Short Commentary

Raman spectroscopy (RS) is an emerging lased based optical technique exploited extensively in the field of bio-medical research [1]. The principle involved in RS is when a monochromatic light is incident on an analyte, originates scattered light after interacting with sample and a small fraction of the incident light will be in elastically scattered and is used to construct Raman spectrum [2]. It is a more powerful tool to characterize any biological system, diagnose disease in tissue, cells and to understand the basics in biology [3]. RS is nondestructive, label free technique that identifies the fingerprint of any molecule and their applications include cell state identification, differentiation of cultivated stem cells, and observation of intra cellular composition and distribution of bio molecules which corresponds too many kinds of cell activities [4]. Currently cells staining and sorting and Biomarkers are used to sort and characterize stem cells. These techniques can be tedious, time consuming and error-prone and talking samples from cells cultures may cause mechanical stress and therefore cause chemistry changes to the cells surface. Furthermore, Biomarkers are limited to specific cells and not easily translated to adult stem cells. RS provides details regarding the structure, chemical bond, nature of its environment (hydrogen bonding, etc.,) plays key role in the field of biology [5].

Practical applications are limited because Raman signals are incredibly weaker compared to fluorescence signals. Numerous methods have been developed to enhance the signal and among them using noble metal nano structures, SERS technique is quite interesting [6]. Surface enhanced Raman spectroscopy (SERS) is a powerful vibrational spectroscopy technique which is nondestructive, good sensitivity and the method of preparation is simple [7]. They have been extensively used for biomedical, cancer markers in live cells, sensing, imaging and therapeutic applications [8,9]. SERS may amplify the intensity of the signal, to target biological molecule in trace amounts [10], and simultaneous examination of chemical structure and capable of detecting the single molecule [11,12] and widely used in different fields of research [13-18]. Gold nanoparticles are safe, biocompatible, chemically inert and for their physiochemical properties used as a promising material for biomedical applications [19]. Gold nanoparticles can be used as surface enhanced Raman scattering (SERS) substrates to amplify the Raman scattering intensities of molecules adsorbed to their surfaces by means of localized surface plasmon resonance (LSPR) [20,21].

Au Nps and Ag Nps shows interest in the emerging field of nano biotechnology and medicine, their efficiency in optical and spectroscopic property, advances in their synthesis and fabrication, have brought these particles in the prominent field of nano technology research and their applications in therapy and imaging [22] and various fields [23]. Application of nanoparticles in the field of biotechnology is increasing interest and their effects exerted on body, there is possible of toxicity and is an issue to be discussed [24] and more research article and reviews have been published on Cytoxicity of nanoparticles [25-29]. Cytoxicity of silver nano particles were studied over human mesenchymal stem cells [30] and studies on PC-12 cells shows changes in gene expression and interference with signal transduction after exposure of silver nano particles [31,32]. Cytoxicity of silver and gold nanoparticles in various cell systems have been studied both in vivo and in vitro conditions [33-35], human skin (HeLa), human leukaemia (K562), human breast carcinoma (SK-BR-3) and HepG2 cells lines [36-38]. Gold nanoparticles shows cytoxicity effect on epithelial cells and human carcinoma cell line [39-41] and their effects of gold nanoparticles were studied in vitro on Balb/3T3 mouse fibroblasts [42], human dermal fibroblasts [43] adipose derived stromal cells [44]. Gold nanoparticles were shown to induce cytoxicity in cos-1 cells [45], HeLa cells [46], human alveolar type II like cell line A 549 and NC1H441 in vitro [47] and toxicity of gold nano particles were studied on human glioma cell line LN-229 and human glioma stem cell line HNGC-2 [48]. Several groups have shown minimal toxicity with gold nanoparticles and more studies need to perform.

Our laboratory in UAEU studied the cytoxic effect of gold nanoparticles on mouse gastric cells for SERS. The stomach is lined by different types of epithelial cells which are generated continuously by gastric stem (GS) cells. These cells are rare and difficult to identify. Previous reports demonstrated the use of gold nanoparticle-based surface-enhanced Raman scattering (SERS) for probing the differentiation of embryonic stem cells [49]. As a first step to use SERS as a tool to identify and characterize GS cells we tested the effect of gold nanoparticles (GNP) on growth and viability of GS cells. Trypsinized mouse GS cells were incubated with GNP either directly or after forming embryoid bodies using the hanging drop method. Transmission electron microscopy (TEM) was used to localize GNP inside cells (Figure 1), whereas Pico Green assay were used to measure cell proliferation. TEM confirmed the intracellular localization of GNP (Figure 2). Pico Green assay showed that the number of GS cells treated with GNP increased by 32.8% within 3 days in comparison to untreated cells. We are currently employing SERS to identify and characterize GS cells. In conclusion, GNP does not affect the viability and cell proliferation. Embryoid bodies cultured in differentiation media show a slight morphological difference (not shown here) which is yet to be determined by Raman.

Figure 1: Treated mGSCs with 20% (v/v) GNPs for 5 h were first fixed karnovsky’s fixative for 30 min, and then washed three times with PBS. Cells were then post fixed in 1% OsO₄ for 30 min, and washed three times with distilled water for 5 min. Cells were dehydrated with a series of ascending alcohol concentrations (30%,50%,70%,80%,90%,100%) followed by incubation with a series of mixtures of Agar 100 Resin with alcohol. Finally 50 nanometre thick sections of the samples were first stained for 60 min in uranyl acetate, then for 25 min in lead citrate. Micrographs were taken with a Philips CM10 transmission electron microscope (Philips, the Netherlands).

Figure 2: The mGSCs culture medium, Hyclone RPMI 1640 (Thermoscientific,Utah) was prepared by the addition of 10% (v/ v)Foetal bovine serum (FBS)(Gibco), and 1% (v/v) penicillin/streptomycin (pen/strep) (Gibco) at concentrations 100 IU/ml and 100 ug/ml respectively. Prior to experiments mGSCs cultured at 37°C, 5% CO₂ overnight until cells attachment with the addition of either 10%,20% ,30% (v/v) autoclaved 100 nm Gold NanoParticles (GNP) (BBInternational, UK) for 3 days. At each time point (days 0,2, and 4) cells were trypsinized for 5 min, then 490 ul of cell suspension was mixed by vortexing with 10 ul of 0.5 ug/ml of propidium iodide (PI) solution. Cell number was counted using Cell Lab Quanta SC flow cytometry (Beckman coulter Inc, USA).

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