Green-synthesized gold nanocubes functionalized with bisdemethoxycurcumin analog as an ideal anticancer candidate

DOI 10.1515/gps-2013-0090 Green Process Synth 2014; 3: 47–61

Rajagopal Aruna Devi, Arul Prakash Francis and Thiyagarajan Devasena*

Green-synthesized gold nanocubes functionalized with bisdemethoxycurcumin analog as an ideal anticancer candidate

Abstract: Novel eco-friendly methods for the synthesis of metal nanoparticles are preferred and have recently been widely researched. In this study, a cost-effective and environmentally friendly method for the synthesis of gold nanoparticles was investigated. Costus pictus, also known as insulin plant due to its antidiabetic activity, was used as the reducing agent. Chloroaurate ions were rapidly reduced by the methanolic extract of Costus pictus, which was indicated by the color change from yellow to ruby red. The presence of a peak at 530 nm in UV-visible spectro-photometry confirmed the reduction of chloroaurate to gold nanoparticles. The reaction kinetics and effects of time, temperature, concentration of extract and substrate were also studied. The size and morphology of gold nano-particles were analyzed using scanning electron micros-copy (SEM) and high-resolution transmission electron microscopy (HR-TEM) and they were found to be cuboidal, with a size of around 20 nm. The size of the particles was also analyzed by the particle size analyzer, and was found to be 45 nm. These particles were functionalized with bis-demethoxycurcumin analog (BDMCA). X-ray diffraction (XRD) analysis of prepared nanoparticles showed that the gold nanoparticles have a face centered cubic structure and that of functionalized nanoparticles showed peaks similar to that of BDMCA. Fourier transform infrared spec-troscopy (FTIR) studies revealed the presence of reducing groups in the plant methanolic extract and the functional-ization of nanoparticles with BDMCA was also confirmed. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that, the BDMCA-function-alized gold nanocubes possess anticancer activity. The mechanism of action is discussed.

Keywords: BDMCA; Costus pictus; cytotoxicity; gold nano-cubes; green synthesis.

*Corresponding author: Thiyagarajan Devasena, Centre for Nanoscience and Technology, A.C. Tech Campus, Anna University, Chennai-600025, Tamil Nadu, India, e-mail: [email protected] Aruna Devi and Arul Prakash Francis: Centre for Nanoscience and Technology, A.C. Tech Campus, Anna University, Chennai-600025, Tamil Nadu, India

1 Introduction

Nanotechnology is an emerging field with applications in almost every domain of science. A particle that has at least one of its dimensions in the 1–100 nm range is called a nanoparticle. Nanoparticles bridge the bulk materials and atomic structures and are hence widely studied for use in various applications. The increase in surface area to volume ratio imparts numerous advantageous proper-ties to nanomaterials when compared to bulk materials. The advantage of a nanoparticle over its bulk counter-part is that it has enhanced optical, magnetic, electronic and thermal properties. Metal nanoparticles are being employed for a wide range of applications. They have dis-crete valence and conduction bands, which imparts spe-cific electronic and conductive properties to them. They can also be modified with functional groups and conju-gated with ligands, antibodies and drugs. These modified or conjugated metal nanoparticles are widely used for drug delivery, gene therapy, imaging and diagnostics [1].

Gold nanoparticles in general, have wide applications in sensor [2], biomedical [3] and imaging [4] applications. They are used in the treatment of various types of cancer [5], diabetes [6], etc. They are used in photodynamic therapy of cancer cells, as the gold nanoparticles get heated up to kill the tumor cells when they absorb light of a specific wavelength region [7]. Due to the high surface to volume ratio, they can be used in the delivery of thera-peutic agents [8]. Gold nanoparticles can scatter light and hence they can produce a wide array of colors under dark field microscopy. This property is used in imaging [9]. They are also used to detect biomarkers in the diagno-sis of heart diseases and cancer [10]. Gold nanoparticles may be synthesized using a wide range of methods that fall under three major classifications: physical, chemical [11] and biological routes. When it comes to biomedical applications, nanoparticles synthesized by physical and chemical means have certain disadvantages, due to the use of hazardous chemicals and toxic by-products. Also, these particles have random size distributions and less stability. A cleaner and greener novel method that utilizes

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48 R. Aruna Devi et al.: Anticancer activity of BDMCA functionalized gold nanoparticles

phytochemicals and microbial enzymes for reduction of the metal ions is used nowadays.

Green synthesis involves the reduction of metal ions by sources like microbial enzymes and plant extracts [12].Green synthesis of gold nanoparticles using flower extract, spice leaf extract etc., has been previously reported [13]. The advantages of green chemistry in nanoparticle synthesis are that, the process is less laborious, no toxic by-products are formed, there is less energy requirement, a fast reduc-tion rate, it is environmentally friendly, cost-effective, easily scalable to a larger scale and is simple. Also, the nanoparti-cles synthesized by this method show uniform and narrow size distribution, high dispersity and high stability [14]. Since no toxic chemicals are used in synthesis, the nano-particles are highly biocompatible and can be used for bio-medical applications and pharmaceutical applications [5]. The phytochemicals in plants, like flavanoids and phenols not only reduce the metal ions, but also stabilize them and act as capping agents. This capping effect produces highly monodispersed nanoparticles, thereby leading to increased stability and reduced agglomeration. Moreover, the coating of these phytochemicals over the nanoparticles can lead to a synergistic effect. This is very advantageous in the case of biomedical applications, since the large surface area of the nanoparticles can enhance the effectiveness of the coated phytochemical [14].

Costus pictus, commonly known as insulin plant, is known to possess antidiabetic activity. The plant is rich in reducing phytochemicals like flavanoids and phenols. A gas chromatography-mass spectrometry (GC-MS) study shows that the plant extract has flavanoids like querce-tin and kaempferol, and phenolic acids like vanillic acid, syringic acid, benzoic acid and coumaric acid. The pres-ence of other phytochemicals like quinines, steroids and tannins has also been detected [15]. The analysis of essen-tial oils of the leaf showed that it contains hexadecanoic acid, pentanol, ionone and farnesyl acetone in a greater amount. Therefore, this plant extract may be an excel-lent agent for the reduction of chloroauric acid and its stabilization. Also, various therapeutic uses like antidia-betic, anticancer, antioxidant, antibacterial and diuretic effects of this plant have been proven. Studies in animal models showed that the extract was non-toxic and that it had no side effects [15, 16]. When the nanoparticles are capped by these phytochemicals, the activity is increased due to the synergistic effect. Therefore, we suggest that the gold nanoparticles synthesized and stabilized by Costus pictus extract may have better biomedical activity when compared to uncapped nanoparticles. A previous studies by Grace et al. suggested that gold nanoparti-cles do not possess antimicrobial activity. However, gold

nanoparticles that are functionalized exhibit antimicro-bial activity [17]. Similarly, gold nanoparticles functional-ized with anticancer agents show enhanced activity due to the increased surface area [3, 6]. Bisdemethoxycurcumin analog (BDMCA), 1,7-bis (4-hydroxyphenyl)-hepta-1,6-di-ene-3,5-dione, is a reported anticancer agent, but suffers from poor solubility and bioavailability. Therefore, coating of BDMCA over gold nanoparticles may result in dual ben-efits: 1) increasing the activity of gold nanoparticles and 2) enhancing the solubility of BDMCA due to the high surface to volume ratio offered by gold nanoparticles. Based on the literature background, we report a novel and intrinsically green approach for the synthesis of gold nanoparticles using the methanolic extract of Costus pictus D. Don. The protocol for synthesis was optimized. The formation kinet-ics, size, shape, and crystallite structure were also studied and the cytotoxicity was evaluated for the naked gold nan-oparticles and BDMCA functionalized gold nanoparticles.

2 Materials and methods

2.1 Materials

The plant material was collected from Green Scape Farm, Madurai, Tamil Nadu and was authenticated by Park Anatomy Research Centre (Reg no: PARC/2012/1419). BDMCA with nanosize was prepared as described by Francis et al. [18].

2.2 Reagents and chemicals

Tetrachloroauric acid was obtained from FINAR reagents Ltd, Mumbai, Maharashtra, India and was of high purity. All of the chemicals and reagents used for this proto-col were of analytical grade, and obtained from Sigma Aldrich, Banglore, Karnataka, India.

2.3 Methods

2.3.1 Preparation of Costus pictus extract

Fresh leaves of Costus pictus were washed thoroughly and shade dried. The dried leaves were finely ground. An amount of 5 g of the leaf powder was mixed with 50 ml of hexane to remove any oil content present in it. This mixture was stirred for 24 h at room temperature. It was then filtered and the residue was mixed with 100 ml of



R. Aruna Devi et al.: Anticancer activity of BDMCA functionalized gold nanoparticles 49

methanol. This mixture was stirred for 24 h at room tem-perature. The resulting solution was filtered and the fil-trate was used as the reducing agent for the preparation of gold nanoparticles.

2.3.2 Preparation of gold nanoparticles

Chloroauric acid solution (20 ml of 0.001 m) was taken in a beaker. It was mixed thoroughly using a magnetic stirrer and 400 µl of plant extract was added. This reaction was carried out at 70°C for 15 min. Color change was observed from yellow to ruby red.

2.3.3 Functionalization of nanoparticles with BDMCA

Gold nanoparticle solution (50 ml) was taken in a beaker. An amount of 200 µl of 1% polyethylene glycol (PEG) was added to the above solution. The above mixture was homogenized at 8000 rpm for 30 min. BDMCA (10 mg, dissolved in 1 ml of acetone) was added to 50 ml of the PEGylated gold nanoparticle solution. The above mixture was homogenized at 8000 rpm for 30 min. The solution was centrifuged at 12,000 rpm and the pellet was lyophi-lized. The lyophilized powder was used for characteriza-tion and to study the biological activity.

2.3.4 Characterization of the as-prepared and functionalized nanoparticles

The UV-visible spectrum of the as-prepared and function-alized gold nanoparticles was recorded using a T90 PG UV/VIS spectrophotometer at room temperature in the wavelength range between 200 and 800 nm.

The average particle size of the prepared gold nano particles and BDMCA coated nanoparticles was estimated by using the Malvern system particle size analyzer.

Scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDAX) were performed, using a Hitachi SU-6600 SEM machine with an accelerating voltage of 15 kV. Thin films of the sample were prepared on an aluminum foil paper by just dropping a very small amount of the sample and allowing to dry. The morphol-ogy of the as-prepared and PEGylated gold nanoparti-cles was studied using field emission SEM (FE-SEM). The surface, size and elemental analysis (EDAX) of the gold nanoparticles was also done using FEI TencaiTM G2 F20 (S-TWIN) high-resolution transmission electron micros-copy (HR-TEM), operating at 200 kV.

The X-ray diffraction (XRD) pattern was measured by drop coated films of gold nanoparticles, extract, BDMCA and BDMCA functionalized characteristic Cu-Kα radia-tion (λ = 1.5406 Å) in the range of 20°–80° at a scan rate of 0.05°/min, with a time constant of 2 s. The functional groups in the extract and on the surface of the gold nano-particles were identified by a Fourier transform infrared spectroscopy (FTIR) spectrum recorded in the range of 4000–400 cm-1 using a Perkin-Elmer Spectrum One spec-trometer at resolution of 4 cm-1.

2.3.5 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay

The anticancer potential of the gold nanoparticles and BDMCA functionalized gold nanoparticles was estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Michigan Cancer Foundation-7 (MCF-7), breast cancer cells were used for the analysis. Cells were cultured in 10% fetal bovine serum in Dulbecco’s Modified Eagle Medium along with 1000 µl of antibiotic mix. The number of cells was counted using a hemocytometer. The cells were seeded in a 96 well plate and incubated for 24 h. Gold nanoparticles and BDMCA functionalized gold nano-particles were added to the wells in varying concentrations and were further incubated for 24 h. MTT (20 µl) was added to the wells and incubated for 4 h. Dimethyl sulfoxide (100 µl) was added to avoid false negatives. The plates were transferred to a plate reader and absorbance was measured.

3 Results and discussion

3.1 Macroscopic observation

After the addition of plant extract to the gold chloride solution, there was a gradual color change from yellow to light pink and then to ruby red as seen in Figure 1. This is in line with previous reports on the synthesis of gold nan-oparticles. Ghosh et al. [12] and Reddy et al. [14] showed that the formation of gold nanoparticles can be followed by observing the color change from yellow to ruby red. This may be due to the surface plasmon effect exhibited by noble metals in the nano scale. This occurs when the oscillation of surface electrons is in resonance with the incident light. The increase in color intensity indicates the increase in the amount of nanoparticles formed. The time taken for the change of color from yellow to pink varied with respect to temperature. At 30°C, color change




occurred after 2 h and at 50°C color change occurred after 60 min. At 70°C, color change occurred after 10 min. The intensity increased rapidly up to 30 min and then remained almost constant. This rapid reduction and sub-sequent color change may be attributed to the presence of reducing phytochemicals in the plant extract. Previous reports by Reddy et al. [14] suggest that the plant phyto-chemicals have the ability to induce rapid reduction of metal substrate solution to generate nanoparticles. This is also in line with our findings.

3.2 UV-visible spectroscopy

The UV spectrum of the gold nanoparticles is shown in Figure 2. The analysis of the UV-visible spectrophotometric

Figure 1 Color change denoting the formation of gold nanoparticles.

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Figure 2 UV spectra of gold nanoparticles showing surface plasmon band at 534 nm.

data confirmed that the surface plasmon resonance (SPR) band was located at 534 nm. This peak is specific to gold nanoparticles as previously reported [14]. Therefore, we confirm the formation of gold nanoparticles in this green synthesis protocol.

3.2.1 Factors influencing the reduction process

From the color change observation (Figure 1) and UV spectra (Figure 2) we suggest that the plant extract is responsible for the rapid reduction of substrate and for-mation of gold nanoparticles. Therefore, we further ana-lyzed the influence of various factors like: (i) time, (ii) extract concentration and (iii) substrate concentration on the reduction of gold chloride.

3.2.1.1 Influence of timeThe progress of the reaction was monitored by taking the UV spectra of the samples since the extract was added to the substrate, until the completion of the reaction (Figure 3A). We observed that the absorption increases with time, thus contributing to the increase in intensity, which in turn is due to the increase in the formation of nanoparticles. We also noted that there is no major shift in the maximum absorbance wavelength. The maximum absorbance wavelength lies in the range of 530 nm–540 nm throughout the course of the reaction. This is specific for gold nanoparticles and our results are further supported by previous reports [17, 19]. A minor blue shift was observed from 538 to 533 nm, which denotes the reduction in size of the particles. Thus, we also infer that our protocol is optimized for the synthesis of nano-sized gold particles. Formation kinetics was studied by recording the absorbance values at 530 nm for differ-ent time intervals. The absorbance intensity increased with increase in reaction time, as seen in Figure 3B. This indicates the formation of more nanoparticles as time increases. This may be due to the reducing property of the phytochemicals, like flavanoids and phenols in the plant extract. Also, we note that the absorbance becomes almost constant after 20 min. This indicates that the reaction is completed by 20 min. No further reaction or agglomeration occurred since the phytochemicals cap the nanoparticles and impart stability.

3.2.1.2 Influence of metal ion concentrationOn adding the extract to serially increasing concentra-tion of the gold chloride solution, we observed a slight red shift (Figure 3C). The shift was from 535 to 546 nm. The




results are tabulated in Table 1. This indicates that the par-ticle size increases with increase in concentration of the substrate. Also, the time taken for the completion of the reaction and the amount of extract added for reduction increased with increase in concentration of gold chloride. Additionally, that there was an increase in peak absorb-ance as the metal ion concentration increased. This may due to the formation of a large number of gold nanopar-ticles. Therefore, 1 mm can be justified as the optimum metal ion concentration to be used for the synthesis of gold nanoparticles.

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Table 1 Influence of metal ion concentration.

Concentration of metal ion (mm) λmax

1 5352 5393 546

3.2.1.3 Influence of extract volumeThe effect of volume of extract on the reduction of gold chloride was studied. Different volumes of extract, e.g., 400 µl, 600 µl, 800 µl, 1000 µl and 1200 µl were added to a constant metal ion concentration (1 mM) and the UV spectrum was recorded (Figure 3D). It was interesting to observe the shift in the peak wavelength, that is, a blue shift. This shift denotes that the particle size decreases as the volume of extract increases, which is tabulated in Table 2. We can also see that the peak is broader for low

Table 2 Influence of extract volume.

Volume of extract (µl) λmax

400 539600 536800 5351000 5341200 533




volumes and increases as the extract volume increases. This denotes that the number of particles formed has increased significantly at a higher volume of extract. As the extract volume increases, the volume of the reducing agent in the extract also increases and hence there may be an enhancement in substrate reduction. It was also noted that the time taken for the reduction was inversely proportional to the volume of extract. From the previous graph, we optimized 1 Mm as the optimum concentration of substrate. To reduce 1 Mm of substrate, 400 µl of extract is the optimum volume. Thus we can take 400 µl as the optimum value of extract to be added.

3.2.2 Functionalized gold nanoparticles

Pegylation of the nanoparticles was also studied using UV-visible spectrophotometry. The maximum absorbance for

PEG lies at around 300 nm, as reported in Figure 4A. The amount of PEG added to the nanoparticle solution was varied and absorbance was studied. It can be observed from the graph (Figure 4B) that, as the amount of PEG increases, the intensity of the peak also increases. The peak for gold nanoparticles is found around 530 nm. The nanoparticle solution containing 200 µl of PEG was found to be more stable, as there was no color change for a long period. It was used for functionalization. The centrifuged sample was used to investigate the binding of BDMCA and PEG to the nanoparticles. It can be seen from Figure 4C that there are no intense peaks in the supernatant curve. This shows that all of the PEG and drug are bound to the gold nanoparticles and have settled in the pellet. The UV spectrum of the pellet shows peaks for PEG and gold nano-particles. The peak for gold nanoparticles is broad and the peak for BDMCA is absent. This may be due to the masking effect of high concentration of gold nanoparticles, when

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Figure 4 UV spectra of (A) polyethylene glycol (PEG), (B) PEGylated gold nanoparticles, (C) functionalized nanoparticles-supernatant and pellet.




compared to BDMCA. It can be explained by the broad peak of gold nanoparticles.

3.3 Particle size analysis

The average particle size of the gold nanoparticles was measured using the particle size analyzer by the dynamic light scattering technique (Figure 5A). The histogram shows that the particle size of the gold nanoparticles ranged from 17 to 130 nm and the average size was found to be around 45 nm. This reduction in size is due to the efficient reducing property of the phytochemicals in the plant extract. Also, the uniform distribution shows that the particles are well dispersed and almost uniform. This is due to the capping effect of the phytochemicals that pre-vents agglomeration of the particles. The size distribution of the functionalized nanoparticles was determined using a particle size analyzer. It can be observed from Figure 5 that the peaks are single and uniform in nature. This shows that the particles are of almost uniform size and are well dispersed. We can observe from that, that the average size of the PEGylated nanoparticles is 71 nm (Figure 5B), which is slightly higher than that of as-synthesized

nanoparticles. The average size of BDMCA functional-ized nanoparticles is 105 nm (Figure 5C) which is, in turn, larger than that of PEGylated nanoparticles. Thus we can confirm that the nanoparticles have indeed been coated with PEG and BDMCA.

3.4 FE-SEM-EDAX analysis

The biosynthesized gold nanoparticles presented variable shapes, most of them being spherical in shape as shown in Figure 6A and B. Gold nanospheres were formed pre-dominantly with diameters ranging from 30 nm to 40 nm. The particles were found to be well dispersed with well-defined edges. This may be due to the protection offered by plant phytochemicals against agglomeration. They act as capping agents and impart stability to the nanoparticles. Also, in a high magnification image (Figure 6C and D), we can see the formation of polyhedral structures of gold nanoparticles. This may be due to the growth of the nanospheres to form structures. Cube-like structures with rounded edges are observed. The rounding of edges might be due to the increase in surface tension. Figure 7 demonstrates the morphology of PEG passivated gold

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Figure 5 Particle size analysis of (A) gold nanoparticles, (B) PEGylated gold nanoparticles, and (C) BDMCA coated gold nanoparticles.




Figure 6 Scanning electron microscopy (SEM) images of gold nanoparticles at high and low resolution.

Figure 7 Scanning electron microscopy (SEM) images of PEGylated gold nanoparticles.




nanoparticles imaged through SEM. The gold nanoparti-cles are found attached to the polymer matrix. It is clearly observed from Figure 7 that the particles in the nano regime are well separated, with almost the same size and shape. The particles with smooth and solid surfaces revealed that the PEG has a considerable effect on the nanoparticles. We can also note that, the particles tend to stay without any agglomeration and the size is less. Such stable par-ticles have wide applications in imaging, cancer therapy and other biomedical applications. A further study was carried out using EDAX to confirm the elements of the particle, and the EDAX pattern obtained is represented in Figure 8. The EDAX analysis displayed signature spectra for gold and it convincingly evidenced the presence of gold in the nanoparticles. The peak for carbon is dominat-ing, since the sample was drop coated over a carbon film for analysis. The peak for copper is also present, as the sample was placed over a copper grid.

3.5 HR-TEM analysis

The TEM images show the formation of anisotropic gold nanoparticles. We can see that the particles are polydis-persed, with varying shapes like spheres, triangles and polyhedrons. We speculate that, in our study, the initially formed nanospheres might have undergone shape trans-formations over time. It might have been mediated by the phytochemicals in plant or the reaction conditions. Similar results have been reported in previous papers [20, 21]. Thus, we may assume that, initially, the nanoparticles

were spherical. Over time, room temperature sintering might have caused the formation of large sized polyhe-dral structures. These results are supported by our SEM results, which show spherical particles at a high resolu-tion and polyhedral shapes at a low resolution. This kind of mechanism has also been previously reported [22]. From Figure 9A to F, we can see pentagonal projections with slightly rounded edges. This might have resulted from decahedral shaped particles [22]. It can be seen from the lattice fringes that each triangular face represents a single crystalline nature. Thus, we can say that the nanoparti-cle is made of five single crystalline units. The rounded edges and truncated shapes might be due to the increase in surface stress as the particle grows. Similarly, the few triangular projections in TEM might be due to truncated or partially developed tetrahedron. The hexagonal projec-tions that are almost spherical in shape may be the result of icosahedrons projection. The triangular faces can be identified by the contrast in the image. Hereby, we can say that polyhedral gold nanoparticles can be synthesized using Costus pictus methanolic extract and the shape and size may be controlled if we fine-tune the reaction condi-tions. Polyhedral gold nanoparticles find wide applica-tions in imaging, sensors and surface enhanced raman spectroscopy (SERS) applications.

3.6 XRD analysis

The formation of gold nanoparticles synthesized using the plant extract was further supported by XRD

800Acquire EDX residualAcquire EDX modeled spectrumAcquire EDX peak fitAcquire EDX NG correctedAcquire EDX

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Figure 8 Representative energy dispersive X-ray analysis (EDAX) profile of gold nanoparticles.




measurements. Diffraction peaks were observed at 27.78°, 32.18°, 35.62°, 38.2°, 44.38°, 46.3°and 64.4°. The Bragg reflections corresponding to the (111), (200) and (220) sets of lattice planes are observed. This may be indexed on the basis of the face-centered cubic structure of gold as seen in Figure 10A. The obtained data matched with Joint Com-mittee for Powder Diffraction Set (JCPDS) Data 04-0784, confirming that the cubic phases of gold nanoparticles synthesized using the methanolic extract of lattice planes are relatively weak and broad when compared to the (111) peak, which is highly intense. This indicates that the gold nanocrystals in the thin film are oriented predominantly in the (111) plane. The crystallite size was calculated using Scherrer’s formula: d = 0.94 λ/βcosθ, where 0.9 is the shape factor, generally taken for a cubic system, λ is the X-ray source wavelength, typically 1.5406 Å, β is the full width at half the maximum intensity in radians, and θ is the

Figure 9 High-resolution transmission electron microscopy (HR-TEM) analysis of (A) to (F) various shapes and projections of gold nanoparticles.

Table 3 Crystallite size of gold nanoparticles calculated using Scherrer’s formula.

2θ Full width half maximum

Crystallite size (nm)

38.2517 0.4847 17.244.4359 0.7812 11.164.4654 0.7241 13.12

Bragg angle. Using the above formula, the crystallite size was calculated to be ~13.8 nm. The results are tabulated in Table 3. The XRD profile of the extract shows peaks at 27°, 32°, 38°, 44°, 46°, 64° and 77° (Figure 10A). These peaks match with the JCPDS Data 03-0250, which corresponds to palmitic acid. It has been previously reported that pal-mitic acid is the primary constituent in Costus pictus [16]. Also, the extract peaks are found in the XRD profile of gold




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nanoparticles. The peaks of the extract, 27°, 32°, 38°, 44°, 46° and 64°, are present in the XRD profile of gold nano-particles as well. However, a slight shift is observed. Hence it proves that the nanoparticles have been capped by the plant phytochemicals. Figure 10B shows that the diffrac-tion peaks for BDMCA are present at 26°, 27°, 30°, 33°, 38°, 39°, 44°, 53°and 65°. The peaks for BDMCA coated gold nanoparticles are present at 26°, 27°, 37°, 39°, 43°, 64° and 77°, respectively. The XRD profile of BDMCA coated nanoparticles was compared with that of pure BDMCA. On comparing the XRD profiles of the drug-BDMCA and drug coated gold nanoparticles, we can observe that matching peaks are present. The major peak at 27° and other peaks at 26°, 38°, 39°, 44° and 64° are almost similar, or show a slight shift. This may be due to the capping of drug over the gold nanoparticles. Thus, it proves the capping of drug over the gold nanoparticles. This functionalized nanopar-ticle can be used for many biomedical applications. Also, functionalization increases the bioavailability of water-insoluble BDMCA and enhances its efficiency.

3.7 FTIR

The FTIR spectra were recorded from 500 cm-1 to 4500 cm-1. The FTIR spectrum of plant extract helps in identi-fying the possible biomolecules which aid in the reduc-tion of the chloroaurate ions and act as capping agents to maintain the stability of the nanoparticles. Figure 11A shows the FTIR spectra of the extract and as-prepared nanoparticles. The intense absorption band at 3435 cm-1 corresponds to the O-H stretch. This may indicate the presence of phenol groups in the extract. There are peaks at 2940 cm-1 and 2842 cm-1, corresponding to the

asymmetric and symmetric stretching vibrations of car-bonyl groups and C-H stretch of methylene, which may indicate the presence of terpenoids. The sharp peaks at 1654 cm-1 correspond to amides or keto-esters and may be due to the quinines released by the extract. The bands at 2042 cm-1 and 1114 cm-1 correspond to the C-N stretching vibration of aliphatic amines or to alcohols/phenols. This may indicate the presence of proteins in the extract. The peak at 1455 cm-1 denotes the aromatic ring stretch which may be due to the presence of flavanoids like querce-tin in the extract. The peaks at 2371 cm-1 and 1092 cm-1 indicate the presence of phosphorus bonding, which is present in the extract and also to large ring C-O stretch which may be due to the presence of quinines and other ketones. Similar to extract, the nanoparticle solution also has a strong stretch at 3441 cm-1, which indicates O-H stretching. The other peaks at 2370 cm-1, 2081 cm-1

and 1637 cm-1 are similar to that obtained in the extract, and may correspond to amines, quinines, ketone or phe-nolic groups. Also the presence of a peak at 711 nm is characteristic of C-Cl stretch, which may be due to the bonding of gold chloride to the extract due to the carbon-chlorine bond. The shift in peak wave number from 2371 cm-1 to 2370 cm-1 and 1654 cm-1 to 1637 cm-1 may be due to the binding of extract with the gold nanoparticles. This indicates that the gold nanoparticles synthesized using the plant extract are surrounded by some proteins and metabolites, such as terpenoids, quercetin, flavonoids having functional groups of amines, alcohols, ketones, aldehydes, and carboxylic acids. Previous studies have reported the reducing and capping ability of phytochemi-cals and proteins during the green synthesis of nanopar-ticles [13]. These results validate our present findings. Our interpretation may further be supported by the GC-MS




and high performance liquid chromatography data of the leaf extract of Costus pictus [15, 16], which reveals the presence of phenols, quinines and flavanoids. Thus, we suggest that the phenols, quinines and flavanoids of the extract may play a role in: 1) reducing the gold ion to gold nanoparticles, 2) capping and stabilizing the nanoparti-cles, and 3) preventing coalescence. Therefore, we may infer that the methanolic extract of Costus pictus may be an effective reducing agent in the green synthesis of gold nanoparticles. The graph (Figure 11B) shows the FTIR spectrum of the PEGylated gold nanoparticles. Peaks are observed at 590 cm-1, 1077 cm-1, 1110 cm-1, 1636 cm-1, 2851 cm-1, 2922 cm-1 and 3427 cm-1. The bend in the region 1000 cm-1 to 1350 cm-1 is representative of the skeletal C-C vibra-tions of the long chain polymer. The peak at 590 cm-1 is specific for OH out of plane bend. This is indicative of the presence of OH groups in the polymer. The broad peak at

2851 cm-1 is specific for the CH stretch which may be due to the stretching vibrations of C-H bonds in the C back-bone. The broad bend in the region 3200 cm-1 to 3500 cm-1 is specific for normal polymeric OH stretch. Thus, it con-firms the capping of gold nanoparticles with the polymer. The other peaks correspond to those of the extract coated gold nanoparticles, as discussed earlier. Figure 11C shows the FTIR spectra of pure BDMCA and BDMCA coated gold nanoparticles. The spectra of BDMCA coated gold nano-particles shows matching peaks with that of pure BDMCA. The peaks at around 940 cm-1, 1024 cm-1, 1640 cm-1, 2345 cm-1, 2923 cm-1 and 3426 cm-1 are found to be matching. The group of peaks lying within 900 cm-1 and 1300 cm-1 is representative of the skeletal C-C vibrations. The strong stretch at 1640 cm-1 is characteristic of conjugated ketones or quinines. This may be due to the presence of the ketone groups present in BDMCA. The peak at 1475 cm-1

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Figure 11 Fourier transform infrared spectroscopy (FTIR) spectrum of (A) extract and as-prepared gold nanoparticles, (B) polyethylene glycol (PEG), (C) bisdemethoxycurcumin analog (BDMCA) and BDMCA coated gold nanoparticles.




is a signature peak for aromatic ring stretch. This may be due to the presence of aromatic rings present in BDMCA. The group of several peaks in 950 cm-1 to 1225 cm-1 may also denote the presence of aromatic C-H in plane bends in the drug. The presence of a strong stretch at 1636 cm-1,

which is characteristic of alkenyl C = C stretch, may denote the presence of the alkenyl groups. It also denotes the presence of aryl substituted C = C groups in BDMCA. The peak at 2927 cm-1 is specific to the asymmetric/symmetric stretch of the methylene C-H group, thus confirming its presence in the drug. The presence of a peak at 670 cm-1 is representative of the aromatic C-H out of plane bend, which may have arisen from the aromatic rings in the compound. The peak at 752 cm-1 might have arisen due to the methylene group rocking in the carbon backbone. The bend in the region from 3250 cm-1 to 3600 cm-1 is due to the presence of a hydroxyl group. H bonded OH stretch corre-sponding to this region may be indicative of the presence of OH groups in the ring. The matching peaks of BDMCA and BDMCA coated gold nanoparticles clearly prove that the nanoparticles have been functionalized with BDMCA.

3.8 MTT assay

Our previous reports have revealed the validity of cytotoxic-ity studies to prove the anticancer activity of nanoparticles [23] and BDMCA [18]. The concentration of the anticancer compound which induces 50% cell death by cytotoxicity is called the IC50 value or IC50 dose. In general, microscopic imaging and MTT assays can be done (in cells treated with the test compound) to assess the cytotoxicity and IC50, respectively. Microscopic morphology of untreated MCF-7 cell lines maintains their integrity and shows normal mor-phology (Figure 12A). Gold nanocubes-treated MCF-7 cell lines (Figure 12B) reveals shrinkage, and disorganization of cell architecture. Interestingly, BDMCA-functionalized gold nanocubes were found to induce well-defined morphologi-cal changes such as membrane damage, blebbing, floating and chromosomal condensation. All of these morphologi-cal features may be attributed to cytotoxic cell death, which was more pronounced in cells treated with BDMCA-func-tionalized gold nanocubes. Our earlier investigations revealed the enhanced cellulolytic activity of bioactive

Figure 12 Microscopic morphology of (A) MCF-7 cells, (B) gold nanoparticle treated MCF-7 cells; and (C) bisdemethoxycurcumin analog (BDMCA)-functionalized gold nanoparticle treated MCF-7 cells.




compounds when coated to metallic nanoparticles like gold and silver [24, 25]. Thus, we infer that coating of BDMCA on the surface of green synthesized gold nanocubes may lead to an effective anticancer candidate. Francis et al. and Durga et al. [23, 26] reported that the anticarcinogenic nan-oparticles are capable of inducing alterations in the cellular morphology. According to Bhawana et al. [27], particles with nanosize show enhanced membrane transport, thereby leading to rapid cellular entry. According to Jaattella [28], cellular shrinkage and membrane damage are the markers of apoptotic cell death caused by anticancer compounds. Correlating these reports with the present findings, we imply that BDMCA coated gold nanocubes easily make way into the breast cancer cell membrane and causes apoptotic cell death. As compared to free gold nanocubes, the BDMCA coated nanocubes may possess enhanced cellular entry. This would have led to the slight differences in the mor-phological changes which are shown in Figure 12B and C. An MTT assay was done in aliquots of cells treated with different doses of BDMCA-functionalized gold nanocubes and free gold nanocubes (50, 75, 100, 125, 150, 175, 200, 225 μg/ml). The relative cytotoxicity is represented in Figure 13. Absorbance of the dye by the cell is directly proportional to the number of viable cells and inversely proportional to the cytotoxicity or anticancer activity of BDMCA-function-alized gold nanocubes. We observed a decrease in absorb-ance in BDMCA-functionalized gold nanocubes treated MCF-7 cells when compared to untreated MCF-7 cells. The concentration of BDMCA-functionalized gold nanocubes that induced 50% cytotoxicity was 100 µg/ml. At the same concentration, the free gold nanocubes induced just 35% cytotoxicity. These findings clearly reveal that the BDMCA-functionalized gold nanocubes exhibit better anticancer efficacy than free gold nanocubes. As the mitochondrial

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Figure 13 Cytotoxicity of gold nanoparticles and bisdemethoxycur-cumin analog (BDMCA) coated gold nanoparticles.

dehydrogenase is the key enzyme that coverts MTT to formazan, we suggest that BDMCA-functionalized gold nanocubes may induce cell death by influencing the mito-chondrial pathway. Our hypothesis is further supported by the morphological analysis shown in Figure 12. Our previ-ous report suggests that BDMCA possesses anticancer activ-ity [29, 30]. We recently reported that BDMCA nanoparticles may also hinder the assembly and function of microtubules of the mitotic spindle in breast cancer cells to exert its anti-cancer effect. We have also reported that BDMCA nanopar-ticles mediate caspase 3 induced DNA fragmentation for promoting apoptosis [18]. The phytochemicals of the Costus pictus plants coated on the surface of the gold nanocubes (as evidenced by the FTIR spectrum) might also contribute to anticancer activity. Therefore, we suggest that BDMCA-functionalized gold nanocubes may synergistically induce breast cancer cell death by: i) cytotoxicity, ii) disorganizing the mitotic spindle, and iii) DNA fragmentation. The activ-ity may be due to the high surface to volume ratio of the gold nanocubes, or due to the phytochemical cap, both of which would have enhanced the activity of BDMCA. Thus, BDMCA-functionalized gold nanocubes could emerge as an anticancer agent against various cancers. Further studies on BDMCA-functionalized gold nanocubes in different cancer models in vivo are underway in our laboratory.

4 Summary and conclusionWe have optimized a facile, novel, rapid and environmen-tally benign green protocol to synthesize gold nanocubes using Costus pictus extract as a reducing and capping agent. Kinetics studies validate the optimization and reli-ability of the green protocol for the production of medici-nally valued gold nanoparticles. The nanocubes were monodispersed with 45 nm size. The nanocubes were functionalized with BDMCA. BDMCA-functionalized gold nanocubes exhibit higher anticancer activity against breast cancer cell lines as compared to unfunctionalized gold nanocubes. The activity may be attributed to: i) the high surface to volume ratio of the gold nanocubes, ii) phytochemical coating on the surface of the nanocubes, which may possess anticancer activity, iii) the BDMCA, which has the capacity to induce cytotoxicity, apoptosis or mitotic spindle disorganization. Thus, we conclude that the green synthesized gold nanocubes that are functional-ized with BDMCA could emerge as an ideal and safe anti-cancer candidate.

Received October 7, 2013; accepted December 8, 2013; previously published online January 20, 2014




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Rajagopal Aruna Devi graduated with a BTech in biotechnology from Anna University, Chennai and obtained her MTech in nanoscience and technology from Anna University, Chennai. Her research areas of interest are nanobiotechnology, green synthesis, and drug deliv-ery. She has presented her research work at various conferences.

Arul Prakash Francis is a research scholar at the Centre for Nanosci-ence and Technology, Anna University, Chennai, India. He obtained his BPharm from Madras Medical College, Chennai and his MTech in nanoscience and technology from Anna University Chennai. His

research areas of interest are nanobiotechnology, nanotoxicology, drug delivery and green synthesis. He has published three articles in reputed journals and presented his research work at various conferences.

Thiyagarajan Devasena is currently working as Associate Professor at the Centre for Nanoscience and Technology, Anna University, Chennai, India. She obtained her PhD in biochemistry from Anna-malai University in 2003. She has contributed to the areas of cancer biology, green chemistry, nanotoxicology, nanobiotechnology and drug delivery. This contribution has led to two patents, four books and 35 publications in renowned research journals with an H-index of 8. As a research guide, Dr. T. Devasena produced 2 PhDs and guided 18 MTech students in various fields. At present, 12 research scholars are pursuing their PhDs under her direct supervision. She is also a member of various professional bodies. She has organized many national and international seminars and workshops. She is honored with various awards and titles.



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Green-synthesized gold nanocubes functionalized with bisdemethoxycurcumin analog as an ideal anticancer candidate