Diastase assisted green synthesis of size-controllable gold nanoparticles

Abstract

Diastase, a natural enzyme, was used for the one pot aqueous synthesis of gold nanoparticles (AuNPs) of tunable size. During the synthetic process, diastase acts concurrently as both a reducing and stabilizing agent, while no additional chemical reagents or surfactants are added. The formation of AuNPs was confirmed by using a UV-visible spectrophotometer, with a characteristic surface plasmon resonance (SPR) band at 530 nm. The size of the diastase-stabilized AuNPs can be easily controlled by changing the quantity of diastase. The produced AuNPs were characterized by using powder X-ray diffraction (XRD), UV-visible spectroscopy, Fourier transform infrared spectroscopy (FT-IR) and transmission electron microscopy (TEM). The FTIR spectrum revealed the capping of diastase on the surface of AuNPs. Furthermore, the formed gold nanoparticles are stable for more than three months. In vitro cytotoxicity studies by MTT assay on HCT116 and A549 cancer cells showed that the cytotoxicity of the as-synthesized Au nanocolloids depends on their size and dose.

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Introduction

Gold nanoparticles have increasingly been paid more attention due to their attractive shape- and size-associated optical, electronic and magnetic properties. Due to these properties, AuNPs have been used in electronics,¹ sensing,^2,3 catalysis⁴ and drug delivery systems.⁵ It is interesting to note that materials in the nanometer range can exhibit a transition between molecular and solid states due to their unique properties, which are ascribed to their surface effects and quantum confinement.⁶

There are two well-known synthetic strategies for the preparation of metal NPs: the top down and bottom up approaches. However, the latter is most effective and common where metal ions are reduced to NPs in the presence of a capping ligand by using a reducing agent. In recent times, there has been a remarkable rise in the biological applications of gold nanoparticles due to their biocompatibility.^7,8 On the other hand, the properties shown by NPs depend on their morphology and dimensions, and hence there is an increase in demand for the size-controlled synthesis of biocompatible NPs.^9,10

Syntheses of biocompatible AuNPs for extensive biomedical applications must be free of the hazardous chemical substances used in other chemical methods. Current trends in the preparation of nanoparticles using green methods have drawn the attention of many researchers due to their environmentally friendly nature, easy preparation mode and exclusion of toxic chemicals and solvents. Several reports have shown the synthesis of nanoparticles using a variety of green reducing agents and microbes.^11–13 Our group has shown the reducing ability of plant extracts for the synthesis of size-controlled gold nanoparticles.¹⁴

Herein we report a green, one pot and size-controlled synthesis of diastase-stabilized gold nanoparticles in aqueous medium without using any additional reducing agent. This work also demonstrates how the size of AuNPs can be tuned by simply changing the quantity of diastase.

Experimental section

Materials

Hydrogen tetrachloroaurate trihydrate (HAuCl₄·3H₂O, 99%) and diastase were purchased from Sigma-Aldrich (Bangalore, India). Diastase powder was solubilised in double distilled water under sonication at 50 °C for 5 minutes and used for further experiments for the preparation of AuNPs. Human lung carcinoma (A549) and human colon carcinoma (HCT116) cell lines were obtained from ATCC (NCCS, Pune, India). Cisplatin (CDDP) was procured from HiMedia, Bangalore, India.

Preparation of AuNPs

0.6 mL of aqueous diastase solution (1%) was added to 2 mL of 0.5 mM HAuCl₄, and the pH was maintained at 12 using NaOH. The mixture was heated for 5 min to obtain a colloid D1. Further, to examine the effect of the quantity of diastase on the size of AuNPs, different volumes of diastase solution (0.5 mL and 0.1 mL) were added to obtain colloids D2 and D3, respectively.

Cytotoxicity evaluation

The cytotoxic effects of the Au nanocolloids D1, D2 and D3 on A549 and HCT116 cancer cells were studied by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with penicillin (100 μg mL⁻¹), streptomycin (100 U mL⁻¹) and 10% heat-inactivated foetal bovine serum (FBS) in tissue culture flasks (T-25) in a humidified atmosphere at 37 °C and 5% CO₂. Cells were seeded one day prior to exposure to AuNPs.

The actively growing A549 and HCT116 cells were seeded at 1 × 10⁴ cells per well in a 96-well microtiter plate and incubated in DMEM/1% FBS with Au nanocolloids D1, D2 and D3 at different volumes (0.4, 0.8, 1.2 and 1.6 mL) for 24 h at 37 °C, 5% CO₂ and a relative humidity of more than 80%. A control experiment was also carried out without AuNPs. Further, to assess the toxicity levels of the AuNPs, the test medium was discarded after 24 h of incubation and cells were incubated again with 20 μL of MTT solution (0.5 mg mL⁻¹ MTT diluted in phenol red-free DMEM without FBS) for 1 h at 37 °C, 5% CO₂. Subsequently, the MTT solution was replaced by 20 μL of DMSO in each well and the optical density was measured by using an ELISA plate reader at 550 nm against a reference at 655 nm. Cell viability for each treatment was calculated as the ratio of the mean OD of 9 replicated wells relative to that of the negative control (only cell culture medium and cells were added). The absorbance values of the negative control and treated cells were used for the determination of the cell viability. The percentage cell viability was calculated by assuming 100% cell viability for the negative control: % cell viability = (OD of test/OD of control) × 100 and expressed as mean percentage ± stdev (n = 6). Alternatively, the cytotoxicity properties of the AuNPs were compared with standard drug Cisplatin (CDDP) as a positive control.

Characterization

The initial characterization of different Au nanocolloids was carried out by using a Jasco V-670 UV-visible double beam spectrophotometer. Spectral data were measured in the range from 200 to 800 nm, while double distilled water was used for blank measurements. Origin 8.1 was used to plot the data obtained. In order to study the size and morphology of the obtained AuNPs, the Au nanocolloids (D1, D2 and D3) were observed by TEM (JEOL JEM 2100 HR-TEM) at an operating voltage of 200 kV. Samples were prepared by diluting 1 mL of colloid to 10 mL with distilled water and ultrasonicating for 5 minutes. A drop of the resulting mixture was placed on a lacey copper grid with ultrathin Cu on porous carbon film and was further allowed to dry under vacuum. The fully dried grids were then used for TEM analysis, and a simultaneous measurement of selected area electron diffraction (SAED) was carried out. The solid AuNPs for XRD were obtained by centrifuging the colloids of D1 at 15 000 rpm. The X-ray diffraction (XRD) study of the AuNP powder was carried out at room temperature using a Bruker D8 Advance diffractometer with Cu Kα radiation (λ = 1.54 Å) over the angle range from 10° to 80°. The instrument was operated with a scanning rate of 4° min⁻¹ and a step size of 0.02°, and was calibrated with lanthanum hexaboride (LaB₆) before the sample analysis. Purified AuNP powder was analysed by using Attenuated Total Reflectance-FTIR (ATR-FTIR) spectroscopy (JASCO ATR-FTIR 4100). To study the surface functionalization, diastase was used as a control. Dynamic light scattering (DLS) analysis for the diastase-reduced Au nanocolloids was carried out by using a Horiba Scientific Nanoparticci (SZ-100) instrument.

Results and discussion

In this work a natural enzyme, diastase, was used as a bioreductant to prepare AuNPs of different sizes. The aqueous solution of diastase plays a key role in the reduction and capping of the formed AuNPs. The effect of the change in the quantity of diastase on the reduction time, size and morphology of AuNPs is discussed in detail. The rapid reaction rate is due to the presence of NaOH, which as a strong base causes the fast hydrolysis of chloroauric acid and increases the rate of reaction.¹⁵ Although various amino acid residues of proteins have the ability to reduce gold ions to nanoparticles, the kinetics of gold ion reduction at room temperature by proteins are very slow.¹⁶ However, it has been reported that enzymes with freely exposed thiol groups have the ability to catalyse the synthesis of gold nanoparticles, while others do not.¹⁷ Since diastase contains two free thiol groups in its cysteine residue, these could be involved in the synthesis of AuNPs and confer them further stability.¹⁷ Thiol group-stabilized nanoparticles have already been reported.¹⁸

Initial conversion of Au⁺³ to Au⁰ was observed with a change of colour from yellow to ruby red, and this was monitored by UV-visible absorption spectroscopy. The intense colour of the AuNPs was due to surface plasmon resonance. The appearance of an intense absorption band at 530 nm in the UV-visible spectrum provided additional confirmation of the reduction, as AuNP colloids are known to reveal a distinct absorption band between 500–600 nm in the visible region.

To determine the effect of the quantity of diastase on the bioreduction process, the volume of diastase in the synthetic mixture was changed while that of HAuCl₄ (0.5 mM) was kept constant. From Fig. 1, it is evident that a blue shift in the SPR was observed with an increase in the quantity of diastase, and the absorption maxima for Au nanocolloids D1, D2 and D3 were found at 530 nm, 540 nm and 570 nm, respectively. It is well known that the SPR band is highly sensitive to the properties of the particles (size and shape), the distance between neighboring NPs, the refractive index of the medium and the environment in which the nanoparticles are dispersed. The blue shift observed with the increase in the quantity of diastase is indicative of the formation of smaller NPs (also confirmed by TEM). Fig. 2 represents the size distribution of nanocolloids D1, D2 and D3 obtained by using different volumes of diastase. It can be seen that a reduction in the size of nanoparticles was obtained with an increase in the quantity of diastase.

Fig. 1 UV-visible spectra of AuNPs synthesized using different quantities of diastase.

Fig. 2 Average particle sizes obtained for Au nanocolloids D1 (A), D2 (B) and D3 (C).

Further, to determine the effect of pH on the size of the NPs, the synthesis of AuNPs was carried out using 0.6 mL of diastase at various pH values. Fig. 3 shows the role of pH in tuning the size of the AuNPs. It is interesting to note that the size of the NPs increases with a decrease in pH from 12 to 2. The formation of smaller nanoparticles with a diastase solution of pH 12 is indicated by the SPR absorption of the nanocolloid at 520 nm.

Fig. 3 UV-visible spectra of AuNPs synthesized using diastase at different pH values.

However, this peak is red shifted to 590 nm for the nanocolloid synthesized with diastase at pH 2, and no NPs were formed when using diastase at pH 1. On the other hand, anisotropy is observed for the colloids prepared with diastase solutions between pH 10 and pH 4, which is confirmed by the appearance of one extra peak between 650 and 800 nm (Table S1†). This may be due to the decreased availability of thiol groups in anionic form (–S⁻) in the diastase with a decrease in the pH of the environment. Although the number of –SH groups is greater in acidic medium, the extent of capping is less because of the reduced availability of –S⁻ groups in the acidic medium for capping the formed Au nuclei, and vice versa.

Additionally, to examine the variations in morphology and size with changing quantities of diastase, the prepared Au nanocolloids (D1, D2 and D3) were subjected to TEM studies. The TEM images at different magnifications for gold nanocolloids D1, D2 and D3 along with their SAED patterns are presented in Fig. 4. TEM images at different magnifications for D3 nanocolloids are shown in Fig. 4A and B. From the TEM analysis, the abundance of hexagon and blunted angle triangular shaped AuNPs is clear, with an average particle size of 148 nm. However, the presence of tail-like structures attached to the sharp edges of nanoplates may be an indication of surface capping of the nanoplates with diastase. TEM images of nanocolloid D2 are presented in Fig. 4D and E, and the images also show the presence of triangles and hexagons along with a greater number of spherical particles, with moderately smaller sizes compared to nanocolloid D3 – the average particle size was 23.2 nm. On the other hand, the TEM images of colloid D1 shown in Fig. 4G and H demonstrate a rich abundance of spherical AuNPs with smaller sizes (average particle size of 9.7 nm) when compared to D2 and D3. From the TEM results, it is clear that uniformity in the size and shape of the NPs can be obtained by increasing the quantity of diastase. The SAED patterns of the nanocolloids (Fig. 4C, F and I) reveal the polycrystalline nature of colloids D1 and D2 and the single crystalline nature of colloid D3, and the corresponding pattern of D2 was indexed to the (111), (200), (202), (311) and (222) reflections. From the TEM analysis, it was concluded that there is a decrease in the particle size of the AuNPs with an increase in the quantity of diastase. However, anisotropy can be overcome by raising the quantity of diastase to obtain uniform and smaller spherical NPs (with 0.6 mL of diastase). It was observed from UV-visible studies that nanocolloids D1 and D2 were stable for more than three months, but nanocolloid D3 was stable for a week (data not shown).

Fig. 4 TEM images and SAED patterns of Au nanocolloids D3 (A–C), D2 (D–F) and D1 (G–I).

The mechanism of size and shape tuning of the Au nanocolloids is shown in Fig. 5. During the synthesis, the development of blunted Au nanotriangles and hexagons was favored when the quantity of diastase was 0.1 mL. In contrast, the formation of spherical nanoparticles was observed when the quantity of diastase was increased to 0.5 and 0.6 mL, indicating the role of diastase as a protective controller for the formation of spherical nanoparticles by reducing Ostwald ripening and favoring a diffusion-controlled process. At low quantities of diastase, the rate of nucleation is much faster, while the rate of coating of AuNPs with diastase is relatively slow, resulting in the formation of large blunted triangles. As the quantity of diastase is increased, the rate of coating gradually increases when compared with the nucleation rate, leading to the effective formation of small spherical AuNPs. Since the diastase enzyme contains two free thiol groups that are arranged spatially close to each other, there is a strong possibility of attachment of the formed nanocrystals to both the thiol groups of diastase.¹⁷ Since the size of the diastase is much smaller than the size of the NPs, the formation of dense shell-like structures with enzymes linked through intermolecular interactions around spherical nuclei is possible.¹⁹ In the case of colloid D1, the nanocrystals that are formed initially are well-capped by diastase shells and are highly stable. In contrast, the gold nanoparticles that are formed later are less protected by diastase and are less stable. Due to room temperature coalescence, the rapid reduction and assembly of these spherical nanoparticles leads to the development of new anisotropic structures such as nanotriangles with high surface energy. The formed nanotriangles undergo a shrinking process in order to minimize the surface energy, resulting in the formation of blunted nanotriangles.¹⁴ Similar results have been reported when honey²⁰ and Cinnamomum camphora leaf extract²¹ were used for the synthesis of AuNPs.

Fig. 5 Mechanism showing how diastase is involved in the reduction and tuning the size and shape of AuNPs.

A plausible mechanism for the formation of AuNPs using diastase could be through the interaction of the thiol moieties of cysteine residues present in diastase with the metal. Since the diastase contains two free surface-exposed thiol groups, these could react with the Au salt and form NPs. The reducing ability of proteins is due to the presence of reducing amino acids (cysteine and histidine) in their structures.²² However, histidine is active only in an organic environment due to its tertiary amine group.²³ Hence, the free thiol groups present in the diastase are responsible for the reduction and stabilization. The mechanism involves the release of electrons and protons from the free thiol groups of the diastase for conversion of Au⁺³ to Au⁰, and the resulting sulphur atoms contribute to the formation of disulphide bridges. The gold nuclei formed are immediately stabilized by the leftover free –SH groups of the diastase through formation of Au–S bonds, as thiol groups have a high affinity towards gold.¹⁷

Fig. 6 shows the XRD pattern of the purified and dried AuNPs. The diffraction peaks at 38.25, 44.48, 64.7 and 77.7 correspond to the (111), (200), (202), (311) and (222) planes of cubic AuNPs, respectively (JCPDS no. 96-901-1614).

Fig. 6 XRD pattern of AuNPs obtained from nanocolloid D2.

The surface capping of diastase molecules on the AuNPs could also be confirmed by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectra of native diastase and the corresponding AuNPs are shown in Fig. 7. For pure diastase, the presence of a band at 1650 cm⁻¹ indicates the alpha helix structure of the diastase.

Fig. 7 FTIR spectra of pure diastase (black) and AuNPs (red) obtained from colloid D1.

The FTIR spectrum of the dried AuNPs shows the presence of bands similar to those for native diastase, indicating the presence of diastase on the surface of the AuNPs. The shifting of bands corresponding to the C=O stretch of the peptide bond from 1650 cm⁻¹ for the native diastase to 1631 cm⁻¹ for the AuNPs after reduction indicates the change in the secondary structure of the enzyme capped on the surface of the AuNPs.^24,25 All these results indicate the surface stabilization of the AuNPs with the diastase enzyme.

In vitro cytotoxicity

An MTT assay was performed to determine the biocompatibility and potential of the AuNPs for biomedical applications. The biocompatibility of the AuNPs towards human gastrointestinal cancer cells such as Panc-1, HepB3 ²⁶ and HeLa cells⁷ was studied. Shukla et al. and Connor et al. have reported the non-toxic nature of citrated and biotinylated AuNPs towards leukemic cells (K562), while smaller AuNPs were more toxic.^7,27 In a few reports, Au nanorods were found to be toxic when compared to spherical AuNPs, which were almost non-toxic.^28–33 This is due to the dependence of cytotoxicity on the shape, size and surface modification of NPs. On the other hand, the effect of the size of AuNPs on in vitro cytotoxicity in HeLa cells has already been proved. Indeed, Hauck et al.³⁴ and Pan et al.³⁵ have shown that the size of the particle, and not the capping agent, is responsible for determining the toxicity of AuNPs. Moreover, there have been no systematic studies comparatively analyzing the size and dose dependent cytotoxic behaviour of AuNPs synthesized using diastase towards two different cell lines.

The comparative cytotoxic behaviour of Au nanocolloids D1, D2 and D3 was analysed for two different cancerous cell lines – HCT116 and A549. Fig. 8A and B represent the cell viability data for Au nanocolloids D1, D2 and D3 and their comparative evaluation against positive and negative controls. From Fig. 8, it can be concluded that smaller AuNPs were found to be more toxic for both cell lines compared to the larger ones. It was observed that cell viability was greater when treated with larger AuNPs, and A549 cells showed greater viability for all the three different sizes compared to HCT116. Notably, biocompatibility is observed below 30 ppm for Au nanocolloid D1 and 35 ppm for Au nanocolloids D2 and D3 for both cell lines. Furthermore, to determine the effect of capping agents on cell viability, a blank MTT assay was carried out with diastase. However, no significant toxicity was found with diastase, indicating the negligible role of the capping agent in the toxicity of AuNPs. Additionally, a decline in the cell viability is observed with an increase in the concentration of AuNPs. The half maximal inhibitory concentration (IC₅₀) values of Au nanocolloids D1, D2 and D3 are 107.5, 139.6 and 166.6 mg L⁻¹, respectively. All these results confirm that the toxic effects of the as-prepared Au nanocolloids are size and dose dependent.

Fig. 8 Cell viability of A549 (A) and HCT116 (B) cell lines induced by diastase-reduced AuNPs.

Conclusions

In this paper, we have shown the efficiency of diastase as a reducing and stabilizing agent for the size-controlled synthesis of AuNPs. The size distribution of the resulting AuNPs can be effectively tuned by changing the quantity of diastase used. The availability of thiol groups in the enzyme may play a key role in tuning the size and shape of the AuNPs. Additionally, the biocompatibility of diastase may lend the synthesized AuNPs towards biomedical applications. In vitro cytotoxicity studies showed that the toxic effects of the as-prepared Au nanocolloids towards HCT116 and A549 cancer cell lines are size and dose dependent.

Acknowledgements

SBM greatly acknowledges VIT University, Vellore-632014, India for the financial help and platform given to do this research. Prof. C. Ramalingam, Dean of SBST-VIT University, acknowledges VIT University, Vellore, India for providing the RGEMS-VC-Fund to carry out research for the nano-food research group (NFRG). NFRG acknowledges the Department of Biotechnology (DBT, India) for the project under consideration with permanent project number BT/PR10414/PFN/20/961/2014. NFRG wishes to acknowledge Veer Kunwar Singh Memorial Trust, Chapra, Bihar, India for partial support – VKSMT/SN/NFNA/2014/014.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra03117f

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