Towards plant-mediated chemistry – Au nanoparticles obtained using aqueous extract of Rosa damascena and their biological activity in vitro

https://doi.org/10.1016/j.jinorgbio.2020.111300Get rights and content

Highlights

  • Controlled synthesis of Au nanoparticles (NPs) with Rosa Damascena extract.

  • Plant-mediated synthesis results in stable and monodisperse colloids.

  • Selective cytotoxicity towards cancer cells over peripheral blood mononuclear lymphocytes.

  • Attractive hybrid materials combining metal NPs platform and plant-derived metabolites.

Abstract

An eco-friendly, efficient, and controlled synthesis of gold nanoparticles with application of the aqueous extract of Rosa damascena (Au@RD NPs) without using any other reducing agents was studied. Au@RD NPs of narrow size distribution were characterized by UV–vis and FT-IR spectroscopies, transmission electron microscopy, X-ray powder diffraction, X-ray photoelectron spectroscopy, particle size analysis, and zeta potential measurements. In vitro stability experiments revealed that the Au@RD NPs were stable for over a year (pH ~ 3.5), proving a significant stabilizing potential of the aqueous RD extract. The high total content of polyphenols, flavonoids, and reducing sugars along with the powerful antioxidant activity of the RD extract was determined by spectroscopic and analytical methods. Colloids prepared from the purified and lyophilized Au@RD NPs (electrokinetic potential of ca. -33 mV) were stable for at least 24 h under terms similar to physiological conditions (pH = 7.4, PBS). The in vitro cytotoxicity of Au@RD NPs was investigated against peripheral blood mononuclear lymphocytes (PBML), acute promyelocytic leukemia (HL60), and human lung adenocarcinoma (A549). Selective cytotoxicity of Au@RD NPs towards cancer cells (HL60, A549) over normal cells (PBML) in vitro was explicitly demonstrated by viability assays. Comet assay revealed a higher level of DNA damages in cancer cells when compared with normal ones. Apoptotic death in cancer cells was proved by measuring caspases activity. Thus, the developed Au@RD NPs, obtained by the plant-mediated green synthesis, are attractive hybrid materials for the medical applications combining two active components – metal nanoparticles platform and plant-derived metabolites.

Graphical abstract

Eco-friendly, efficient, and controlled synthesis of gold nanoparticles with application of the aqueous extract of Rosa Damascena (Au@RD NPs) resulted in the stable colloid exhibiting selective cytotoxicity towards acute promyelocytic leukemia (HL60) and human lung adenocarcinoma (A549) over peripheral blood mononuclear lymphocytes (PBML) in vitro.

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Introduction

Green chemistry-based synthesis of metal nanoparticles (NPs) is becoming now an increasingly popular eco-friendly method, even instead of chemical synthesis. In particular, plant extracts as reducing and stabilizing agents are progressively used to synthesize metal NPs [[1], [2], [3], [4], [5]]. The interest in their application is great due to the relative ease of synthesis, good control of sizes and shapes, optical characteristics, and high biocompatibility of the resulting NPs.

Gold nanostructures are of a great interest for scientific research because of their wide applications in photonics, electronics, catalysis, optical sensing and imaging, and drug delivery [[6], [7], [8]]. These remarkable applications are built upon the surface plasmonic resonance (SPR) phenomenon that originates from the collective oscillation of conduction electrons relative to the lattice of gold ions [9]. The physicochemical and biological characteristics of Au nanostructures varies as a function of their size, shape, hollowness/porosity, and other properties. Variety of different shapes including nanospheres, nanorods, nanoshells, and nanocages is possible to obtain and various synthetic strategies have recently been proposed in the literature and successfully implemented in laboratories [1,4,[10], [11], [12]]. Importantly, apart from the above-mentioned hallmarks stability and reactivity can also be nowadays precisely controlled [13]. Based on various in vitro studies it is well-documented that overall toxicity exhibited by anisotropic Au NPs can be derived from capping agents or ligands used in their synthesis [12]. However, this is primarily the case of syntheses using toxic organic reagents as capping, reducing and/or stabilizers agents. In such cases, there is concern about inaccurate purification of the samples in the post-treatment or during dialysis exchanging surface ligands. Thus, the remarkable SPR properties together with biocompatibility of gold make its nanostructures promising both as a contrast agent for in vivo optical imaging, and as a therapeutic agent for chemotherapeutic and/or photothermal treatment of cancer [14].

Despite a wide scope of possibilities offered by a plant-mediated synthesis, this mode of synthetic protocols is still in its infancy, when considered in-depth understanding of the actual course of the reduction, nucleation, and growth of nanoparticles. In most of the cases reported in the literature to date, there is a little attention paid to elucidation of the mechanistic pathway. Reduction of metal ions is mostly explained by a mixture of several different compounds. To the best of our knowledge, there are very few reports focused on precise explanation of synthetic route. For instance, Ahmad et al. suggested that free reactive hydrogen generated during the tautomeric transformation from the enol-form to the keto-form of flavonoids (i.e., luteolin and rosmarinic acids) is responsible for metal ions reduction [15]. In the case of flavonoids, other reduction scenario is also plausible – the internal mechanism of the conversion of ketones to carboxylic acids. The carbonyl groups or any π-electrons of flavonoids are able to chelate metal ions and then reduce them. This has been proven by Rajendiran et al. on the example of quercetin capable of reduction of Au3+ ions [16]. Furthermore, another mechanism can be connected with oxidation of hydroxyl groups of polyphenols to carbonyl groups or quinones, when metal ions are bioreduced [[17], [18], [19]]. Alternatively, in the case of terpenoids, the reducing potential is connected with the hydroxyl groups and their proton-releasing ability. This was proved by Srivastava et al. for eugenol with its sole hydroxyl group together with the inductive effect induced by the electron-withdrawing methoxy and allyl functional groups of the para and ortho position, respectively [20]. In general, hydroxyl and carboxyl groups of any natural macromolecules are suggested as the reactive sites allowing for the noncovalent interactions with metal ions and their efficient reduction [[21], [22], [23]]. Noteworthy, various functional groups of these compounds not only make them capable of triggering NPs formation but also strongly influence the physicochemical characteristics of the resulting NPs. Thus, plant extracts, containing both primary and secondary metabolites, can be considered as reducing, protecting (prevention of any further growth and particles aggregation during the initial stages of synthesis), and stabilizing agents (prevention of aggregation and agglomeration of the formed nanoparticles).

What is noteworthy, more attention has been currently devoted to the development of biocompatible and biodegradable functional assemblies emerging new directions for the further development of plant metabolites-based materials. These highly hybrid biomaterials can be promising vectors in the field of controlled-release biomedical applications, notably in cancer therapy [24,25]. In particular, biosynthesized Au NPs can become new-generation “magic bullets” against various types of tumors. Engineered multifunctionality on metal nanoparticles based on oxidative coupling assembly of plant metabolites assures selective activity against neoplasms over normal cells. Oxidative DNA damage preventive activity and antioxidant potential of plant extracts have been documented in-depth [26,27]. As well, anticancer activity for both various plant metabolites and Au NPs has been proved [[28], [29], [30]]. These findings offer enormous opportunities to design of novel hybrid or composite materials combining two active components. This, in turn allows discovery of unpredicted and unknown additive or synergistic biological effects.

Herein, we propose a development of energy-efficient, cost-effective and eco-friendly synthesis of Au NPs with application of the aqueous extract of Rosa damascena (Au@RD NPs). Deliberate use of Au NPs is aimed at their potential dual function as a carrier and a contributor to overall activity. Moreover, a final biomaterial, proposed for selective and effective fight against cancer and metastasis, is also composed of plant metabolites as active reagents, that modify surface of Au NPs. The morphology, size, crystal structure and composition of the resulting Au@RD NPs were precisely investigated by scanning and transmission electron microscopies (SEM, TEM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), thermogravimetric analysis (TGA), UV–vis and FT-IR spectroscopies, particle size analysis, as well as zeta potential measurements. The emphasis was put on controlling the size of the gold NPs by varying the synthesis conditions and thereby modulating their physicochemical and biological properties. The cytotoxicity of Au@RD NPs was evaluated in vitro on selected normal cell line: peripheral blood lymphocytes (PBML) and two cancer cell lines: acute promyelocytic leukemia (HL60), human lung adenocarcinoma (A549) based on the assessment of cell viability via double-staining fluorescence assay. To gain more insight into the mode of action of Au@RD NPs the DNA damages in the treated cells were studied by the single-cell gel electrophoresis (comet assay). As well, activity of initiator caspases 3 and 7 was measured by a luminescent assay. The main purpose of biological research was to show selective cytotoxic action of Au@RD NPs towards cancer cells over normal cells in the studied experimental model in vitro.

Section snippets

Materials

Tetrachloroauric(III) acid (HAuCl4 × 3H2O) was used as a source of Au(III) ions required for the synthesis of Au NPs and was purchased from MERCK Co. The other chemicals: Folin−Ciocalteu reagent, gallic acid, quercetin (≥95%, HPLC grade), and quercetin-3-glucoside (≥90%, HPLC grade) were also purchased from MERCK Co. Before use, glass and all laboratory vessels were thoroughly washed in aqua regia and thoroughly dried. All reagents were of analytical grade and only double-distilled deionized

Controlled synthesis of Au@RD NPs using RD extract as a green multifunctional agent

The synthesis of Au@RD NPs was realized through a simple procedure based on reduction of tetrachloroauric(III) acid by the aqueous RD extract. The reduction of HAuCl4 in aqueous solution was carried out with a constant previously selected volume and concentration ratios of reactants (HAuCl4: RD; 1: 9 V: V) at different temperatures (22, 40, and 90 °C). The progress of Au@RD NPs formation was monitored by observation of surface plasmon resonance (SPR) band centered at around 520 nm,

Conclusions

A systematic and detailed study on synthesis of Au NPs using aqueous extract of RD flowers along with evaluation of their biological activity in vitro towards peripheral blood lymphocytes (PBML), acute promyelocytic leukemia (HL60), and human lung adenocarcinoma (A549) cell lines was presented. The progress of Au@RD NPs synthesis was monitored by UV − Vis, while powder XRD, XPS, SEM, and TEM techniques were applied for in-depth characterization of the resulting nanostructures. We showed that

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was partially funded by the Scientific Fund of Andrzej Frycz Modrzewski Krakow University, Poland grant number 26/2018. The authors are very grateful to Małgorzata Kalemba-Drożdż from Faculty of Medicine and Health Sciences, Andrzej Frycz Modrzewski Krakow University for providing biological material (Rosa damascena) for research. The authors would also like to thank Paulina Musielak for her work on optimization of Au@RD NPs synthesis within realization of MSc thesis. Finally, the

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