Facile bio-synthesis of gold nanoparticles by using extract of Hibiscus sabdariffa and evaluation of its cytotoxicity against U87 glioblastoma cells under hyperglycemic condition

https://doi.org/10.1016/j.bej.2015.09.021Get rights and content

Highlights

  • Extracts of plant Hibiscus sabdariffa was used for synthesis of gold nanoparticles.

  • Stability of the gold nanoparticles was studied in presence of glucose.

  • Cytotoxicity of nanoparticles was studied against U87 Glioblastoma multiforme.

  • Nanoparticles were investigated for cyctotoxicity under hyperglycemic condition.

Abstract

In the present paper, a facile synthesis of gold nanoparticles is reported with leaf and stem extract of Hibiscus sabdariffa. Structural features of as synthesized nanoparticles are characterized by UV–vis spectroscopy, XRD, FTIR, and XPS. Morphology of the above synthesized gold nanoparticles is investigated by electron microscopy. The stability of the nanoparticles is studied in different concentrations of glucose which suggested their possible application under hyperglycemic condition. As synthesized nanoparticles has shown selective toxicity towards U87 glioblastoma multiforme cell line under normal and hyperglycemic condition, indicating their potential to be used in the development of value-added products in the biochemical industries. The possible mode of activity of the above nanoparticles has been studied by in vitro molecular techniques.

Introduction

Malignant gliomas are heterogeneous, highly invasive primary brain tumors in adults, with an incidence of ≈78% of all primary malignant brain tumors [1], [2]. Glioblastoma multiforme (GBM), classified by World Health Organization (WHO) as a grade IV glioma is one of the most aggressive cerebral tumors due to its rapid proliferation and tendency to infiltrate brain tissues [3]. Usual treatment of gliomas can involve chemotherapy, radiation and surgery. Despite sincere efforts, treatment of GBM remains one of the most challenging tasks in clinical oncology. While new molecular pathways responsible in the biology and invasiveness of glioma are being constantly discovered, translation of achievements in basic science into clinical trial is quite slow [4], [5]. Situation become more complicated if the patient diagnosed with GBM have a previous history of hyperglycemia. A recent study shows that if hyperglycemic patients with good Karnofsky performance score (KPS) are diagnosed with GBM, there are rare chances of survival and recovery [6]. Thus, much more significant developments need to be made before we can witness positive outcomes. In this regard, nanomaterials based strategies have received much attention by the scientific community owing to their unique ability to target malignant cells and tissues [7], [8]. Out of various nano-structured materials under investigation, gold nanoparticles (Au NPs) are of particular interest to scientific community owing to their stability, chemical inertness, and potential biotechnological applications [9], [10], [11]. Additionally, Au NPs can effectively bind to amine and thiol groups which help in surface modification and increase their effectiveness in biomedical applications [11]. Although Au NPs processed by physical and chemical protocols have shown great potential in killing tumor cells, their clinical trial has been delayed due to their possible toxic behavior towards healthy cells and tissues [12]. Hence there has been a growing interest to find bio-inspired strategies for synthesis of bio-compatible Au NPs. It is suggested that biogenic nano-systems could be biocompatible and therefore may portray effective anticancer property. Microorganisms have been extensively studied for synthesis of Au NPs [13], [14], [15]. However, microbial synthesis of NPs have usually shown in general following process drawbacks, (1) may require longer time for reduction, which leads to unwanted growth and/or agglomeration of nano-crystallites which in turn may affect the nano-size dependent biological applications, (2) may require tedious downstream processing for recovery of purified metal NPs with sufficient yield, (3) may be difficult to optimize and scale up the process parameters [16], [17]. To overcome the above mentioned process disadvantages, plants and their extracts have been investigated as an alternative source for biological synthesis of Au NPs [18], [19], [20]. Research has shown that biomolecules like amino acids, organic acids, phenol and flavonoids present in plants play an important role in reducing the metal ions to their respective nanomaterials as well as aid in capping of the metal NPs which keep them stable for a longer period of time [21], [22]. In addition to this, plant extracts are safe to handle, easily available and possess a broad variety of metabolites that may aid in reduction of metal salts to NPs [18], [22], [23].

Keeping this in view, in the present study a systematic approach has been made to synthesize Au NPs from the extract of Hibiscus sabdariffa plant which is widely used as a flavouring agent in food industry and also has a herbal medicinal property [24]. The cytotoxic activity of as synthesized biogenic Au NPs has been investigated against U87 GBM cell line in the presence and absence of glucose. To the best of our knowledge the leaf and stem extract from the above mentioned plant has never been exploited for synthesis of Au NPs. However, bio-synthesis of Ag NPS using the flower extract from the above mentioned plant was communicated [25], [26]. In addition to this, hardly any report is available on the application of Au NPs in targeting GBM under hyperglycemic condition. Hibiscus, in general, is supposed to have the highest level of antioxidants present in any of the widely available food. It has been reported that protocatechuic acid present in this plant is found to inhibit tumor promotion on mouse skin and hence it could be inferred that protocatechuic acid is effective as a cancer chemopreventive agent [27]. It is expected that, presence of the above acid in the plant extract might help in the surface modification of the Au NPs thereby enhancing the anticancer property of the later. Additionally, we have investigated the stability of the as synthesized Au NPs in presence of different concentration of glucose (at ambient and normal body temperature) which makes them useful for potential application under hyperglycemic condition. We have performed the above experiment with the mentioned plant extract because from previous reports it has been well studied that extract from the particular plant plays significant role in inhibiting the high glucose stimulated migration and proliferation of vascular smooth muscle cells through regulation of CTGF, AGE and other downstream signals which constructively signifies protection against diabetic vasculopathy [28].Cytotoxicity of synthesized Au NPs has also been studied against 293 normal cells to ascertain their bio-compatibility. To understand the possible mode of action of Au NPs against U87 GBM cells in-vitro, further experiments have been performed by using fundamental molecular techniques such as DAPI, DNA degradation by agarose gel electrophoresis and protein degradation analysis.

Section snippets

Extraction of plant exact (bio-reductant)

H. sabdariffa was purchased from the local market of Bhubaneswar, Odisha, India. The leaves were separated from the stem. A solution was prepared by grinding 700 g of leaves or stem in 1200 ml distilled water. The as mentioned experimental data with the particular amount of leaves or stems was optimized through performing sets of reactions by conducting a set of preliminary screening process. First, the solution obtained was passed through muslin cloth and the filtrate was filtered using filter

Results and discussion

Bio-synthesis of Au NPs has been followed by change in the surface Plasmon resonance (SPR) band of the aqueous dispersion. Metal NPs have been synthesized with LE and SE of H. sabdariffa. Fig. 1(a) and (b) shows the UV–vis spectra of the corresponding aqueous dispersion of Au NPs obtained by using LE and SE respectively. From the above figure it is suggested that SE did not yield well dispersed NPs [13], [14]. The change in the color of the gold salt solution from light yellow to purple is the

Conclusion

In summary, cytotoxicity of biogenic Au NPs is investigated against U87 GBM cells under normal and hyperglycemic conditions. From experimental observations, present report communicates a promising consequence. Water soluble well dispersed Au NPs is synthesized via a novel route by using the LE of medicinal plant H. sabdariffa. Following process optimization, it is observed that maximum yield of the as synthesized Au NPs is noted at an acidic pH (4.0–6.0) and temperature of 100 °C. An initial

Acknowledgement

This work is supported by Department of Biotechnology (DBT), Government of India.

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