Biocompatible synthesis of peptide capped copper nanoparticles and their biological effect on tumor cells
Highlights
► Protein capped copper nanoparticles were obtained by simple and rapid biosynthetic approach. ► In vitro Cytotoxicity of nanoparticles assessed in human lung carcinoma cells. ► Elevated indices of oxidative stress and cellular damages in A549 cells were recorded. ► This biocompatible nanoparticle formulation effective as delivery system against cancer cells.
Introduction
The synthesis of metallic nanoparticles is an active area of academic and, more importantly, “application research” in nanotechnology. A variety of chemical and physical procedures have been used for synthesis of metallic nanoparticles [1]. However, these methods are accompanied by many problems including use of toxic solvents, generation of hazardous by-products, and high energy consumption. Accordingly, there is an essential need to develop environmentally benign procedures for synthesis of metallic nanoparticles. A promising approach to achieve this objective is to exploit the array of biological resources in nature [2], [3]. Indeed, over the past several years, plants, algae, fungi, bacteria, and viruses have been used for production of low-cost, energy-efficient, and nontoxic metallic nanoparticles [4], [5]. The main disadvantage of this process is it is very tedious to maintain cell culture. To overcome this drawback plant extracts were used for the synthesis of nanoparticles [6], [7].
Some metallic NPs are showing increased toxicity, even if the same material is relatively inert in its bulk form (e.g., Ag, Au, and Cu). NPs also interact with proteins and enzymes within mammalian cells and they can interfere with the antioxidant defense mechanism leading to reactive oxygen species generation [8]. The initiation of an inflammatory response and perturbation and destruction of the mitochondria can cause apoptosis (programmed cell death) or necrosis. Toxicity of metal nanoparticles also depends on the capping agent. For instance, chitosan loaded copper nanoparticles [10] and Ag-Dendrimer nanocomposites [12] were reported to exhibit cytotoxicity while Ag-polysaccharide nanocomposites were shown to be non-cytotoxic [11]. As compared to metals like Ag and Au cytotoxicity of Cu has been less studied. This cytotoxic activity of nanoparticles (NPs) can be utilized to target tumor cells [9].
However, there are significant challenges with regard to the delivery of NPs at the cellular levels and in vivo stability. One of the approaches for improving the in vivo stability, circulation lifetime, and cellular uptake of NPs is to incorporate the particles into or on the surface of liposomes [13], [14]. Another approach is capping the NPs with non-toxic biocompatible macromolecules like polysaccharides or proteins [15], [16].
In this work, an easy and rapid synthesis of copper nanoparticles capped with peptides present in the latex of plant Euphorbia nivulia (Euphorbiaceae) is reported. The plant commonly known as ‘Patrasnuhi’ has innumerable medicinal applications [17] including antimicrobial activity. The latex-capped-nanoparticle formulation being biocompatible was directly used for in vitro cytotoxicity studies in human lung carcinoma cell line (A549) with an aim to assess the suitability in induction of in vitro cytotoxicity at various doses. Possible cytotoxic mechanisms were evaluated using specific parameters for cytotoxicity and oxidative stress viz. cell viability assay (MTT), extracellular lactate dehydrogenase (LDH) release, reactive oxygen species (ROS) generation, intracellular reduced glutathione (GSH) content, malondialdehyde (MDA) content as a indicator of membrane lipid peroxidation, superoxide generation and acridine orange/ethidium bromide (AO/EB) staining for evaluation of cell death were performed in the dose range of 1–100 μg ml−1 of LCuNPs [18], [19].
Section snippets
Materials
Copper nitrate (Cu (NO3)2·3H2O), and ascorbic acid were purchased from Merck Mumbai, India. All the solutions were prepared using double-distilled deionised water. Crude milky white latex obtained from the green stem of E. nivulia was stored under refrigeration at −20 °C.
Cell culture
Human lung carcinoma cells (A549, passage no. 10) were obtained from National Centre for Cell Sciences, Pune, India), seeded (1.0 × 105 cells ml−1 in T-25 Flask) and cultured in DMEM (Himedia, India Pvt. Ltd.) containing 10% FBS
Characterisation of LCuNPs
Fig. 1A shows the digital photograph of LCuNPs synthesized with different precursor concentration. It can be seen that the red color deepens with increasing precursor concentration. At the same time there is a nominal increase in size as seen from the results of DLS (Fig. 1B) which also showed a single narrow distribution with average particle size in the range of 12–16 nm. A representative TEM image (Fig. 2A) shows the particles observed are spherical with average particle size of 5–15 nm. This
Conclusion
Simple, biosynthesis of copper nanoparticles was successfully carried out in a short span in aqueous solution of plant latex. The penetration of LCuNPs in the cell membrane was clearly revealed by dark field and fluorescent microscopy imaging. The dose dependent cytotoxicity of the as synthesized LCuNPs against lung carcinoma cells was evident in the form of gross structural damages and elevated indices of oxidative stress in a dose dependent manner. Thus, it can be inferable form this study
Acknowledgements
The authors are grateful to the University Grants Commission, New Delhi for financial assistance. Special thanks to Dr. P.S. Nagar for providing Euphorbia nivulia latex for the investigation and Dr. Geeta S. Padate, Head, Department of Zoology for her encouragement.
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