The chemical inertness of gold has been used internally in humans for the past 50 years, from its use in teeth to implants to radioactive gold used in cancer treatment. The primary rationale for selecting gold nanoparticles is their biocompatibility, very high surface area (large amount of drugs can be loaded), ease of characterization, and surface modification (i.e., organic molecules such as drugs, peptides, antibodies, etc. can be easily conjugated to gold nanoparticles). Choosing the right ligand for nanoparticle synthesis is key in forming AuNPs with desirable properties. In this study, we chose the naturally occurring peptide ligand, GSH, and thiol-rich ligand, lipoic acid, because of the favorable properties such as the presence of thiol, carboxylic acid and amino groups, water solubility at relevant biological pH, biological compatibility, and ease of functionalization, thereby making water-soluble nanoparticles for biological applications.
The presence of carboxylic acid and amino groups on GSH ligand complexed with Au (I) has the potential advantage of being a pH-sensitive compound, which can adopt different conformational states and sizes depending on the pH of solution. Whetten et al. synthesized the Au NPs via sodium borohydride reduction of the mixture of tetrachloroauric acid and GSH in methanol–water (2:3) and obtained gold nanoparticles with most abundant component having a diameter of 0.9 nm. We have initiated to design such nanoparticles on the direct intervention of phytochemicals for the production of gold nanoparticles which may provide a new method and an important opportunity for improvement in breast cancer treatments, the most common form of cancer in women worldwide. Such nanoparticles coupled with the specific targeting agents have the ability to track and eliminate breast cancer cells. In this paper, we present grape-gold-based nanoparticles for breast cancer diagnosis and treatment for which we used human breast lymphoma cells.
3.1 Synthesis of the biocompatible nano gold from V. vinefera
Figure 1, a–c depicts the change in color of GAuNPs before and after capping. The color of the grape extract changed pink to wine red (1a), to blue upon addition of glutathione (1b), and to dark blue after capping with lipoic acid (1c). Synthetic conditions have been optimized for the quantitative large-scale conversions of HAuCl4 to the corresponding AuNPs using grape extract. The foremost phytochemicals present in grape extract consist of water-soluble catechins (catechin, epicatechin, epicatechin gallate, epigallocatechin, epigallocatechin gallate, etc.,) and thearubigins which are oligomers of catechins of unknown structure. As the generation of AuNPs using grape extract involves aqueous media, the water-soluble phytochemicals of grape extract may be playing a major role in the overall reduction reactions of HAuCl4. Interestingly, the systematic investigation of Satish et al. (2009) granted the role of polyphenols (catechins and theaflavins) for the generation and stabilization of AuNPs through independent experiments. Normally, thiols containing organic compounds are employed to stabilize AuNPs against agglomeration and strong interaction (Brust et al. 1994). It has been shown that all the catechins act as outstanding reducing agents to reduce the Au (III) to the corresponding gold nanoparticles (Satish et al. 2009). The nanoparticles thus generated were coated with GSH and lipoic acid stabilizing agent and showed significant stability. These experiments have decidedly confirmed that catechin and epigallocatechin gallate hand out dual roles as reduction and stabilizing agents, whereas epigallocatechin and epicatechin can be used only for the reduction of gold salts and require GSH and lipoic acid as an external stabilizing mediator. Thus, our study provided an evidence for the better stability by GSH and lipoic acid when coupled to biomolecules to obtain new delivery platforms (Roux et al. 2005).
3.2 UV–visible spectroscopy studies
The gold nanoparticles synthesized from grape extract were relatively monodisperse in colloidal solution, which was confirmed by a single peak in the absorbance spectra (Fig. 1). Gold nanoparticles exhibit some special optical properties such as Plasmon resonance, which is primarily a quantum phenomenon operative on the nanoscale. Absorption measurements indicated that the Plasmon resonance wavelength of GAuNPs was 535 nm. As shown in Fig. 1, the peaks 2 and 3 are shifted towards the higher wavelength after capping with glutathione (540–580 nm) and lipoic acid (560–620 nm), respectively. The λmax shift in the absorbance spectra was mainly due to the surface modification of the gold nanoparticles (Figs. 2, 3). Fascinatingly, the surface Plasmon resonance, the major cause for the absorption, may be affected by surface modification with covalent coupling thereby increasing their size (Sheetal et al. 2008). The covalent coupling may also be due to the protective coating of the organic molecule, glutathione—a tripeptide (glutamic acid, cysteine, and glycine), which has many binding points for the gold nanoparticles (two carboxylic groups, one thiol group, and three amino groups). More effectively, the thiol group is involved in the attachment with the AuNP. In the case of lipoic acid-capped nanoparticles, the disulfides are reduced by polyphenols to two thiol groups (−S–S– → –SH + −SH), which are involved in the binding of lipoic acid to gold nanoparticles. And the coupling can be extended via either the carboxylic or the amino groups (of glutathione/lipoic acid). It is conceivable that the cocktail of phytochemicals in grapes along with nontoxic antioxidants lipoic acid and GSH are acting synergistically in stabilizing gold nanoparticles from any agglomeration in solution. A similar study by Gautham et al. (2009) created borohydride-reduced AuNPs capped with glutathione and lipoic acid that was covalently linked to horse radish peroxidase, provided some insight in the application of biosensors as tools for diagnostics.
3.3 Size, morphology, and stability properties
The techniques for the characterization of nanoparticle size and morphology are SEM (Bilati et al. 2005) and TEM (Teixeira et al. 2005). Figures 4 and 5 indicate the size and morphology as observed under SEM and TEM on AuNPs synthesized using grapes as spherical in shape within the size range of 20–45 nm (Figs. 3a and 4a). Investigations on experiments using commercially available catechins have unambiguously confirmed that catechins are excellent reducing and stabilizing agents to reduce Au (III) to the corresponding gold nanoparticles (Satish et al. 2009). In addition, the effect of pH (Gardea et al. 2003b), time (Huang et al. 2007), temperature (Groning et al. 2004), and measurement of charges (Shankar et al. 2004a) may also play a major role in the determination of shape and size of nanoparticles. However, the presence of some phytochemicals (Brust et al. 1994) might render a minimum stability by failing to provide effective coating to shield the nanoparticles from agglomeration studies to GAuNPS. In order to capitalize on the reduction powers of such phytochemicals, we have utilized GSH, a tripeptide and lipoic acid, an antioxidant as naturally available stabilizing agent in our reactions. Thus, GSH-GAuNPs (Figs. 3b and 4b) and LA-GAuNPs (Figs. 3c and 4c) sizes as measured by SEM and TEM are in good agreement and are in the range of 40–80 nm suggesting that thiols and peptides are capped on grape phytochemically reduced gold nanoparticles. Such size distribution analysis of capped and non-capped GAuNPs confirms that particles are well dispersed. This extra stability rendered by GSH and lipoic acid-capped AUNP arises due to the chemical inertness by the complete coverage of the gold core by GSH ligands. Gold nanoparticles can be stabilized by anionic ligands such as carboxylic acid derivatives like citrate, tartrate, and lipoic acid (Peng et al. 2007). Earlier studies showed that glutathione used for capping gold quantum clusters (AU-n-SG-m) (−SG, glutathione thiolate) is one such group of compounds which has been well known for the stability of the AUNPs synthesized chemically (Habeeb and Pradeep 2007). Moreover, he confirmed in his results that the bigger clusters, n > 25, can be converted into AU25SG18 by adding excess GSH. In addition, the six free electrons present in the conduction band of nanoparticulate gold make them potential candidates to bind with thiols and amines. Therefore, by changing the size and shape of AuNps, the SPB and scattering may be tuned for application in cellular imaging, drug delivery, and therapy.
3.4 In vitro uptake and localization characterization
Characterization of in vitro nanoparticle uptake and localization is intrinsically linked to cytotoxicological studies because uptake provides evidence of nanoparticle–cell interaction, wherein the delicate intracellular machinery is exposed to nanoparticles. Compared to in vivo studies, in vitro studies benefit from being faster, lower cost, allowing greater control, and minimizing ethical concerns by reducing the number of laboratory animals required for testing. The most commonly used in vitro assessment techniques generally evaluate either viability (live/dead ratio) or toxicity mechanism. Herein, the major viability-based assays are organized into the categories of proliferation, necrosis, or apoptosis DNA damage detection techniques. In our present communication, the cytotoxicty of GAuNPs, GSH-GAuNPs, and LA-GAuNPs under in vitro conditions in HBL-100 cells was examined using two cytotoxicity markers, including MTT reduction, and LDH leakage is used to study the effects of gold nanopatricle conjugates on cellular viability. To check the cytotoxicity of cells treated with GAuNPs (10–150 μL), GSH-GAuNPs (10–150 μL), and LA-GAuNPs (10–150 μL) for 24 h, and a number of viable cells were enumerated by colorimetric MTT assay. After 24 h of post-treatment, HBL-100 cells showed excellent viability even up to 150 μL of GAuNPs, GSH-GAuNPs, and LA-GAuNPs (Fig. 5). Results of MTT assays clearly revealed the cytotoxic effect of GAuNPs in a dose-dependent manner for HBL-100 cell lines and LA-GAuNPs exerted slightly better cytotoxic effect towards HBL-100 cells in comparison to GSH-GAuNPs. Although GAuNPs also showed cytotoxicity towards cancer cells, the effect was much less compared to GSH-GAuNPs and LA-GAuNPs. Treatment with 150 μL or higher doses of GAuNPs and GSH-GAuNPs and LA-GAuNPs to HBL-100 cell increased LDH leakage into culture media (Fig. 6), signifying an AUNP-induced compromise of plasma membrane integrity, and the IC50 of AgNPs was 150 μL. Henceforth, the release of LDH (Fig. 6) in our study is in agreement with the excellent viability of the cells treated with GAuNPs and GSH-GAuNPs and LA-GAuNPs as proven by MTT assay. It is also important to recognize that a vast majority of gold (I) and gold (III) compounds exhibit varying degrees of cytotoxicity to a variety of cells (Basset et al. 2003; Hamer 2007). GAuNPs pretreatment at a concentration of 150 μL reduced the LDH leakage to a minimum, and this concentration is used in subsequent studies.
3.5 Induction of apoptosis by GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells
Surface reactivity, chemical composition, and large specific surface area have been deemed important properties in nanoparticle-mediated toxicity (Wallace et al. 2007). HBL-100 cells, after treatment with nanometer-sized GAuNPs, GSH-GAuNPs, and LA-GAuNPs, exhibited ultra structure and biochemical features that are characteristic of apoptosis, as shown by chromatin condensation and inter nucleosomal DNA fragmentation. The phase-contrast microscopic pictures of altered morphology of HBL-100 cells which is characteristic of apoptotic cell stage when treated with GSH-GAuNPs, and LA-GAuNPs (40–80 nm) are shown in Fig. 7a–d. In addition, the nuclear fragmentation, a hallmark of cellular apoptosis, was clearly exhibited by fluorescent microscopic studies after DAPI staining of untreated and GSH-GAuNPs and LA-GAuNPs (40–80 nm)-treated HBL cells (Fig. 8a–d). A minimum of 200 cells were counted and classified as follows: (1) live cells (normal nuclei: blue chromatin with organized structure); (2) stressed cells (bright-blue chromatin, which is highly condensed, margined, or fragmented).
Metal complexes have been extensively studied for their nuclease-like activity using the redox properties of the metal and dioxygen to produce reactive oxygen species to promote DNA cleavage by direct strand scission or base modification in cancer cells (Burrows and Muller 1998). A more current development in this area has been testing of metal nanoparticles such as gold and platinum nanoparticles for DNA degradation studies (Shen et al. 2009; López et al. 2010). Use of metal nanoparticles can be in particular advantageous in generating singlet oxygen [42 and 43] (Lipovsky et al. 2009; Portolés et al. 2010)
A recent report by Geddes and coworkers demonstrated that the presence of metal nanoparticles can enhance singlet oxygen generation (Zhang et al. 2008). The enhanced electromagnetic fields in proximity to metal nanoparticles are the basis for the increased absorption predicts the extent of absorption and the relative increase in singlet oxygen generation from photosensitizers (Barber et al. 1983; Yang et al. 1995)
A very recent study by Midander and coworkers reported the effect of metal nanoparticles inducing single-stranded breaks in the human lung cells (Midander et al. 2009). Previous studies illustrated the potent cytotoxic, genotoxic, and toxicological activities of nanoparticles in vivo (Midander et al. 2009; Chen et al. 2006) and in cultured cancer cell lines (Sengupta et al. 2007). However, a methodical study using GAuNPs on DNA degradation and cytotoxicity towards breast cancer cells are missing up to date to the finest of our information. Thus, 40–80-nm-sized GSH-GAuNPs and LA-GAuNPs (lanes 2 and 3) treated HBL-100 cells in our study displayed a ladder pattern of inter-nucleosomal DNA fragmentation on TBE-agarose gel electrophoresis in DNA ladder assay (Studer et al. 2010) as revealed in Fig. 9 which is also another characteristic of apoptosis. All these results confirm that treatment with GSH-GAuNPs and LA-GAuNPs induce apoptosis in human breast cancer cells compared to GAuNPs.
3.6 Cellular internalization studies of GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells
Membrane integrity is another cellular characteristic commonly used to determine viability during in vitro nanotoxicology experiments. Surprisingly, cancer cells are highly metabolic and porous in nature and are known to internalize solutes rapidly compared to normal cells (Sun et al. 2007). Do the cells internalize the particles or do the particles remain bound to the cell membrane? Results of cellular internalization studies of AuNps solutions are keys to provide insights into their use in biomedicine. Their selective cell and nuclear targeting will provide new pathways for their site-specific delivery as diagnostic/therapeutic agents. To address these issues, confocal microscopic studies confirmed the uptake of GSH-GAuNPs and LA-GAuNPs inside the HBL-100 cells (Fig. 10a–c) with the presence of agglomerated gold nanoparticles; a similar observation was also reported by Stark and coworkers with copper nanoparticles for Hela cells (Studer et al. 2010).
Further, TEM images of breast tumor (HBL-100) cells treated with GSH-GAuNPs and LA-GAuNPs unequivocally validated our hypothesis. Significant internalization of GSH-GAuNPs and LA-GAuNPs via endocytosis within the HBL-100 cells was observed (Fig. 11). GSH-GAuNPs and LA-GAuNPs were detected within larger endocytic compartments of diverse morphology. These include peripherally both early and late endosomes and lysozomes. The internalization of nanoparticles within cells could occur via processes including phagocytosis, fluid-phase endocytosis, and receptor-mediated endocytosis. The viability of HBL-100 cells post-internalization suggests that the phytochemical coating and the size of the nanoparticles renders the nanoparticles nontoxic to cells. A number of studies have supported our study, demonstrating that phytochemicals have the ability to penetrate the cell membrane and internalize within the cellular matrix (Sun et al. 2007; Mizuno et al. 2007).
Therefore, we hypothesized that grape-derived phytochemicals and other antioxidants, if coated on gold nanoparticles, will show internalization within cancer cells. Such a harmless internalization of gold nanoparticles will provide new opportunities for probing cellular processes via nanoparticulate-mediated imaging.
The present investigation resulted in the development of environment-friendly green methodology to produce biologically benign gold nanoparticles stabilized with biologically relevant thiol-rich antioxidants. Here, the gold nanoparticles are generated by reduction of gold precursor (a source of Au3+ ions) by a reducing agent (grape polyphenols) in the presence of stabilizers (GSH and Lipoic acid) that keeps nanoparticles apart, thus avoiding their aggregation. Although there are reports for the anticancer activity of phytochemically stabilized AUNPs, this is the first report to investigate the cytotoxicity of GSH and lipoic acid (in addition to phytochemicals) stabilized AUNPs. Herein, the major viability-based assays, such as proliferation, necrosis, or apoptosis and DNA damage detection of GSH-GAuNps and LA-GAuNps in HBL-100 cells have been proved as keys to provide insights into their use in biomedicine. In addition, GSH-GAuNps and LA-GAuNps selective cell and nuclear targeting compared to GAuNps will provide new pathways for their site-specific delivery as diagnostic/therapeutic agents.