Torti and S. V. Torti, Cell. Mol. Life Sci., 2008, 65, 1631.
26. M. H. Teiten, S. Eifes, M. Dicato and M. Deiderich, Toxins, 2010, 2, 128.
27. H. Itokawa, Q. Shi, T. Akiyama, S. L. M. Natschke and K. H. Lee, Chin. Med., 2008, 3, 11.
28. M. Balasubramanyam, A. A. Koteswari, R. S. Kumar, S. F. Monickaraj, J. U. Maheswari and V. Mohan, J. Biosci., 2003, 28, 715.
29. S. V. Jovanovic, S. Steenken, C. W. Boone and M. G. Simic, J. Am. Chem. Soc., 1999, 121, 9677.
30. K. I. Priyadarsini, D. K. Maity, G. H. Naik, M. S. Kumar, M. K. Unnikrishnan, G. J. Satav and H. Mohan, Free Radicals Biol. Med., 2003, 35, 475.
31. M. H. Pan, S. Y. L. Shiau and J. K. Lin, Biochem. Pharmacol., 2000, 60, 1665.
32. X. Gao, J. Kuo, H. Jiang, D. Deeb, Y. Liu, G. Divine, R. A. Chapman, S. A. Dulchavsky and S. C. Goutham, Biochem. Pharmacol., 2004, 68, 51.
33. F. Zsila, Z. Bikadi and M. Simonyi, Org. Biomol. Chem., 2004, 2, 2902.
34. S. Quideau, D. Deffieux, C. Douat-Casassus and L. Pouysegu, Angew. Chem., Int. Ed., 2011, 50, 586.
35. M. A. Bill, J. R. Fuchs, C. Li, J. Yui, C. Bakan, D. M. Benson, E. B. Schwartz, D. Abdelhamid, J. Lin, D. G. Hoyt, S. L. Fossey, G. S. Young, W. E. Carson III, P. Li and G. B. Lesinski, Mol. Cancer, 2010, 9, 165.
36. S. Bisht, G. Feldmann, S. Soni, R. Ravi, C. Karikar, A. Maitra and A. Maitra, J. Nanobiotechnol., 2006, 5, 3.
37. S. Wan, Y. Sun, X. Qi and F. Tan, AAPS PharmSciTech, 2012, 13, 159.
38. V. R. Yadav, S. Suresh, K. Devi and S. Yadav, AAPS PharmSciTech., 2009, 10, 752.
39. L. Li, F. S. Braiteh and R. Kurzrock, Cancer, 2005, 104, 1322.
40. D. K. Singh, R. Jagannathan, P. Khandelwal, P. M. Abraham and P. Poddar, Nanoscale, 2013, 5, 1882.
41. R. K. Gangwar, V. A. Dhumale, D. Kumari, U. T. Nakate, S. W. Gosavi, R. B. Sharma, S. N. Kale and S. Datar, Mater. Sci. Eng., C, 2012, 32, 2659.
42. B. K. Pong, H. I. Elim, J. X. Chong, W. Ji, B. L. Trout and J. Y. Lee, J. Phys. Chem. C, 2007, 111, 6281.
43. M. H. M. Leung, H. Colangelo and T. W. Kee, Langmuir, 2008, 24, 5672.
44. J. Niu, T. Zhu and Z. Liu, Nanotechnology, 2007, 18.
45. J. L. Gardea-Torresdey, J. G. Parsons, E. Gomez, J. Peralta-Videa, H. E. Troiani, P. Santiago and M. J. Yacamán, Nano Lett., 2002, 2, 397.
46. M. J. Yacaman, M. M. Almazo and J. A. Ascencio, J. Mol. Catal. A: Chem., 2001, 173, 61.
47. S. I. Stoeva, B. L. V. Prasad, S. Uma, P. K. Stoimenov, V. Zaikovski, C. M. Sorensen and K. J. Klabunde, J. Phys. Chem. B, 2003, 107, 7441.
48. P. Zhang, J. Li, D. Liu, Y. Qin, Z. Guo and D. Zhu, Langmuir, 2004, 20, 1466.
49. C. Ziegler and A. Eychmuller, J. Phys. Chem., 2011, 115, 4502.
50. K. Sindhu, R. Indra, A. Rajaram, K. J. Sreeram and R. Rajaram, J. Biomed. Nanotechnol., 2011, 7, 56.
51. F. Zsila, Z. Bikadi and M. Simonyi, Tetrahedron: Asymmetry, 2003, 14, 2433.
52. X. Ji, X. Song, J. Li, Y. Bai, W. Yang and X. Peng, J. Am. Chem. Soc., 2007, 129, 13939.
53. X. Qu, Z. Peng, X. Jiang and S. Dong, Langmuir, 2004, 20, 7.
54. E. Hutter and J. H. Fendler, Adv. Mater., 2004, 16, 1685.
55. D. Ghosh, D. Sarkar, A. Girigoswami and N. Chattopadhyay, J. Nanosci. Nanotechnol., 2011, 11, 1141.
56. J. Kimling, M. Maier, B. Okenve, V. Kotaidis, H. Ballot and A. Plech, J. Phys. Chem. B, 2006, 110, 15700.
57. T. M. Kolev, E. A. Velcheva, B. A. Stamboliyska and M. Spiteller, Int. J. Quantum Chem., 2005, 102, 1069.
58. V. T. Bich, N. T. Thuy, N. T. Binh, N. T. M. Houng, P. N. D. Yen and T. T. Luong, in Structural and spectral properties of curcumin and metal curcumin complex derived from turmeric (Curcuma longa), ed. D. T. Cat, A. Pucci and K. Wandelt, Springer, Berlin, 2009, vol. 127, pp. 271–278.
59. B. Tang, L. Ma, H. Y. Wang and G. Y. Zhang, J. Agric. Food Chem., 2002, 50, 1355.
60. M. K. Chow and C. F. Zukoski, J. Colloid Interface Sci., 1994, 165, 97.
61. L. Pei, K. Mori and M. Adachi, Langmuir, 2004, 20, 7837.
62. P. H. Bong, Bull. Korean Chem. Soc., 2000, 21, 81.
63. Y. Huang and D. H. Kim, Langmuir, 2011, 27, 13861.
64. N. R. Jana, L. Gearheart and C. J. Murphy, Langmuir, 2001, 17, 6782.
65. A. Chompoosor, K. Saha, P. S. Ghosh, D. J. Macarthy, O. R. Miranda, Z. Jhu, K. F. Arcaro and V. M. Rotello, Small, 2010, 6, 2246.
66. Y. Pan, S. Neuss, A. Leifert, M. Fischler, F. Wen, U. Simon, G. Schmid, W. Brandau and W. Jahnen-Dechent, Small, 2007, 3, 1941.
67. S. Krajewski, R. Prucek, A. Panacek, M. Avci-Adali, A. Nolte, A. Straub, R. Zboril, H. P. Wendel and L. Kvitek, Acta Biomater., 2013, 9, 7460.
68. P. Khullar, V. Singh, A. Mahal, P. N. Dave, S. Thakur, G. Kaur, J. Singh, S. S. Kamboj and M. S. Bakshi, J. Phys. Chem. C, 2012, 116, 8834.
69. I. M. M. Piano, V. S. Marangoni, R. C. S. de Oliveira, L. M. G. Antunes and V. Zucolotto, Toxicol. Lett., 2012, 215, 119.
70. M. Sharma, R. L. Salisbury, E. I. Maurer, S. M. Hussain and C. E. Sulentic, Nanoscale, 2013, 5, 3747.
71. R. Bhttacharya, C. R. Patra, R. Verma, S. Kumar, P. R. Greipp and P. R. Mukherjee, Adv. Mater., 2007, 19, 711.
72. C. Basset, J. Vadrot, J. Dennis, J. Poupon and E. S. Zafrani, Liver Int., 2003, 23, 89.
73. J. Lee, H. Y. Kim, H. Zhou, S. Hwang, K. Koh, D. Han and J. Lee, J. Mater. Chem., 2011, 21, 13316.
74. S. K. Nune, N. Chanda, R. Shukla, K. Katti, R. R. Kulkarni, S. Thilakavathy, S. Mekapothula, R. Kannan and K. V. Katti, J. Mater. Chem., 2009, 19, 2912.
75. K. Katti, N. Chanda, R. Shukla, A. Zambre, T. Suibramanian, R. R. Kulkarni, R. Kannan and K. V. Katti, Int. J. Green Nanotechnol. Biomed., 2009, 1, B39.
76. N. Chanda, R. Shukla, A. Zambre, S. Mekapothula, R. R. Kulkarni, K. Katti, K. Bhattacharyya, G. M. Fent, S. W. Casteel, E. J. Boote, J. A. Viator, A. Upendran, R. Kannan and K. V. Katti, Pharm. Res., 2011, 28, 279.
77. S. C. Gupta, S. Patchva and B. B. Aggarwal, AAPS J., 2013, 15, 195.
78. N. S. Rejinold, M. Muthunarayanan, K. P. Chennazhi, S. V. Nair and R. Jayakumar, J. Biomed. Nanotechnol., 2011, 7, 521.
79. M. M. Yallapu, S. F. Othman, E. T. Curtis, N. A. Bauer, N. Chauhan, D. Kumar, M. Jaggi and S. C. Chauhan, Int. J. Nanomed., 2012, 7, 1761.
80. X. Z. Zhao, T. Jiang, L. Wang, H. Yang, S. Zhang and P. Zhou, J. Mol. Struct., 2010, 984, 316.

---

Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3ra45345f

---