Rhamnolipids functionalized AgNPs-induced oxidative stress and modulation of toxicity pathway genes in cultured MCF-7 cells

https://doi.org/10.1016/j.colsurfb.2015.05.034Get rights and content

Highlights

  • Biosurfactant rhamnolipids were extracted from P. aeruginosa culture extract.

  • Non-toxic concentration (0.005%) of rhamnolipids was used for synthesis of Rh-AgNPs.

  • Rh-AgNPs exhibited more cytotoxicity to MCF-7 cells than normal PBMN cells.

  • Rh-AgNPs caused ROS generation, membrane damage and apoptosis in MCF-7 cells.

  • Oxidative stress pathway genes are implicated in Rh-AgNPs induced MCF-7 cells death.

Abstract

Rhamnolipids extracted from Pseudomonas aeruginosa strain JS-11 were utilized for synthesis of stable silver nanoparticles (Rh-AgNPs). The Rh-AgNPs (23 nm) were characterized by Fourier transform infra-red (FTIR) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The cytotoxicity assays suggested significant decrease in viability of Rh-AgNPs treated human breast adenocarcinoma (MCF-7) cells, compared with normal human peripheral blood mononuclear (PBMN) cells. Flow cytometry data revealed 1.25-fold (p < 0.05) increase in the fluorescence of 2′,7′-dichlorofluorescein (DCF) at 0.25 μg/mL. However, at Rh-AgNPs concentrations of 0.5 and 1.0 μg/mL, much lesser fluorescence was noticed, which is attributed to cell death. Results with the fluorescent probe Rh123 demonstrated change in inner mitochondrial membrane and dissipation of membrane potential. The cell cycle analysis suggested 19.9% (p < 0.05) increase in sub-G1 peak with concomitant reduction in G1 phase at 1 μg/mL of Rh-AgNPs, compared to 2.7% in untreated control. The real-time RT2 Profiler™ PCR array data elucidated the overexpression of seven oxidative stress and DNA damage pathways genes viz. BAX, BCl2, Cyclin D1, DNAJA1, E2F transcription factor 1, GPX1 and HSPA4, associated with apoptosis signaling, proliferation and carcinogenesis, pro inflammatory and heat shock responses in Rh-AgNPs treated cells. Thus, the increased ROS production, mitochondrial damage and appearance of sub-G1 (apoptotic) population suggested the anti-proliferative activity, and role of oxidative stress pathway genes in Rh-AgNPs induced death of MCF-7 cancer cells.

Introduction

Development of a reliable, eco-friendly and toxicity-free synthesis of metal nanoparticles (NPs) is an important aspect of nanotechnology research [1]. Lately, the biomimetic and green synthesis of AgNPs using polymer matrices such as starch [2], chitosan [3], cyclodextrins [4], and microbial biomass [5], [6], [7] has been extensively pursued. Microbial synthesis of NPs yields stable particles due to protein capping and interaction with other reducing agents such as nitrate reductase [8], naphthoquinones [9], and anthraquinones [10], secreted by the organisms. Furthermore, synthetic chemicals such as amine and carboxylate surfactants [11], cationic cetylpyridinium, or anionic sodium dodecyl sulphate, or non-ionic Brij 56 [12] have also been used for NPs synthesis. These surfactants are tension-active molecules, amphipathic in nature with both hydrophilic and hydrophobic moieties, and exhibit surface-active properties. With the increasing demand for greener bioprocesses and novel enhancers for NPs synthesis, the biosurfactants, and/or biosurfactant producing microbes are emerging as an alternate source. Thus, biosurfactants with the high surface activity and low critical micelle concentrations (CMC) are regarded as promising substitutes for synthetic surfactants [13]. Several microorganisms like bacteria, fungi, yeasts, and algae are good sources of biosurfactants and offer many advantages over their chemical counterparts. Therefore, the biosurfactant mediated synthesis of NPs is regarded as a clean, non-toxic, and environmentally acceptable “green chemistry” procedure, resulting in reduced NPs aggregation and uniform morphology. Furthermore, the lower toxicity, higher biodegradability, better environmental compatibility, higher foaming, high selectivity and specific activity at extreme temperatures, pH, and salinity [14] are some added advantages over the chemical surfactants.

In this context, the natural rhamnolipids, a subclass of glycolipids produced by bacteria, could serve as simple and economical stabilizer for AgNPs synthesis. Rhamnolipids from Pseudomonas aeruginosa strain BS-161R and its mutant EBN-8 have earlier been used for synthesis of silver NPs (AgNPs) in reverse micelles and composite rhamnolipids-gold NPs microtubules [15], [16]. Pseudomonas species are well known for their capability to produce rhamnolipid biosurfactants on different carbon sources [17], [18]. This has prompted us to develop a simple one-pot method for synthesis of highly stable and dispersible Rh-AgNPs. In this study, we report the role of natural rhamnolipids extracted from the culture supernatant of P. aeruginosa strain JS-11, as a stabilizing agent in synthesis of AgNPs. The synthesized Rh-AgNPs were characterized using the analytical techniques, viz., UV-visible spectrophotometry, XRD spectroscopy, TEM, FTIR and AFM. Rh-AgNPs induced antiproliferative activity and involvement of oxidative stress and toxicity pathways genes have been investigated in human breast adenocarcinoma (MCF7) cells, as an in vitro model.

Section snippets

Bacterial strain characterization and screening for rhamnolipid production

The soil bacteria P. aeruginosa strain JS-11 has been obtained from culture collection of our laboratory [19]. The strain JS-11 was screened for rhamnolipids biosynthesis using the mineral salt-CTAB-methylene blue agar plates, following the method of Inka and Fritz [20]. Briefly, the cell-free culture supernatant (30 μL) was loaded into each pre-cut wells in methylene blue agar plate. The plate was then incubated at 37 °C for 72 h. A dark blue halo zone around the culture was considered as

Synthesis and stability of Rh-AgNPs

The rhamnolipids released by P. aeruginosa strain JS-11 in LB and mineral salt medium containing 4% glucose resulted in decreased surface tension of the medium from 69 mN m−1 to 31 mN m−1, which confirms the presence of biosurfactant in the culture medium. This corroborates with the observations of Rahman et al. [27]. The concentration of the extracted rhamnolipids from the strain JS-11 was determined to be 325 mg/L. The rhamnolipids emulsion in Milli Q water (Supplementary Fig. 1) has been used as

Conflict of interest

There is no conflict of interest.

Acknowledgments

The Chair for DNA research, King Saud University, Riyadh, for this study, is greatly acknowledged. JM is also grateful to the Visiting Professor Program (VPP), King Saud University for all support to carry out this collaborative research.

References (43)

  • J.P. Saikia et al.

    Possible protection of silver nanoparticles against salt by using rhamnolipid

    Colloids Surf. B: Biointerfaces

    (2013)
  • S. Dwivedi et al.

    Synthesis characterization and toxicological evaluation of iron oxide nanoparticles in human lung alveolar epithelial cells

    Colloids Surf. B: Biointerfaces

    (2014)
  • C.G. Kumar et al.

    Extracellular synthesis of silver nanoparticles using culture supernatant of Pseudomonas aeruginosa

    Colloids Surf. B: Biointerfaces

    (2011)
  • T.R. Daniels et al.

    The transferrin receptor part I: biology and targeting with cytotoxic antibodies for the treatment of cancer

    Clin. Immunol.

    (2006)
  • B. Thanomsub et al.

    Chemical structures and biological activities of rhamnolipids roduced by Pseudomonas aeruginosa B189 isolated from milk factory waste

    Bioresour. Technol.

    (2006)
  • S. Dwivedi et al.

    Butachlor induced dissipation of mitochondrial membrane potential, oxidative DNA damage and necrosis in human peripheral blood mononuclear cells

    Toxicology

    (2012)
  • S. Jaiswal et al.

    Enhancement of the antibacterial properties of silver nanoparticles using beta-cyclodextrin as a capping agent

    Int. J. Antimicrob. Agents

    (2013)
  • S. Dwivedi et al.

    Biomimetic synthesis of selenium nanospheres by bacterial strain JS-11 and its role as a biosensor for nanotoxicity assessment: a novel se-bioassay

    PLoS ONE

    (2013)
  • S. Anil Kumar et al.

    Nitrate reductase mediated synthesis of silver nanoparticles from AgNO3

    Biotechnol. Lett.

    (2007)
  • P. Mukherjee et al.

    Fungus mediated synthesis of silver nanoparticles and their immobilization in the mycelial matrix: a novel biological approach to nanoparticle synthesis

    Nano Lett.

    (2001)
  • A.S. Malik et al.

    Mesostructured iron and manganese oxides

    J. Mater. Chem.

    (2003)
  • Cited by (0)

    View full text