Facile synthesis of biocompatible gold nanoparticles from Vites vinefera and its cellular internalization against HBL-100 cells
© Springer-Verlag 2011
Received: 13 August 2011
Accepted: 28 August 2011
Published: 15 September 2011
The remarkable health benefits of the chemical cocktails occluded within Vites vinefera (grapes) have been broadly used as dietary supplements and as natural pharmaceuticals in the treatment of various diseases including human cancer. Current discovery demonstrates the rapid formation of gold nanoparticles with the phytochemicals present in grapes, which serve a dual role as synergistic reducing agents to reduce gold salts into gold nanoparticles and also as stabilizers to provide a robust coating on the gold nanoparticles in a single step. Furthermore, the grape-generated gold nanoparticles (GAuNPs), have demonstrated remarkable in vitro stability on specific functionalization with peptides (GSH) and thiol-containing compounds (lipoic acid) followed by the induction of cell-specific response. In addition, the grape-generated gold nanoparticles (GAuNPs, GSH-GAuNPs, LA-GAuNPs) have demonstrated remarkable affinity towards human breast cancer cells (HBL-100) in the present study. These studies thus signified the cellular internalization of GAuNPs and its conjugates by transmission electron microscopy through endocytosis into cancer cells. Notably, at higher concentration of gold nanoparticles conjugate, there was an asymmetric accumulation of gold nanoparticles in the periphery of the cell nucleus of the HBL-100 cells which was confirmed by fluorescence microscopy. Other than gold salts, no “manmade” chemicals are used in this truly biogenic, green nanotechnological process which thereby paves the way for outstanding opening for their application in molecular imaging and cancer therapy.
Cancer induction, growth, and progression are multistep events, and numerous studies have demonstrated that various dietary agents interfere with these stages of cancer of various organ sites thereby sharing great promise in our conquest to control human malignancies. Based on these encouraging observations, research efforts from across the globe have focused on identifying, characterizing, and providing scientific basis to the efficacy of various dietary phytonutrients in an effort to develop effective strategy to block malignancies. Fruits and vegetables represent untapped reservoir of various nutritive and nonnutritive phytochemicals with potential cancer chemopreventive activity. From the clue of “French paradox,” polyphenolics from grapes (Vites vinefera) and red wines attracted the attention of scientists to define their chemical composition and their properties for human health (Urpi et al. 2009). The reported evidences of beneficial health effects of phenolic compounds (Chacona et al. 2009) include inhibiting some degenerative diseases, such as cardiovascular diseases (Olas et al. 2008) and certain types of cancers (God et al. 2007) reducing plasma oxidation stress and slowing aging (Meyer et al. 1997). The antioxidative characteristics of phenolic compounds are mainly ascribed to their free-radical scavenging and metal-chelating properties, as well as their effects on cell signaling pathways and on gene expression (Soobrattee et al. 2005). Although grape polyphenols are widely used for the prevention and treatment of cancer, its therapeutic effects are always limited by severe adverse effects, such as the stability of biological activity in tissue, bioavailability in vivo, etc. (Chen and Dou 2008). There has been reported that the biological activity of polyphenols might depend on the form of their administration (Henning et al. 2005). To overcome these disadvantages and improve chemotherapeutic activity, researchers have focused on the development of nano-sized drug carriers (Forrest and Kwon 2008).
Nanoparticles have a specific capacity for drug loading, have efficient photoluminescence ability, and are therefore important materials in the targeted delivery of imaging agents and anticancer drugs. (Horcajada et al. 2010; Sajja et al. 2009). Drug carriers made up of nanoparticles (NP) are able to overcome biological barriers, accumulate preferentially in tumors, and specifically recognize single cancer cells for detection and treatment. As the nanorevolution in the realms of medical and technological applications unfolds, it is imperative to develop environmentally benign and biologically friendly green chemical processes (Studer et al. 2010). The utility of plant-based phytochemicals in the overall synthesis and the architecture of nanoparticles and various nanoparticle-embedded products are highly attractive as they bring an important symbiosis between natural or plant sciences and nanotechnology. Continuous demand for new anticancer drugs has stimulated chemotherapeutic research based on the use of metals since potential drugs developed in this way may be less toxic and more prone to exhibit antiproliferative activity against tumors (Shankar et al. 2004). The naturally grown plants which occlude phytochemicals may serve as long-lasting and environmentally benign reservoirs for the production of a myriad of gold nanoparticles. Recently, synthesis of Au and Ag nanoparticles using extracts of Cinnamomum camphora leaf (Huang et al. 2007), phyllanthin (Kasthuri et al. 2009), and Alfalfa sprouts (Gardea et al. 2003b) as a reducing and capping agent has been reported.
The utilities of NP strongly depend upon their physiochemical characteristics and their interaction with various surface moieties. Nanoparticles have to be surface modified to make them stable, biodegradable, biocompatible, and with high specificity for preparation of bioconjugate and some functional groups, such as cyano, thiol (Yonezawa et al. 2006), glutathione (Basu and Pal 2007), and amino groups (Subramaniam et al. 2005; Aslam et al. 2004) which are known to have high affinity for gold can be used as capping agents for gold nanoparticles. Such systems can limit the release of encapsulated materials more effectively (Chen et al. 2007). A major advantage of using these short peptide motifs is that they home in to the tumor vasculature, which is less dependent on the variability of receptors expressed directly on the tumor cell surface (Ruoslahti 2000). Hence, by incorporating appropriate peptides and thiol-rich molecules into a linkage between carrier and drug, it is possible to develop rapid release without appreciably contributing to drug loss during circulation in the central blood compartment.
While the tremendous health benefits of chemical cocktails present within grapes is beyond doubt, the actual applications of the chemical reduction power of the myriad of chemicals present in herbs and spices is still in infancy. Therefore, we investigated the synergistic potentials of polyphenols, flavonoids, catechins, and various phytochemicals present grape extract for the reduction reactions of gold salts to produce AuNps which have potential applications in the diagnosis and therapy of various deadly diseases including cancer. In this study, we synthesized a kind of novel gold nanoparticles conjugates using glutathione and lipoic acid as encapsulant materials for entrapment of grape polyphenols. The morphology, structure, and characteristic of the glutathione capped grape gold nanoparticles and lipoic acid capped grape gold nanoparticles were confirmed by UV–spectroscopy, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and Fourier transform infrared (FTIR). We also extended our communication to study the application of gold nanoparticles as carriers of grape polyphenols by determining its cytotoxicity in vitro against human breast cancer (HBL-100) cell lines and such studies are missing up to date to the best of our knowledge.
2.1 Synthesis of GAuNPs, GSH-GAuNPs, and LA-GAuNPs
Grapes were washed with distill water to remove any traces of contaminants. Grapes were cut into small pieces and added into a conical flask containing 100 ml of Millipore water and boiled for 10 min and filtered using Whatmann filter paper. To 10 ml of grape extracts, 10 ml of 1-mM aurochlorate was added and heated to 75°C for 10 min. The color of the mixture changes from pale purple to dark purple. To the grape-gold colloid solution (25 mL), glutathione (20 mg) was added, and the mixture was stirred for 10–15 min. After the stirring was completed, the mixture was centrifuged at 4,500 rpm to separate the capped gold nanoparticles. The pellet obtained was resuspended in 1 mL of phosphate buffer (pH 7). A 600-μmol portion of α-lipoic acid in 10 mL of NaOH 0.5 M solution was added to 25 mL of freshly prepared citrate-capped Au NPs under stirring at room temperature (20–23°C). After 24 h, AuNPs capped with dihydrolipoic acid (DHLA), obtained from α-lipoic acid reduction, were dialyzed against phosphate-buffered saline (PBS) for 48 h using a 10-kDa cut-off dialysis bag (Interchim®, France). The dialysis medium was changed once to fresh PBS after 24 h. The resulting Au at DHLANPs solution was stored in the dark at 4°C for a maximum period of 2 months. The gold nanoparticles thus formed were separated immediately using a 5-μm filter and were characterized by UV–vis absorption spectroscopy FTIR and SEM analysis.
2.2 Characterization of GAuNPs, GSH-GAuNPs, and LA-GAuNPs
The stability and the identity of the grape-initiated gold nanoparticles (GAuNPs) were measured by recording UV absorbance after 30 min. The Plasmon resonance band at ∼535 nm confirmed the retention of nanoparticulates in all the above mixtures. X-ray diffraction pattern of dry nanoparticles powder was obtained using Siemens D 5005 X-ray diffractometer with CuKa radiation (l = 0.1542 nm).The FTIR spectra were obtained on a Nicolet 5700 FTIR instrument with the sample as KBr pellets. The morphology of the nanoparticles was analyzed using the high-resolution images obtained with a JEOL 3010 transmission electron microscope and scanning electron microscope.
2.3 In vitro studies: HBL-100 cell culture and maintenance
The cells were maintained in Dulbecco’s modified eagle’s medium (DMEM, Invitrogen, Carlsbad, CA) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/mL penicillin, and 5 mg/mL streptomycin—at an incubator setting of 5% CO2 and 37°C. All the experiments were carried out 24–48 h after cells were seeded. The cells were routinely harvested by trypsinization 0.25% when the cells approached sub-confluent stage and were placed on 25-cm culture flasks split into 1:6. Human breast cancer cell (HBL-100) cell line was purchased from NCCS Pune. GAuNPs was dissolved in PBS followed by ultra-sonification for five mints, and concentrations of 8 mg/10 ml were used for the various experiment.
2.4 Assay of in vitro cytotoxicity of GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells
The 3-(4,5-dimethylthazol-2-yl)-2,5-diphenyltetrazolium bromide blue-indicator dye (MTT)-based assay is a simple nonradioactive colorimetric assay to measure cell cytotoxicity, proliferation, or viability. MTT reduction was examined according to the methods of Mosmann (1983), with modification. HBL-100 cells were used for the analysis of cytotoxicity in vitro. The cells (1 × 106cells/mL) were placed into 96-well tissue-culture plates and incubated at 37°C. After 24 h, cells were treated with three different concentrations of GAuNPs, glutathione-capped grape gold nanoparticles (GSH-GAuNPs), and lipoic acid-capped-grape gold nanoparticles (LA-GAuNPs) (10, 50, 100, and 150 μg/mL, respectively). Untreated cells were used as controls. Plates were incubated in a humidified 5% CO2 balanced-air incubator at 37°C for 24, 48, and 72 h, respectively. Then, 10 μL of 5 mg/mL MTT solution was added to each well, and the plates were incubated for another 4 h, and then the medium was discarded. Dimethyl sulfoxide (100 μL) was added to each well, and the solution was vigorously mixed to dissolve tetrazolium dye. The absorbance of each well was measured by enzyme-linked immunosorbent assay reader (BioTek; Austria) at a test wavelength of 570 nm. Percentages of surviving cells to untreated controls were calculated by using the formula as % viability = [(At/As) × 100] %, where At and As indicate the absorbance of the sample and control, respectively.
2.5 Determinination of the integrity of cell membranes: the LDH leakage
2.6 TEM analysis of surface characteristics of HBL-100 cells during internalization of GAuNPs, GSH-GAuNPs, and LA-GAuNPs
HBL-100 cells with a density of 1 × 106 cells/mL were grown in DMEM (Invitrogen) supplemented with 12.5% horse serum, 2.5% fetal bovine serum, 50 U/mL penicillin, and 5 mg/mL streptomycin—at an incubator setting of 5% CO2 and 37°C. After trypsinization and centrifugation, cell pellets were resuspended in Hank’s balanced salt solution for the exposure of 100 μg/mL of GAuNP’s and further incubated for 24 h at 37○C. The medium was then aspired, and the cell layer was rinsed three times with growth medium to remove any traces of uninternalized GAuNPs. About 0.5 mL of 0.1 M Trypsin-EDTA solution was added to each well to detach the cell layer from the plastic. Detached cells were dispersed in 4 mL of complete growth medium and gently pipette out of the well. The cell suspension was transferred into a centrifuge tube and centrifuged at approximately 125×g for 5 min. Supernatant was discarded, and cell pellet was embedded in paraffin, sectioned, and examined under transmission electron microscopy.
2.7 Assay of apoptosis induced by GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells DAPI staining assay
The fluorescent dye 4′,6-diamidino-2-phenylindole (DAPI) was used to detect the nuclear fragmentation that is a characteristic of apoptotic cells. PC12 cells (5 × 103 cells/well in 12-well plates) were incubated at 37°C with GSH-GAuNPs and LA-GAuNPs (50 or 100 μg/mL) and then washed with PBS and fixed with 70% ethanol for 20 min. The fixed cells were washed with PBS and stained with the DNA-specific fluorochrome DAPI (1 μg/ml). Following 10 min of incubation, the cells were washed with PBS, and the plates were observed under a fluorescence microscope (Olympus Optical, Tokyo, Japan).
2.8 DNA fragmentation analysis
An apoptotic cell is characterized by its unique ladder of nucleotide fragments in DNA-agarose gel electrophoresis. HBL cells (5 × 103 cells/well in a 12-well plates) were lysed with lysis buffer (10 mM Tris–HCl, 5 mM EDTA, 200 mM NaCl, 0.2% SDS, and incubated at 60°C for 5 min); the sample was digested with 2.5 μl of proteinase K (more than 3 μl−1; Sigma) and 5 μl of RNase A(Iu μl−1) (Fermentas) and was further incubated at 50°C for 1 h. After centrifugation at 1,000×g for 15 min, the supernatants were extracted with an equal volume of phenol, chloroform, and isoamyl alcohol. The DNA was then mixed with 4 M sodium chloride and 100% ethanol and stored at −70°C overnight. Each DNA sample was loaded onto a 1.8% Tris–boric acid–EDTA agarose gel and electrophoresed at 100 V for 30 min.
3 Results and discussion
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
3.2 UV–visible spectroscopy studies
3.3 Size, morphology, and stability properties
3.4 In vitro uptake and localization characterization
3.5 Induction of apoptosis by GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells
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)
3.6 Cellular internalization studies of GAuNPs, GSH-GAuNPs, and LA-GAuNPs in HBL-100 cells
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.
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