J Pharm Innov DOI 10.1007/s12247-013-9166-x
RESEARCH ARTICLE
Biogenesis of Gold Nanoparticles Using Plant Powders and Assessment of In Vitro Cytotoxicity in 3T3-L1 Cell Line Duraisamy Kalpana & P. B. Tirupathi Pichiah & Arunachalam Sankarganesh & Whoa Shig Park & Seok Myon Lee & Rizwan Wahab & Youn Soo Cha & Yang Soo Lee
# Springer Science+Business Media New York 2013
Abstract Gold nanoparticles were used in various biological applications for their structural and functional properties. For the application of gold nanoparticles in biological applications, the cytotoxicity of gold nanoparticles should be validated. Gold nanoparticles were biosynthesized using the three plant powders obtained from leaves of Torreya nucifera , Cinnamomum japonicum , and Nerium indicum . UV–vis spectroscopic analysis ensured the formation of gold nanoparticles and Bio-TEM analysis revealed the size and spherical shape of gold nanoparticles. XRD pattern with the reflection planes (111), (200), (220), and (311) confirmed the face cubic centered structure of biosynthesized gold nanoparticles. Aromatic compounds and proteins were found to be responsible for the bioreduction of gold salt to gold nanoparticles and stabilization of synthesized gold nanoparticles from the FTIR analysis. The synthesized nanoparticles were tested for in vitro cytotoxicity using 3T3-L1 cell lines. The nanoparticles, D. Kalpana : Y. S. Lee (*) Department of Forest Science and Technology, Institute of Agricultural Science and Technology, Chonbuk National University, Jeonju, South Korea e-mail:
[email protected] P. B. T. Pichiah : Y. S. Cha Department of Food Science and Human Nutrition, Chonbuk National University, Jeonju, South Korea A. Sankarganesh Department of Pediatrics, Medical School, Chonbuk National University, Jeonju, South Korea W. S. Park : S. M. Lee Jeonnam Forest Resource Research Institute, Naju 520833, South Korea R. Wahab College of Science, Department of Zoology, King Saud University, Riyadh, Saudi Arabia
synthesized using ecofriendly plant powders exhibited low level of cytotoxicity even at higher concentrations of 10 μg/ ml. This validates that the synthesized molecules are not toxic and they can be analyzed for various biomedical applications. Keywords Gold nanoparticles . XRD . FTIR . Cytotoxicity . 3T3-L1 cell line
Introduction Nanotechnology, one of the technological innovations of twenty-first century is developing rapidly throughout the world. The nanomaterials have been used as fluorescent biological labels [1, 2], in drug delivery [3], detection of proteins [4], tissue engineering [5], separation, and purification of biological molecules and cells [6–10]. Gold nanoparticles are as well used in the biomedical applications such as regulation of intracellular gene [11], electrochemical biosensor for detection of DNA sequence [12], chemotherapeutic agents [13], drug delivery [14, 15], other applications like detection of gene mutation [16] and identification of bacteria from clinical specimens [17]. The surface plasmon absorption property along with the technique of conjugation of gold nanoparticles with DNA can be applied in various biomolecular applications like labeling, detection, transfer of drugs, and even the genetic materials. By optimizing spectroscopic, fluorescence, luminescence, and electrochemical characteristics of the gold nanoparticles with biological molecules such as DNA sugars, many sensors are designed [18]. Spherical nanoparticles are generally produced in a controlled manner by simple reduction of metal salt solutions by using reducing agents. Spheres are considered as the lowest energy shapes and the spherical gold nanoparticles are commonly prepared by one of these methods; Turkevich method, where citrate is used to reduce gold chloride in boiling water
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to produce nanoparticles of around 15 nm, Frens method, Brust method, where the aqueous solution of gold ions are transferred to an organic phase by a phase transfer agent and simultaneously reduced with borohydride, microemulsion method, involving reduction of gold salts in aqueous core of inverse micelles, and seeding method, where gold seed particles synthesized by other methods, are utilized to produce more gold nanoparticles along with addition of weak reducing agents [19]. Gold nanoparticles synthesized using chemicals or by chemical methods have the chemical substances adsorbed onto their surfaces which could be toxic, eliminating their potential use in biomedical applications. Biological production of nanoparticles were carried out using bacteria Lactobacillus [20], Escherichia coli [21], Thermomonospora sp. Rhodo coccus sp., [22, 23] Pseudomonas aeruginosa [24], fungi Fusarium oxysporium [25], and plants Azadirachta indica, lemon grass [26, 27], Avena sativa [28], aloe vera [29], additionally these, gold and silver nanoparticles can also be produced from Dalbergia sissoo [30] in previous studies. In this connection, Sanghi et al. describes the biosynthesis of gold nanoparticles with the use of fungus Coriolus versicolor [31]. Avasthi et al. presents the matrices of gold and silver nanostructures by atom beam co-sputtering technique [32]. In another report, Mishra et al. presented the synthesis of gold nanoparticles embedded in SiO2 via atom beam co-sputtering method for the detection of human ovarian cancer [33]. Pissuwan et al. also checked the surface modification on gold nanoparticles on macrophage cells [34]. Among the various resources, plant-mediated biological synthesis is more economic, simple, and clean as compared to the microbiological synthesis. Gold nanoparticles exhibit both toxicity [35, 36] and nontoxicity [37]. Gold nanoparticles are widely used in various biomedical applications, but in certain fields of applications where they are used as tracers, in drug delivery or gene delivery, they should not be toxic to the target cells, whereas when used as chemotherapeutic agents against cancer cells, the cytotoxic nature of gold nanoparticles is utilized. In the present study, we have utilized Torreya nucifera , Cinnamomum japonicum , and Nerium indicum leaf powders to synthesize the gold nanoparticles using gold salt solution. T. nucifera exhibits neuroprotective, antioxidant, antiviral property, and N . indicum , grown for ornamental purpose, belongs to Apocynaceae with antiulcer property [38–42]. T. nucifera belonging to Taxaceae family is a coniferous tree native to Jeju Island, South Korea and Southern Japan. It has been traditionally used to treat stomach ache, rheumatoid arthritis, and hemorrhoids. C . japonicum belongs to Lauraceae family and are well known for their aromatic oils. The synthesized gold nanoparticles were confirmed by UV– vis spectroscopy and characterizations of the nanoparticles were performed using bio-transmission electron microscopy
(Bio-TEM), X-ray diffraction (XRD), and Fourier transform infrared spectroscopy (FTIR). The in vitro cytotoxicity of the gold nanoparticles was tested using 3T3-L1 cell lines. 3T3-L1 cell line is derived from 3T3 cells. The cells have morphology similar to fibroblast and when the cells are induced or under appropriate conditions, they differentiate into adipocytes and could store the triglycerides.
Materials and Methods Materials Gold (III) chloride trihydrate was purchased from SigmaAldrich, the plants T. nucifera , C . japonicum , and N . indicum were collected from the Wando Arboretum, Wando, South Korea. Synthesis of Gold Nanoparticles Leaves of T. nucifera, C. japonicum , and N. indicum were washed separately under running water to remove the adhered dust particles. The leaves were air dried completely and made into fine powder. One gram of the leaf powder was added into 250-ml volumetric flask containing 100 ml of 1 mM gold (III) chloride trihydrate solution. The flasks were completely covered with aluminium foil to provide dark conditions. The flasks were incubated at 30 °C in a shaking incubator and shook at 200 rpm. The change in color of solution from pale yellow to pink or purple was checked at regular intervals. UV-Visible Spectroscopy The samples were collected at regular intervals of every 1 h and they were filtered to remove the leaf powders. The clear solution was used for spectrum analysis between the wavelengths 400 and 700 nm in Shimadzu UV-3101PC spectrophotometer. The absorbances were taken after 24 h of incubation for culture medium of untreated 3T3-L1 cells, culture medium containing nanoparticles without 3T3-L1cells, and cells treated with nanoparticles at concentrations of 10 and 100 μg/ml. Measurement of Size of Biosynthesized Gold Nanoparticles by TEM Analysis Samples were filtered through Whatman filter paper to remove the leaf powders. The filtrate was centrifuged at 10, 000 rpm for 15 min; the pelleted gold nanoparticles were resuspended in water and washed by centrifugation at 10, 000 for 15 min. The washed gold nanoparticles were resuspended in distilled water for Bio-TEM analysis. Bio-TEM samples were prepared by spotting a drop of aqueous solution of gold nanoparticles over the carbon coated copper grids.
J Pharm Innov Fig. 1 UV–vis absorption spectra of gold nanoparticles at various time intervals bio synthesized using a Torreya nucifera , b Cinnamom japonicum, and c Nerium indicum leaf powder
a 1.0 Torreya nucifera
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Samples were allowed to stand on the grid for 2 min and the excess of solution was removed by blotting with blotting paper and grids were dried well for microscopic analysis. The morphology was analyzed using HITACHI-JP/H7600 instrument functioning at an accelerating voltage of 100 kV. Isolation and Purification of Gold Nanoparticles The solutions were filtered through Whatman filter paper 1 and then centrifuged at 10,000 rpm for 30 min at 4 °C. The pelleted gold nanoparticles were washed twice with sterile distilled water to remove excess of gold salts and additional biomolecules found along with the gold nanoparticles. The washed gold nanoparticles were then dispersed into 2 ml of sterile Millipore water and then freeze dried. The freeze-dried gold nanoparticles powder were used for XRD, FTIR, and cytotoxicity assay.
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The dried gold nanoparticles powdered were fixed over silicon-made zero background holder. The X-ray diffraction pattern were acquired from Rigaku X-ray diffractometer using the monochromatic radiation Cu Kα of wavelength 1.540 A, and diffraction pattern was observed between the angle 30– 80° with a steep angle of 0.05°. Crystalline sizes of the biosynthesized gold nanoparticles were evaluated using Scherrer formula with JADE 9 software. The dried nanoparticles were mixed with pure KBr at a ratio of 5:95 and samples were fixed in the sample holder. FTIR pattern was obtained from FTIR spectrophotometer (PerkinElmer model PE1600) within the spectral range of 400–4,000 cm−1 at a scan speed of 16 cm/s. The FTIR spectrum for leaf powders were also obtained similarly as for the gold nanoparticles.
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3T3-L1 cell line was cultured in Dulbecco's modified eagle medium (DMEM) supplemented with 10 % (v/v) fetal bovine serum (FBS), and 1X of penicillin and 1X of streptomycin. The cells were maintained under standard cell culture conditions of 5 % CO2, 95 % humidity, and 37 °C in CO2 incubator.
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The cytotoxicity induced by gold nanoparticles, synthesized using three different leaf powders, were examined by 3-(4, 5dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide (MTT) assay. 3T3-L1 cells were seeded in a 96-well plate at
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J Pharm Innov Fig. 2 TEM images of the gold nanoparticles biogenesised using a Torreya nucifera, b Cinnamomum japonicum, and c Nerium indicum leaf powder at various magnifications
a density of 2×103 cells per well and incubated for 24 h in humidified incubator with 5 % CO2. The working standard of freeze-dried nanoparticles was made at a concentration of 1 mg/ml in DMEM and then filter sterilized for performing cytotoxicity assay. Cells were exposed to gold nanoparticles ranging between the concentrations of 0.1 ng and 10 μg/ml. The nanoparticles were thoroughly dispersed in DMEM cell culture medium supplemented with 10 % FBS. The cells were treated with blank solution without gold nanoparticles for control and different concentrations of gold nanoparticles for 24 h at 37 °C and 5 % CO2. After the exposure of nanoparticles for 24 h, the medium was removed and cells were
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washed with phosphate-buffered saline. The wells were then replaced with culture medium containing 10 μl of MTT reagent along the sides of the well and incubated for 4 h at 37 °C in the CO2 incubator. After incubation, the tetrazolium crystal formed was dissolved in solubilizing buffer and the absorbance was measured at 570 nm using ELISA reader. The morphology of cells without nanoparticles treatment and nanoparticles treated cells were viewed under microscope after 24 h of incubation period at 37 °C.
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Bragg angle (2 ,Degree) Fig. 3 XRD pattern of dried powder of gold nanoparticles biosynthesized with a Torreya nucifera, b Cinnamomum japonicum, and c Nerium indicum leaf powder
The bulk materials and as well as individual atoms do not possess any intense color but the colloidal gold nanoparticles possesses a very intense color. Visible change in the color from yellow to purple and pink were observed during gold nanoparticles synthesis. The UV–vis spectroscopy is useful and important technique to determine the formation and stability of nanoparticles in aqueous solutions. The spectrum analysis done between the wavelengths 400 and 700 nm revealed maximum absorption peaks within 536–540 nm for the nanoparticles synthesized using T. nucifera, and between 536–545 and 530–533 nm for the gold nanoparticles synthesized with the leaf powder of C . japonicum and N. indicum , respectively. The maximum absorption was found to be within the range of characteristic absorption peaks of gold nanoparticles (Fig. 1a–c). The surface plasmon resonance arises due to collective oscillation of free conduction electrons induced by
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Fig. 4 FTIR spectrum of a leaf powder of Torreya nucifera and b gold nanoparticles synthesized using Torreya nucifera
an interactive electromagnetic field [43]. The synthesis of gold nanoparticles occurred within in 1 h with T. nucifera leaf powder and C. japonicum leaf powder, but it took more than an hour for the N . indicum leaf powder to initiate the synthesis of gold nanoparticle. The nanoparticles were found to be synthesized rapidly by T. nucifera comparatively than the C. japonicum and N. indicum . The absorbance maximum increases as synthesis of gold nanoparticles increases; the
intensity was observed to increase with time with C . japonicum and N . indicum leaf powders, whereas for T. nucifera, the intensity was found to decrease with increase in time and stable after 3 h of synthesis. So the optimum production of gold nanoparticles occurred within 1 h in the case of T. nucifera. Similarly, the optimum production of gold nanoparticles occurred between the third–fourth hour and fourth–sixth hour using the C. japonicum and N. indicum
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Fig. 5 FTIR spectrum of a leaf powder of Cinnamomum japonicum and b gold nanoparticles synthesized using Cinnamomum japonicum
leaf extracts. After the second, third, and fourth hours, respectively, for the gold nanoparticles synthesized using leaf powders of T. nucifera, C. japonicum, and N . indicum, further intensity in absorption was not noticed which indicated the completion of the synthesis. After these time intervals, intensity was found to decrease which could be attributed by the decrease in presence of gold nanoparticles in aqueous solution; after synthesis, the gold nanoparticles were found to start adhering to leaf powders as the time increases. The synthesis
of nanoparticles using leaf powders had both the advantages and disadvantages in separation and purification of nanoparticles. The advantage is that easy separation of leaf powder by filtration and the disadvantage is when time increases or after the completion of synthesis the nanoparticles starts to adsorb on the surface of leaf powders. So it is important to optimize the time interval for completion of nanoparticle synthesis and recover as soon as nanoparticle synthesis is completed.
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Fig. 6 FTIR spectrum of a leaf powder of Nerium indicum and b gold nanoparticles synthesized using Nerium indicum
Determination of Size and Shape by Bio-Tem Analysis Morphology and size of the nanoparticles were determined by using the transmission electron microscopy (TEM) images (Fig. 2). Bio-Tem images of the synthesized nanoparticles indicated that the gold nanoparticles were at most well dispersed and had uniform size. Gold nanoparticles synthesized using the leaf powder of T. nucifera had uniform shape of sphere. Size of the produced spherical nanoparticles was found
to be within the range of 8–42 nm. Spherical nanoparticles were found to be predominant, but few nanoparticles with pentagonal shapes and nanotriangles were also found within the nanoparticles synthesized using leaf powder of C . japonicum . The nanoparticles produced by C . japonicum varied in size between 4 and 35 nm. The shape of nanoparticles obtained using leaf powder of N. indicum was observed to be spherical, ranging between the sizes of 4 and 38 nm. Some nanorods and nanotriangles were also found to be produced by
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C. japonicum
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Fig. 7 Cytotoxicity of gold nanoparticles exhibited on 3T3-L1 cell line synthesized using leaf powder of Torreya nucifera , Cinnamom japonicum, and Nerium indicum
the leaf powder of N. indicum. The gold nanoparticles synthesized using leaf powders of C. japonicum and N. indicum was well dispersed compared to nanoparticles synthesized using leaf powder of T. nucifera (Fig. 2). XRD Analysis XRD patterns of the gold nanoparticles displayed Braggs reflections representative of the face cubic centered (fcc) structure of gold corresponding to JCPDS #04-0784 (Fig. 3). Four peaks at 38.2, 44.2, 64.7, 77.9, 37.95 44.2, 64.7, 77.4° and 38.35, 44.45, 64.7, 77.8° in the two theta range of XRD pattern revealed the (111), (200), (220), and (311) reflections of fcc structure of metallic gold for the gold nanoparticles obtained using T. nucifera, C. japonicum, and N. indicum, respectively. The intensity of the (111) plane was found to be predominant. The average crystallite size was calculated according to Scherrer equation using JADE 9 software for all the reflection peaks. The crystallite structure was calculated to be 11.6, 16.0, 16.7, and 15.4 nm for the reflections obtained for gold nanoparticles synthesized with T. nucifera. For gold nanoparticles synthesized with C . japonicum leaf powder, the size was measured as 21.7, 23.1, 100, and 19.9 nm for the reflections (111), (200), (220), and (311) reflections, respectively. Crystallite sizes of 12.9, 18.9, 19.2, and 15.1 nm were obtained for the gold nanoparticles prepared using N. indicum leaf powder. FTIR Analysis FTIR analysis was performed to identify the biomolecules that are potentially involved in reduction of gold salt to gold, in capping, and stabilization of the formed gold nanoparticles. FTIR analysis was carried out for both the plant powder and the biosynthesized gold nanoparticles (Figs. 4, 5, and 6). Absence of certain peaks in the FTIR spectrum of gold nanoparticles from that of native plant powder indicates that not all the biomolecules present in plant powder were responsible for
gold nanoparticle synthesis and only certain particular biomolecules were involved in formation and stabilization of gold nanoparticles. A peak at range of 3,700–3,770 cm−1 were observed in all the three spectrum obtained for the gold particles synthesized using three plant powders, which was not observed in the spectrum of plant powders. These peaks indicate the C-OH stretch of alcohols including phenol; this indicated that when the plant powders are added to aqueous gold salt solution, aromatic compounds were liberated into aqueous solution from the plant powders. Peaks formed within 3,260–3, 285 cm−1 indicated the normal polymeric OH stretch of alcohol and hydroxy compounds. The C-H stretch of alkanes was revealed out by peaks at 2,920 and 2,850 cm−1 for all the three biosynthesized gold nanoparticles. Formation of strong peak between the ranges of 1,600 and 1,690 cm−1 supported the involvement of proteins in capping and stabilization of gold nanoparticles. Peaks within 1,600–1,690 is due to the C=O stretching and indicated the amide I linkage of proteins. The amide II bond were prominent by formation of peaks between the wave number 1,480 and 1,575 cm−1, which is formed either due to CN stretching or NH bending. The amide III linkage due to CN stretching of NH bending between the range of 1,229 and 1,301 cm−1 was observed with gold nanoparticles synthesized using T. nucifera and N. indicum, but no peak was formed in the case of gold nanoparticle synthesized using C. japonicum. Similarly, strong peaks formed between 1,000 and 1,260 cm−1 indicated the C-O bond of carboxylic acids. FTIR studies revealed that proteins and aromatic compounds present in the plant powders were liberated into solution during the biosynthesis of nanoparticles, and they aid in the stabilization of formed gold nanoparticles. 0.3
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Fig. 8 Visible spectroscopy analysis of cultured cell culture medium, nanoparticles in culture medium without cells, and culture medium of cells after treatment of nanoparticles in medium. 3T3-L1 cells absorbance of culture medium of cells without the treatment of gold nanoparticles, NP absorbance of gold nanoparticles suspended in culture medium without the addition of 3T3-L1 cells, Cells + NP absorbance of culture medium after incubation of cells with gold nanoparticles suspended in culture medium at a concentration of 100 (A1, B1, C1) and 10 μg/ml (A2, B2, C2). Absorbances were measured at 543, 545, and 535 nm for samples A1, A2; B1, B2; C1, C2, respectively, where A, B, C corresponds to the nanoparticles synthesized using T. nucifera, C. japonicum, and N. indicum, respectively
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Fig. 9 Microscopic images of control 3T3-L1 cell lines and cells treated with various concentrations of gold nanoparticles synthesized using different plant powders. Control 3T3-L1 cells untreated with gold
nanoparticles A1, B1, C1, and A2, B2, C2 are 3T3-L1 cells treated with 10 and 100 μg/ml of gold nanoparticles synthesized using T. nucifera, C. japonicum, and N. indicum, respectively
In Vitro Cytotoxicity of Gold Nanoparticles
respectively, synthesized using T. nucifera, C. japonicum, and N. indicum. The absorbance of culture medium measured at maximum absorption for all the three synthesized nanoparticles at concentrations of 10 and 100 μg/ml after 24 h of treatment on 3T3-L1 cells were found to completely decrease as in Fig. 8, compared to the culture medium containing nanoparticles without inoculation of cells and underwent the same period of incubation. These results suggest that the gold nanoparticles were either attached onto the cells or entered into the cells. The 3T3-L1 cells were adherent and absorbance was also measured for culture medium in which only the cells were grown without the nanoparticles, to analyze whether the cells incubated in culture medium affects the absorbance values; controls with only cells and without nanoparticles were given same conditions of treatment and absorbance was measured. The negative absorbance values (nearly zero) indicated that the culture medium does not interfere with the absorbance maximum of gold nanoparticles. It also suggests that decrease in absorbance maximum in culture medium of the cells treated with nanoparticles is because of absence of nanoparticles in the culture medium after treatment. To find whether gold nanoparticles have altered the morphology of cells, they were observed under microscope and the images are presented in Fig. 9. No significant changes in morphology were seen with all the three gold nanoparticles synthesized using three different plant powders at concentrations of 10 and 100 μg/ml. Differentiation of cells
The toxicity of the gold nanoparticles synthesized using various plant leaf powders were analyzed using 3T3-L1 cell line by the widely used MTT cytotoxicity assay. The cytotoxicity exhibited by different concentrations of gold nanoparticles was found to be dose dependent (Fig. 7). The gold nanoparticles synthesized using T. nucifera at concentrations 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, and 10 μg/ml showed cytotoxicity of 12.06, 14.85, 15.34, 17.1, 18.34, and 19.57 %, respectively. And 26.87, 26.03, 26.81, 27.18, 30.15, and 34.83 % of cytotoxicity was seen, respectively, with the cells treated with 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, and 10 μg/ml of gold nanoparticles produced using the leaf powder of C . japonicum. Cytotoxicity of 18.12, 18.78, 18.26, 20.02, 20.5, and 22.78 % was exhibited by the gold nanoparticles synthesized using N. indicum at concentrations 0.1 ng, 1 ng, 10 ng, 100 ng, 1 μg, and 10 μg/ml, respectively. The nanoparticles synthesized by using these three leaf powders were found to be not highly toxic, except for the gold nanoparticles synthesized using C. japonicum leaf powder. As very low cytotoxicity was shown by the gold nanoparticles synthesized using three leaf powders, the uptake of nanoparticles by cells were tested using visible spectroscopy analysis [37]. The gold nanoparticles synthesized were checked for their maximum absorption wavelength and it was observed at 543, 545, and 535 nm,
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into adipocytes and storage of lipids were also clearly seen as observed with the untreated control cells. Studies by Goodman et al. revealed that the cationic particles are moderately toxic and the anionic particles are nontoxic [35]. It is related with the interactions of the nanoparticles with the cell membrane which is mediated by their electrostatic attraction to the negatively charged bilayer. The surface modified gold nanoparticles using citrate and biotin surface modifiers were found to be nontoxic at concentrations up to 250 μM for an exposure of nanoparticles to 3 days on the K562 leukemia cell line. It was also found that the gold nanoparticles were nontoxic, though they are taken inside by the cells [37]. The capping molecules of gold nanoparticles were found to be responsible for cytotoxicity of the tested cells and they were not found to influence the uptake of nanoparticles in human ATII-like cells lines [44]. The nanoparticles synthesized by using three different leaf powders did not differ much in their cytotoxic activity which could be attributed by the same kind of capping molecules of gold nanoparticles and not much difference in either the shape or size was found between the three different gold nanoparticles.
Conclusion The gold nanoparticles of uniform shape were successfully synthesized rapidly using three different leaf powders of T. nucifera, C. japonicum, and N. indicum. Including synthesis, the prepared nanoparticles were characterized with the standard characterization techniques such as X-ray diffraction spectroscopy, TEM, and FTIR spectroscopy. The X-ray diffraction spectroscopy used to check the crystalline property of the materials showed that the nanoparticles had a face-centered cubic structure and was stabilized by proteins and aromatic compounds. The TEM is used to determine the general morphology of biogenesised spherical nanoparticles with T. nucifera, C. japonicum, and N. indicum leaf powder. The chemical composition of the biomolecules involved in the reduction of salt to nanoparticles was analyzed with FTIR spectroscopy, which justifies that the plant powder were responsible for the formation of gold nanoparticles. The nanoparticles were produced by green methods and found to be nontoxic to 3T3-L1 cell line suggesting its potential use in the biomedical applications. Acknowledgments The project was funded by National Research Foundation of Korea (Grant No: 1101000369). Authors thank National Research Foundation of Korea for the financial support.
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