Anti-cancer studies of noble metal nanoparticles synthesized using different plant extracts
© Springer-Verlag 2011
Received: 10 February 2011
Accepted: 26 April 2011
Published: 10 May 2011
Biofunctionalized gold and silver nanoparticles synthesized using different plant extracts of guava and clove in vitro anti-cancer efficacy against four different cancer cell lines human colorectal adenocarcinoma, human kidney, human chronic myelogenous, leukemia, bone marrow, and human cervix have been studied and reported. The present experimental study suggests that flavonoids functionalized gold nanoparticles synthesized using aqueous clove buds extract are more potential than guava leaf extract towards anti-cancer activities. The microscopic and 2,3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) assay infer that the functionalized irregular shaped gold nanoparticles synthesized with aqueous clove bud extract showed a satisfactory anti-cancer effect on all the cell lines. The silver nanoparticles synthesized using same extracts are devoid of anti-cancer activity. The XTT assay revealed dose-dependent cytotoxicity to cancer cell lines. The study revealed that the free radicals generated by gold nanoparticles are responsible for anti-cancer effect. To confirm the free-radical scavenging efficacy of gold nanoparticle, nitric oxide assay is followed. We observed that the gold nanoparticles swabbed the free radicals in dose-dependent manner. With continued improvements, these nanoparticles may prove to be potential anti-cancer agents.
Cancer is observed as the most dangerous class of disease categorized by uncontrolled cell growth (Chow 2010; Suriamoorthy et al. 2010). There is a marginal increase in cancer cases in the last few years, and most of the time, it ends up with taking life (Dite et al. 2010; Parveen and Sahoo 2010; Smith et al. 2010). In many types of cancer, we are yet to find a satisfactory medicine or carrier of medicine as in case of drug delivery to be used as a satisfactory chemotherapeutic agent. Nanotechnology, an interdisciplinary research field comprising chemistry, engineering, biology, and medicine, has great potential for early detection, accurate diagnosis, and tailored treatment of cancer (Sakamoto et al. 2010). Nanoparticles are usually smaller than several hundred nanometers in size, comparable to large biological molecules such as enzymes, receptors, of a size about 100 to 10,000 times smaller than human cells. These nanoparticles can offer unprecedented interactions with biomolecules both on the surface and inside the body cells, which may bring revolution in cancer diagnosis and treatment (Seigneuric et al. 2010; Liu et al. 2010).
Nanotechnology is a burgeoning arena which takes along with it a myriad of prospects and possibilities for advancing disease treatment in pharmaceutical and medical field. At nanometric scale, the physico-chemical and biological properties of materials differ fundamentally from their corresponding bulk counterpart because of the size-dependent quantum effect. The noble metal nanoparticles like gold nanoparticles (AuNP), especially surface functionalized represent smart and promising candidates in the drug delivery applications due to their unique dimensions, tunable functionalities on the surface, and controlled drug release (Datar and Richard 2010). Another essential aspect while working with AuNP in bio-applications is safety and biocompatibility (AuNP is already approved by the US Food and Drug Administration.) Biologically synthesized and functionalized, AuNP provide many desirable attributes for use as carriers in drug delivery systems as the functionalized AuNP core is essentially inert and nontoxic 1 reported in recent studies (Han et al. 2007; Kim et al. 2009). Monodispersed nanoparticles can be formed with a core size from <30 nm and also with metal–core–organic–shell morphology; the mono-metal layer can be tailored with a range of biological ligands, help in effective cellular uptake, controlled drug release, and targeted drug delivery (Ghosha et al. 2008; Salmaso et al. 2010). These functionalized nanodelivery systems can be used directly as promising lead molecules in the detection of cancer cells. In this applied research work, we have deduced and detailed the use of synthesized bio-functional noble metal nanoparticles application as an anti-cancer drug and demonstrate the anti-cancer effect of these functionalized AuNP using nitric oxide method. It gives a strong speculation, for the influence of free electrons generated by the surface of the functionalized AuNP has a lethal effect on the electronegative surface membrane of the cancer cells. The free-radical scavenging effect of AuNP is compared with the well-known anti-oxidant butyl hydroxy anisole (BHA), which is determined by nitric oxide method. The end results confirm that functionalizing AuNP with the water-soluble organic moieties can show the synergic anti-proliferative effect in various cancer cell lines, and thus prove to be useful in various types of anti-cancer control systems.
We have already worked for the synthesis of different functionalized Au and Ag nanoparticles using different plant extracts of clove and guava (Raghunandan et al. 2009; Raghunandan et al. 2010a; Raghunandan et al. 2010b). The characterization is elaborately discussed with respect to size, shape stability, and functionalization using different spectroscopic and microscopic techniques. The shape of silver nanoparticles (AgNP) found to be roughly spherical and in 30–60 nm and 20–30 nm range in case of guava leaf and clove buds-mediated synthesis, respectively. The clove-mediated AuNP yielded highly unpredictable irregular-shaped particles in the range of 5–100 nm, whereas guava leaf-mediated synthesis produced poly-shaped nanoparticles in the narrow range of 25–30 nm. In all the cases of noble nanoparticle synthesis, the preliminary studies using FTIR (Fourier transformed infrared spectroscopy) confirms that the different polyphenols of the flavonoids of the respective plants are responsible for the biosynthesis, and the same are capped on the surface of the nanoparticles. Though the further studies on the exact chemical moiety responsible for bioreduction pathway is still underway, in this paper, we are making an effort to understand the anti-proliferative effect of these functionalized “lead” in different cancer cell lines. The encouraging results of functionalized AuNP as an anti-cancer agent using different plant extract has a promising lead for further exploitation.
2.1 Biosynthesis of nanoparticles
2.2 Anti-cancer activity study
Four different cells lines namely HT-29 (human colorectal adenocarcinoma), HEK-293 (human kidney), K-562 (human chronic myelogenous, leukemia, bone marrow), and HeLa (human cervix) cell lines were obtained from the American Type Culture Collection, USA. The growth media: RPMI-1640, sufficient minimum essential medium (MEM), and the phosphate buffer solution (PBS) tablets were obtained from Sigma Chemical Co., St Louis, USA. Standard quality fetal calf serum, penicillin–streptomycin, and trypsin were obtained from Sigma labs. The XTT kit, which consists of the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide (XTT) labeling reagent and the solubilisation solution was procured from Sigma labs, USA. Trypsin (0.25% + EDTA, 1 mM in PBSA), XTT dye–50 mg/mL, sterilized Sorensen's glycine buffer (0.1 M glycine, 0.1 M NaCl adjusted to pH 10.5 with 1 M NaOH), DMSO, growth medium is procured from Aldrich labs. XTT labeling reagent and electron-coupling reagent were thawed, respectively, in a water bath at 37°C. Each vial was mixed thoroughly to obtain a clear solution. To perform a cell proliferation assay (XTT) with one microplate (96 wells), 5 mL XTT labeling reagent was mixed with 0.1 mL electron-coupling reagent.
2.3 Free-radical scavenging activity of AuNP
2.4 Cell culture
A 96-well micrometer plate equipment (Nunc, Denmark) is used for this experiment. Two hundred microliters of MEM is added in the required amount of wells; in each cell, 50 μL of different cell lines are added. Biofunctionalized AgNP and AuNP of different concentrations (10, 20, 30 μg/L) were added into the wells separately. The cells were cultured in the appropriate medium supplemented with 5–10% fetal calf serum and 1% penicillin–streptomycin, using 25 cm2 flasks in a 37°C incubator with 5% CO2. To subculture the cells, the cells were separated, and the fresh culture medium was used with fresh medium as follows: in the first step, the old medium was removed, and then the cells were rinsed briefly with PBS. After adding 1–2 μL of trypsin, the flask was incubated at 37°C using 5% CO2 for 5 min. The upper part of the liquid is discarded, and the detached cells, which are present in the lower part of the flask, are taken in another flask, and after adding 20 μL of medium to it, the culture was divided in two parts. One part was then transferred to a new flask. The cells were grown till they become turbid and confluent. Then, trypsin is added to these cells to prepare a cell suspension, and the numbers of viable cells were counted with a hemocytometer. One hundred microliters of suspension containing 1 × 105 cells was seeded in each well of a 96-well microtiter plate. The plate was then incubated overnight at 37°C with 5% CO2. Periodically, the medium was replaced.
2.5 Cell proliferation assay
Each cancer cell line was grown in a 96-well microtiter plate of a final volume of 100 μL culture medium per well. The cells were then treated with functionalized AgNP and AuNP developed with different plant extract and bio-excretories at a dose of 10, 20, and 30 μg/mL and maintained them in incubator at 37°C with CO2 for 48 h. Fifty microliters of the saturating solution (Roche Diagnostics, USA) was then added into each well. The plate was kept overnight in the incubator. Viability of the cells was counted using an ELISA reader (ELx 800) at 550 nm. The experiment was carried out in six replicates.
2.6 Measurement of growth-inhibitory effect
Cells were grown in 96-well plates in a final volume of 100 μL of culture medium per well. Each well contained 1 × 105 cells/mL and was incubated for 24 h in a 5% CO2 incubator at 37°C. As detailed above, the same procedure was adopted for cell culture. The HeLa cell line was used as it has the lowest IC50 (the concentration of test compound that can inhibit 50% of the cancer cells from proliferating) for functionalized AuNP and the same was observed using microscope. The cells were treated with different nanoparticles at a concentration that was similar to the IC50 value. The plate was then incubated again in the 5% CO2 incubator at 37°C for 48 h. The untreated cells (control) were also incubated for 48 h. The growth of the cells was photographed using a phase contrast microscope (Olympus, USA). Cells, grown in a 96-well tissue culture plate, are incubated with the yellow XTT solution (0.3 mg/mL) for additional 4 h.
The cells were grown till they become turbid and confluent. Trypsin is then added to the cells to prepare a cell suspension, and the number of viable cells was counted with a hemocytometer. Fifty microliters of suspension containing 1 × 105 cells was seeded in each well of a 96-well microtiter plate. The plate was then incubated overnight at 37°C with 5% CO2. Periodically, the medium was replaced. A 96-well micrometer plates are used for this experiment. Two hundred microliters of MEM is added in the required number of wells; to this, 50 μL of different cell lines are added in each well. Biofunctionalized AuNP of different concentrations (10, 20, 30 μL) were administered into the wells separately. The cells were cultured in the appropriate medium, supplemented with 5–10% fetal calf serum and 1% penicillin–streptomycin, using 25 cm2 flasks in a 37°C incubator with 5% CO2. To subculture the cells, the cells were separated, and the fresh culture medium was used with fresh medium as follows: in the first step, the old medium was removed, and then the cells were rinsed briefly with PBS to wash the cells. After this, 1–2 μL of trypsin was added, and the flask was incubated at 37°C and 5% CO2 for 5 min. The upper part of the liquid is discarded, and the remaining detached cells, present in the lower part of the flask, are taken in a separate flask; 20 μL of medium was added, and the culture was divided in two parts. One part of this was transferred to a new flask.
3 Result and discussion
Functionalized AuNP developed with clove buds extract (AuNP-Clo) inhibit 50% of the proliferation of HeLa, HEK-293, and HT-29 cancer cells at 20 μg/mL concentration. The same nanoparticles inhibited the K-562 cancer cell line at 30 μg/mL concentration. This colorimetric assay is based on the capacity of mitochondria succinate dehydrogenase enzymes in living cells to reduce the yellow water-soluble substrate XTT into an insoluble, colored formazan product, and therefore, this conversion only occurs in viable cells (Berridge et al. 1996). Additional incubation period for 4 h after the addition of XTT labeling reagent, the change in the color of the solution in different wells is observed. A solution of orange formazan is formed, which is quantified spectrophotometrically using an ELISA plate reader at 490 nm (Sacconi et al. 2009). For this, a graph of absorbance against drug (AuNP) concentration was plotted and the inhibitory effect (IC50) was calculated. The reduction of XTT can only occur in metabolically active cells; the level of activity is a measure of viability of cells. Absorbance values that are lower than the control cell lines reveals decline in the rate of cell proliferation. Conversely, a higher absorbance indicates an increase in the cell proliferation. Untreated microtiter plates of cell lines with only vehicle (0.3% v/v DMSO in water) is considered as proliferative control.
Growth-inhibitory effect of functionalized AuNP on different cell lines
Inhibitory effect on cell lines (IC50)
19.88 ± 0.006* (62.89%)
20.05 ± 0.002* (58.77%)
20.12 ± 0.007* (56.78%)
28.56 ± 0.010** (51.55%)
56.78 ± 0.001 (32.56%)
31.22 ± 0.011 (48.56%)
36.11 ± 0.001 (42.45%)
39.44 ± 0.010 (38.54%)
45.67 ± 0.002 (35.66%)
61.23 ± 0.005 (28.44%)
3.1 Statistical analysis
All the results are expressed as mean ± SD of triplicate. The difference in inhibitory effect at different doses between treated and corresponding controls was analyzed for statistical significance by performing Student's t test. P < 0.05 implies significance. Nil inhibitory effect on cancer cell lines was observed by both extracts at dilutions less than 10 μg/mL. Anti-proliferative activity of ethanol extract on Vero cell line was constantly less at experimented dilutions as compared to cancer cell lines.
3.2 Growth-inhibitory effect
The freely water-soluble flavonoids present in the guava leaf and clove buds solution that could have been adsorbed on the surface can stimulate or suppress the immune system due to the presence of −OH groups. Presence of such phenolic moieties may be assumed to have synergic effect for the anti-proliferative activities of these bio-adsorbed metal nanoparticles (Kawaii et al. 1999; Salucci et al. 2002; Lee and park 2010). The anti-proliferative activity is also attributed to the irregular shape of the nanoparticles (Chen et al. 2008). These shape-dependent properties of AuNP have different behaviors and make them suitable for therapeutic utilization (Greenfield 2005; Gobin et al. 2007; Kevin 2008; Sperling et al. 2008; Bertussi et al. 2005). AuNP of certain non-regular shapes can readily be adsorbed to the surfaces of the biomolecules which show higher surface plasmon resonance and will have a greater contrast effect than those of photothermal dyes that are used regularly in detection of cancer cells (Giljohann et al. 2010; Katti et al. 2006). Here, the bio-detection sensitivity and biocompatibility parameters become very important. The bio-detection sensitivity of nanoparticles often is associated with their physical and chemical properties, which in turn depend on the shape of the particles (Rinaldo et al. 2006; Hu et al. 2007). Nanoparticles with different dimensions have been applied widely to detect biological molecules. Colloidal AuNP is used to detect specific DNA sequences and single-base mutations in a homogeneous format. AuNP synthesized with biological base are interesting, predominantly because they exhibit the best compatibility with biomolecules. But, bio-detection sensitivity derived from spherical nanoparticles is not strong enough to trace the interaction of biomolecules (Maxwell et al. 2002; Orendorff et al. 2006). Looking into all these aspects, it is reasonable to infer that the biosynthesis of irregular-shaped nanoparticles hopefully might reach this aim because they display novel properties and may improve biological detection sensitivity greatly. The shape of the noble nanoparticle will also play important role as an anti-cancer agent. On the basis of our results, we can speculate on the mechanisms that govern shape-dependent extracellular effect of nanoparticles.
As such, the clove extract itself has reported anti-malignant effect at higher concentration in different types of cancer. The morphology of HeLa, HEK-293, and HT-29 cell line that were treated with AuNP-Clo at 20 μg/mL was studied to prove the effect of these AuNP on the cell growth. Our observations showed that the growth of the cells was inhibited. However, further study is needed to understand the exact mechanism of anti-cancer activity of these nanoparticles. AuNP-Clo proved to possess anti-proliferative properties against all the cancer cell lines tested. The prominent anti-proliferative effect of functionalized AuNP on HEK-293, HeLa, and HT-29 cancer cell lines, as revealed by its IC50 based on XTT assay was found to be 19.88 ± 0.006, 20.05 ± 0.002, 20.12 ± 0.007, and 28.56 ± 0.010, respectively. IC50 of AuNP-Clo was specifically less significant on Vero cell line, i.e., 55.3 ± 2.74. Therefore, it can be said that AuNP-Clo is a promising anti-cancer “lead”. However, the exact mechanism behind the anti-proliferative effects of AuNP-Clo needs to be studied to determine whether the effect is due to an increase in apoptosis. The plasma membrane of the cancer cell defines the separation between the internal constituents of a cell and the outside environment. This semi-permeable membrane allows free diffusion of small and non-polar molecules. However, bigger ones like nanomaterials are incapable of crossing the plasma membrane which requires uptake mechanisms such as endocytosis. Most of bio-functionalized AuNP are easily taken up by the cells through endocytotic mechanisms, but they remain in endosome vesicles, become incapable of reaching the cytosol system. Although endocytotic uptake is the normal phenomenon for a variety of nanomaterials, the efficiency predominantly depends on shape, size, the dispersivity, and the other physico-chemical parameters. This is important, because there may be many factors that affect the anti-proliferative activity in the in vivo study. Further study is underway.
AuNP-Gua has shown activity against HEK-293 but the IC50 > 30 μg/mL. These are devoid of anti-proliferative activity against other three cell lines. AuNP developed with cow urine as a reducing agent has shown activity against HeLa, HEK-293, and HT-29 at 30 μg/mL. Though the inhibitory effect is higher than that of AuNP-Clo, but looking in to the beneficial effects of cow urine, the AuNP synthesized with using cow urine as a reducing medium can be a promising anti-cancer agent. Poly-shaped nanoparticles synthesized using guava extract has shown cytotoxic effect on HEK-293 and the IC50. Functionalized AgNP using other different plant extract and bio-excretory have not shown any cytotoxic effect even at 30 μg/mL. This also proves that the adsorbed bio-moieties alone are devoid of cytotoxic effect at that concentration.
The development of functionalized targeted gold nanoparticles as therapeutic agents has generated great interest in both academy and industry. Targeted gold nanoparticles have shown promising results in in vitro studies, signifying that they are potential as therapeutic carriers. This exploration creates the new avenue to a new standard where the different flavonoids-functionalized gold nanoparticles can be a powerful weapon against cancer.
Financial supports from BRNS (Grant no. 2009/34/14/BRNS) UGC (D.O.No. F.14-4/2001 (Innov. Policy/ASIST)) are acknowledged. We acknowledge SAIF, IIT Mumbai for TEM and Biogenics. Raghunandan Deshpande thank his father Jagannathrao M. Deshpande for editing and Dr. Appala Raju, principal of H.K.E.S’s Matoshree Taradevi Rampure Institute of Pharmaceutical Sciences, Gulbarga for encouraging the research program.
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