Method development for optimised green synthesis of gold nanoparticles from Millettia pinnata and their activity in non-small cell lung cancer cell lines

Abstract

Green synthesis of gold nanoparticles (GNPs) has received substantial attention, because nanoparticles are produced in an eco-friendly way using biomolecules present in plant extracts in a single step reaction. This research article highlights GNPs obtained using shade-dried leaf extracts of Millettia pinnata (L.) with aqueous auric chloride (HAuCl₄) at ambient temperature. In the present study, GNPs with average particle size 37 nm in size were fabricated. Furthermore, the synthesis method to obtain stable and monodispersed GNPs was advanced by optimising enzyme concentration 100 μg/ml, pH 5.4, substrate concentration 0.45 mM and 12 h time of reaction. The confirmation of GNPs formation and characterisation was followed by UV-vis-absorption spectroscopy, dynamic light scattering (DLS), and zeta potential (ZP) for the analysis of shape, size, and stability, respectively. TEM images and powder XRD revealed the GNPs synthesis of spherical-shaped nanoparticles in the face-centred cubic arrangement. Cytotoxicity of GNPs was studied against A549 lung cancer cells with IC₅₀ 14.76 μg/ml and found lower as compared to doxorubicin IC₅₀ 11.23 μg/ml but significant enough to be used as a vehicle GNPs produced using green source can be used as significant therapeutic agents and drug delivery carriers.

Fig. 1

Plant protein quantification and enzymatic activities (a) Protein concentration of enzyme extracted from the different plant using Bradford protein assay; (b) Ascorbic oxidase activity confirming maximum activity by M. pinnata leaf extract; (c) Peroxidase activity displayed maximum activity by M. oleifera leaf extract. The enzyme was extracted from plant leaves with 100 mM phosphate buffer containing 1% polyvinylpyrrolidone and at pH 7.4

Fig. 2

Visual colour change observation of synthesized GNPs and Surface plasmon resonance (SPR) of GNPs (a) Colour change after GNPs synthesis; (b) Spectrophotometry of C. procera (L.) – Yellow, M. oleifera (Lam.) – blue, M. pinnata (L.) – Black (Best suitable GNPs, selected on the basis of spectra range for its application on drug delivery), B. alba (L.) – Red, E. neriifolia (L.) – Pink; (c) Spectrophotometry of GNPs (a) plant enzyme concentration – 50 μg/ml Black, 100 μg/ml Pink, 150 μg/ml Blue, 200 μg/ml Green, 250 μg/ml Red; (b) pH 5.4 Red, pH 6.4 Black, pH 7.4 Pink, pH 8.4 Green, pH 9.4 Blue; (c) Gold concentration – 0.015 mM Blue, 0.03 mM Black, 0.15 mM Pink, 0.3 mM Red, 0.45 mM Green; (d) Sampling time – 1 h Fluorescent Green, Sample 2 h Green, Sample 3 h Yellow, Sample 4 h Red, Sample 5 h Pink, Sample 6 h blue, Sample 12 h black

Fig. 3

Cell viability assay of GNPs synthesised in various conditions shown potentiality and change in their effectiveness when synthesised in different conditions (Doxorubicin is used as a standard). (a) GNPs synthesised at different concentration (µg/mL) of enzyme; (b) GNPs synthesised at different pH of enzyme; (c) GNPs synthesised at different concentration (mM) of HAuCl₄; (d) GNPs synthesised at regular time interval of 1, 2, 3, 4, 5, 6 and 12 h

Fig. 4

Apoptosis is visible in apoptosis study from GNPs synthesised in most optimised condition. Acridine orange/Ethidium bromide staining of (a) Living cells, (b) Early apoptotic cells, and (c) Late apoptotic. Nuclear fragmentation can be visualised

Fig. 5

Microscopic images of synthesized GNPs for structural determination (a) Spherical shapes of the GNPs are clearly visible in TEM micrographs; (b) Spherical shape is confirmed in Atomic Force Microscopy with three dimensional Visualisation and Topographic view

Fig. 6

Size and nature determination of GNPs (a) XRD of GNPs; crystalline FCC structure is confirmed using Bragg's angle analysis. (b) DLS of GNPs for size determination. GNPs synthesised are 100 nm of average size thus suitable for use in biomedical applications

Fig. 7

Stability determination of GNPs (a) Spectrum scan of GNPs after 3 months for stability; (b) Zeta potential of freshly synthesised GNPs; (c) Zeta potential of GNPs after 3 months. Zeta potential confirms that synthesised GNPs are not agglomerating and stable in nature

Fig. 8

FTIR and proposed chemical mechanism involved in nanoparticle synthesis (a) FT‐IR spectrum of GNPs synthesised by M. pinnata (L.) leaf extracts (a – Ascorbic acid; b – GNPs after wash; c – GNPs before wash); (b) The chemical mechanism involved in the reduction of HAuCl₄ into Au^o nanoparticle