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Cytotoxicity and Cellular Internalization Studies ofBiogenic Gold Nanotriangles in Animal Cell LinesAmit Singh a d , Ravi Shukla b , Shabir Hassan b , R. R. Bhonde b & Murali Sastry ca Physical Chemistry Division, National Chemical Laboratory, Pune, Indiab Tissue Engineering and Banking Lab, National Center for Cell Sciences, Pune, Indiac Tata Chemicals Innovation Center, Mulshi, Pune, Indiad National Institute for Nanotechnology, Edmonton, Albert, CanadaVersion of record first published: 16 Dec 2011.

To cite this article: Amit Singh , Ravi Shukla , Shabir Hassan , R. R. Bhonde & Murali Sastry (2011): Cytotoxicity and CellularInternalization Studies of Biogenic Gold Nanotriangles in Animal Cell Lines, International Journal of Green Nanotechnology,3:4, 251-263

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International Journal of Green Nanotechnology, 3:251–263, 2011Copyright c© Taylor & Francis Group, LLCISSN: 1943-0892 print / 1943-0906 onlineDOI: 10.1080/19430892.2011.633479

BIOMEDICINE

Cytotoxicity and Cellular Internalization Studies ofBiogenic Gold Nanotriangles in Animal Cell Lines

Amit SinghRavi Shukla

Shabir HassanR. R. BhondeMurali Sastry

ABSTRACT. Biogenic gold nanotriangles have been used in this study to understand their cytotoxicityand biocompatibility in animal cells. These gold nanotriangles were synthesized using the leaf extractof the lemon grass (Cymbopogan flexuosus) plant. Cancerous as well as non-cancerous cells were usedto study their dose dependent viability on exposure to the gold nanotriangles. Additionally, it has beenshown that gold nanotriangles are internalized inside the cells and are compartmentalized into thecytoplasm. Thus, it has been inferred that the biologically synthesized gold nanotriangles are indeedbiocompatible and, thus, are promising candidates as scaffolds for delivery of drug, genes, or growthfactors inside the cells. Additionally, their unique optical properties make them a promising candidatefor hyperthermic treatment of cancer. The AFM analysis of the cells treated with gold nanotrianglesshowed pits on their surface, which could be the probably point of entry of these nanotriangles into thecells.

KEYWORDS. biogenic nanotriangle, cytotoxicity, biocompatibility, internalization, human cells

Received 2 March 2011; accepted 3 October 2011.A.S. thanks Council of Scientific and Industrial Research (CSIR), Government of India for financial

assistance.Amit Singh is affiliated with the Physical Chemistry Division, National Chemical Laboratory, Pune, India.Ravi Shukla, Shabir Hassan, and R. R. Bhonde are affiliated with the Tissue Engineering and Banking

Lab, National Center for Cell Sciences, Pune, India.Murali Sastry is affiliated with the Tata Chemicals Innovation Center, Mulshi, Pune, India.Amit Singh is currently at the National Institute for Nanotechnology, Edmonton, Albert, Canada.Address correspondence to R. R. Bhonde, Tissue Engineering and Banking Lab, National Center for Cell

Sciences, Pune 411007, India. E-mail: rrbhonde@nccs.res.in

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INTRODUCTION

Nanotechnology is revolutionizing human lifein a major way over the past decade. Metalnanomaterials have specially been investigatedfor several applications in the field of cataly-sis,[1,2] fuel cells,[3] heavy metal detection,[4,5]

photonic band-gap materials,[6] non-linear op-tical devices,[7] surface-enhanced Raman spec-troscopy,[8] chemical sensing,[9,10] biology, andmedicine.[11] Colloidal gold has been of primeinterest to researchers and several works thathave led to its potential application in the fieldof biodiagnostics,[12] therapeutics,[13] drug deliv-ery,[14] bioimaging,[15] immunostaining,[16] andbiosensing.[17] Thus, the future use of goldnanostructures for various biological and clin-ical applications has been envisioned.[18]

Owing to the fact that metal nanoparticleshave found a strong niche in the world ofnanotechnology, there has been a constant con-science effort to develop recipes for synthesis ofmetal nanostructures of varying sizes, shapes,and properties. To this date, synthesis of rods,[19]

disks,[20] triangular prisms,[21] multipods,[22]

cubes,[23] and nanoshells[24] have successfullybeen reported using several physical andchemical methods. However, recent biologicalsyntheses of metal nanostructures have gainedtremendous popularity due to the environmen-tally friendly green chemistry approach. Afterthe first report on the use of a microorganism(Pseudomonas stutzeri AG259) by Klaus etal.[25] for synthesizing silver nanoparticles intra-cellularly, there have been numerous attemptsto use biological materials. To this date, reportsare available on successful use of several bacte-ria,[26] S-layer,[27] fungi,[28] algae,[29] and plantsystems[30] for the synthesis of metal nanos-tructures. However, precision over synthesisof nanoparticles of an anisotropic shape usingbiological systems has been an unrealized dreamfor a long time. Shao et al. had used asparticacid for synthesis of gold nanotriangles.[30]

Yet, the major breakthrough was the report bySastry and coworkers on the synthesis of a largepopulation of gold nanotriangles by a roomtemperature method, using leaf extract of lemongrass plant (Cymbopogon flexuosus).[31] Thenanotriangles thus formed show very interesting

optical properties with a strong absorbance inthe near-IR (NIR) region due to their anisotropicshape. Sastry and coworkers have shown thatthe NIR optical absorbance peak of these goldnanotriangles can be tuned at will by controllingthe rate of reduction through variation in theamount of reducing agent[32] or the temperatureof the reaction.[33] Due to this strong opticalabsorbance in the NIR region, these triangleshave been shown to have potential applica-tion in the field of optical coatings to blockhigh-energy radiations.[32] Additionally, onefuturistic application that can be envisaged is inthe hyperthermic treatment of tumorous cancer.

However, the biocompatibility of the goldnanotriangles is a key issue to be addressedbefore its use in any clinical or biologicalapplication. One of the important aspects to beconsidered here should be the cytotoxicity of thegold nanotriangles due to their size,[34] shape,and property;[35] chemical composition;[36] orinteraction with cell surface,[37] which havebeen known to affect the biocompatibility ofa material. The initial studies on cytotoxicityof nanomaterials have been mainly focused onaerosols and their intake and concentration inthe lungs.[38] Later, the toxicity effects of heavymetals[35] and quantum dots[39] were studied ingreat detail. However, with a proven potentialin the biomedical field, gold has also be usedas a system to study the biocompatibility andcytotoxicity effect. There are numerous reportson the cytotoxicity studies of gold (I)[40] andgold (III)[41] precursors, apart from the goldnanoparticles itself.[42]

Although a lot of work has been done toascertain the cytotoxicity levels and biocom-patibility issues related with gold nanoparticles,Sastry and coworkers gave the first detailedaccount of the immunological response ofcells on exposure to the gold nanoparticles andelucidated the mode of internalization.[43] Theyreported gold nanoparticles synthesized bysodium borohydride reduction did not show anyvisible cytotoxicity up to 100 µM concentrationin macrophages. Furthermore, it was shownthat gold nanoparticles did not elicit anystress-induced production of pro-inflammatorycytokines TNFα or IL-1β in the macrophages.At higher concentrations, the gold nanoparticles

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inhibited the secretion of reactive oxygenspecies (ROS) and reactive nitrite species(RNS) and, thus, indicated that they havemuch to offer in the gold nanoparticle basedbiomedical applications. It was observed thatthe gold nanoparticles are internalized inside thecells by the mechanism of pinocytosis and arecompartmentalized in the lysosomes to arrangein the perinuclear space without entering thenucleus. Thus, this report, in particular, hasrationalized the attempts from different groupsacross the world on the use of gold nanoparticlesfor various biomedical applications.

In the work presented in this paper, anattempt has been made to perform similarexperiments on the biogenic anisotropic goldnanotriangles. Biological synthesis has beenlooked upon as an environmentally benignreplacement to the toxic chemical methods forsynthesis of nanostructures. It is believed thatthe biogenic nanostructures should be better atbiocompatibility and, thus, will have immenseapplication for biological and clinical prospects.However, no sincere attempt has been madeto scientifically confirm this hypothesis. Thefollowing sections are devoted to address theseissues using gold nanotriangles. The goldnanotriangles were synthesized by the reductionof chloroaurate ions using the leaf extract oflemon grass plant. Selectively, small-sized goldnanotriangles were synthesized for this work,which have NIR absorbance maxima centeredat approximately 950 nm. This was done withthe anticipation that these triangles could be anexcellent candidate for hyperthermic treatmentof cancer cells, if the optical properties aretuned such that the live cells are transparentto that wavelength.[44] The cytotoxicity studieswere performed on cancerous as well as non-cancerous human cell lines, which revealed thatgold nanotriangles show up to 80% cell viabilitywhen cells were exposed at 800 µM concentra-tions for 24 h. This result is comparable to thedata obtained from chemically (sodium boro-hydride) reduced spherical gold nanoparticlescytotoxicity data at 100 µM concentration,[43]

thus confirming better biocompatibility withbiogenic gold nanotriangles. The flow cytometryanalysis and phase contrast microscopy imagesconfirm that the gold nanotriangles are indeed

internalized inside the cells in the cytoplasmicspace but do not enter the cell nucleus.

MATERIALS AND METHODS

Materials

All chemicals and materials were obtainedfrom Sigma-Aldrich Chemicals, St. Louis, MO,and used as-received unless mentioned other-wise.

Synthesis of Gold Nanotriangle

The gold nanotriangles were synthesized asdescribed elsewhere.[31] In a typical experiment,lemon grass (Cymbopogon flexuosus) broth wasprepared by boiling 100 g of freshly cut and thor-oughly washed leaves of the plant in 500 mL wa-ter for 5 min. Amounts of 1, 1.4, 1.8, 2.2, 2.6, and3 mL of this broth was then added to 10 mL of10−3 M aqueous solutions of chloroaurate ionsand the reduction process was monitored by UV-vis-NIR spectroscopy measurements. The vary-ing amount of broth helps to control the rateof chloroaurate ion reduction. It has been ob-served that higher the amount of the broth (re-ducing agent), the higher is the rate of reductionand, thus, the smaller is the size of nanotrianglesformed. The optical absorption of these test so-lutions helped in choosing the concentration ofthe broth, which formed gold nanotriangles withNIR optical maxima at approximately 950 nm.It was observed that when 3 mL of broth wastaken in 10 mL of 10−3 M chloroaurate ions, weobtained a solution with the desired optical ab-sorption characteristics. The process was scaledup by making 5 batches of the solutions, where30 mL of the broth was added to 100 mL of10−3 M chloroaurate ions in each batch to main-tain the same ratio as in the test solution. Thesolution gives a ruby-red color rather than thetypical brown-red color obtained for the solu-tion prepared by standard procedure, indicatingthat it contains more of spherical nanoparticlesrather than the triangular ones. The triangularnanoparticles were purified from the solution bytwo rounds of centrifugation at 3000 rpm for 30min each, followed by two rounds at 2000 rpm

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for 30 min. The pellet was thoroughly washedwith deionized water after each round of cen-trifugation and was finally resuspended in 5 mLof deionized water by sonication for 10 min. Thepellets from all the batches were pooled togetherto get the final solution of purified gold nanotri-angles, which was characterized by UV-vis-NIRand transmission electron microscopy (TEM).Simultaneously, atomic absorption spectroscopyof the solution was also done, which revealedthat the concentration of gold in the final solu-tion was 2.4 mM.

Cell Culture

The cells were cultured in Dulbecco’s mod-ified Eagle’s medium (DMEM). The mediumwas supplemented with 10% fetal bovine serum(Gibco BRL, Carlsbad, CA), 100 units/mL peni-cillin, 100 ı́g/mL streptomycin, and 2 mM glu-tamine in a humidified atmosphere of 5% CO2and 95% air at 37◦C.

Gold Nanotriangle Treatment andCytotoxicity Determination by MTT Assay

The cytotoxicity studies were performedin the cancerous (RAW264.7 macrophages &MCF-7 human breast cancer cell line) as wellas non-cancerous (NIH 3T3 mouse embry-onic fibroblast cell line) cells. The RAW264.7macrophages were cultured in RPMI-1640 cul-ture medium while the MCF-7 and NIH 3T3 cellswere cultured in Dulbecco’s modified Eagle’smedium (DMEM). Both the culture mediumswere supplemented with 10% fetal calf serum(FCS), 100 units/mL penicillin, 100 µg/mLstreptomycin, and 2 mM glutamine and the cellswere incubated in a humidified atmosphere of5% CO2 and 95% air at 37◦C. Actively growingcells were seeded with density around 1 × 105

cells/well in a 96-well tissue culture plate. Thecells were treated with different concentrationsof gold nanotriangles (5, 25, 50, 100, 200, 400,and 800 µM) for 24 h. The control cells were nottreated with gold nanotriangles and were keptin the same volume of phosphate buffer saline(PBS, pH = 7.4) for the same period of time. Af-ter the end of the exposure time, the cell viabil-ity was checked using the 3-(4,5-dimethylazol-2-yl)-2,5- diphenyl-tetrazolium bromide (MTT)

assay.[45] The MTT assay is based on the opticaldetection of the purple colored formazan at 570nm, which is formed by the enzymatic reduc-tion of yellow tetrazolium MTT. All experimentswere performed 3 times in quadruplets, and theiraverage has been shown as cell-viability percent-age in comparison with the control experiment.The gold triangles untreated controls were con-sidered to be 100% viable. As a control, MTTassays were also performed with gold nanotri-angle solution only at all concentrations, whichshowed no formation of formazan crystals and,thus, eliminated the possibility of a false posi-tive.

Enzyme-Linked Immunosorbent Assay(ELISA)

The RAW264.7 cells were seeded at the celldensity of 1 × 106 cells/well, in the 6-well tis-sue culture plates and grown overnight. The cellswere treated with 250 µM concentration of goldnanotriangles for 24 h. Simultaneously, the cellstreated with 5 µM concentration of bacteriallipopolysacchride (LPS) were taken as the pos-itive control for this experiment while the un-treated cells served as the standard control. Theculture supernatants in all the cases were assayedfor the pro-inflammatory cytokines TNF-α (tu-mor necrosis factor-α) by enzyme-linked im-munosorbent assay (ELISA) using the Opt-EIAkits (BD Biosciences). The ELISA experimentswere performed as per the instructions given bythe manufacturer. The RAW264.7 macrophagecells were chosen for this experiment due to thefact that macrophages are primary immune ef-fector cells and, thus, there would be maximumprobability of particle internalization by themthrough active phagocytosis. Therefore, they arethe most suitable candidates for study of stressinduction.

UV-vis-NIR Spectroscopy Measurements

The synthesis of gold nanotriangles and theirpurification by centrifugation was monitoredbyUV-vis spectroscopy on a Jasco dualbeamUV-vis-NIR spectrophotometer (model V-570)operated at a resolution of 2 nm.

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Biogenic Gold Nanotriangles in Animal Cell Lines 255

TEM Measurements

Samples for TEM measurements were pre-pared by drop-coating films of the different goldnanoparticle solutions on carbon-coated cop-per TEM grids followed by measurements ona JEOL model 1200EX instrument operated atan accelerating voltage of 120 kV.

AFM Measurements

Cell imaging was done in the tapping and con-tact modes by AFM on a VEECO Digital Instru-ments multimode scanning probe microscopeequipped with a Nano- Scope IV controller. Forsample preparation, NIH 3T3 cells were seededat a density of 5 × 104 cells/mL per glass cov-erslip and grown for a period of 24 h. The cellswere exposed to 100 µM gold nanoparticles for15 min followed by several washings with PBS(pH 7.0). The cells were fixed with freshly pre-pared 2% chilled paraformaldehyde (ICN, Au-rora, OH) in PBS for 10 min. After fixation, thecells were washed 5 times with PBS (pH 7.0)and coverslips were attached to metallic packswith conducting double-sided tape. The metallicpacks were mounted on a 6399e-piezoscanner(100 µm) for AFM analysis. Phase and frictionimages were collected in the tapping and contactmode, respectively, at a scanning frequency of 1Hz.

Flow Cytometry Analysis

The NIH 3T3 fibroblast cells were treatedwith 50, 100, and 250 µM concentration of goldnanotriangles and incubated for 24 h in a humidi-fied atmosphere of 5% CO2 and 95% air at 37◦C.Thereafter, the cells were washed thoroughly inphosphate buffer saline (PBS, pH 7.4) to removeany surface bound or uncoordinated gold nan-otriangles. These cells were then checked forchange in their cellular granularity using flowcytometry. Additionally, the cells were also im-aged under an inverted phase contrast micro-scope to confirm that the gold nanotriangles areinternalized inside the cells.

FIGURE 1. (A) UV-vis-NIR spectra of the goldnanotriangle solutions. Curves 1–6 correspondto 1, 1.4, 1.8, 2.2, 2.6, and 3 mL of broth, respec-tively, in 10 mL of 10−3 M chloroaurate ions. (B)UV-vis-NIR spectra of as-synthesized solution(curve 1) and purified gold nanotriangle solution(curve 2).

RESULTS AND DISCUSSION

The test solutions were analyzed by UV-vis-NIR spectroscopy and Figure 1A shows the op-tical spectra of the different gold nanoparticlesolutions obtained. Curves 1–6 show the absorp-tion spectra of the as-synthesized gold nanotri-angle solution using 1, 1.4, 1.8, 2.2, 2.6, and3 mL of broth, respectively, in 10 mL of 10−3

M concentration of chloroaurate ions. The po-sition of the absorption maxima in the NIR re-gion (longitudinal plasmon peak) clearly showsa blue shift with increasing concentration of thelemon grass broth (reducing agent) in the solu-tion. Additionally, a transverse plasmon peak isobserved in the visible region centered at 530nm in all the curves. Also, the plot clearly re-veals that curve 6 shows the NIR plasmon cen-tered at 950 nm, which corresponds to the goldnanoparticle solution synthesized using 3 mL oflemon grass broth. As has been discussed, thesame recipe for synthesis of gold nanotriangleswas used for all the further analysis. The reasonbehind choosing the solution with NIR opticalabsorption maxima at 950 nm is that the humancells are optically transparent at this wavelengthand, thus, the gold nanotriangles in this solutioncan serve as excellent candidates for hyperther-mic treatment of cancerous cells.

Figure 1B shows the UV-vis-NIR spectra ofthe as-synthesized gold nanoparticle solution

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FIGURE 2. (A) Representative TEM image of as-synthesized gold nanotriangles. (B) TEM imageof the purified gold nanotriangles. The scale bars in A and B correspond to 200 and 100nm. (C)Particle size distribution plot of the gold nanotriangles. The average edge length was found to be113 nm.

(curve 1) prepared by reducing 100 mL of 10−3

M concentration chloroaurate ions using 30 mLof the lemon grass broth. It can be clearly con-cluded from the nature of the curve that the ab-sorption characteristic of the gold nanoparticlesolution remains essentially the same when thereaction was carried out using 100 mL of ini-tial volume of chloroaurate ions (compared tocurve 6, Figure 1). However, it is worthwhilementioning that this does not hold true for largervolumes, where we observe change in the ab-sorption profile of the final solution. Therefore,it was necessary to prepare 5 small batches of100 mL each, rather than one batch of 500 mL ofchloroaurate ions with corresponding amount ofthe lemon grass broth as per the ratio followed.Curve 2 in Figure 1B corresponds to the puri-fied gold nanotriangles after the centrifugation.It can be seen from the intensity of the longi-tudinal to transverse plasmon peak that the tri-angular nanoparticle population increased in thesolution after centrifugation, as compared to theas-synthesized solution (curve 1). Thus, the as-synthesized solution was indeed enriched in thepopulation of gold nanotriangles after centrifu-gation. However, some spherical gold nanopar-ticles do remain in the solution along with thetriangles, as can be realized from the intensepeak at 535 nm in the optical spectrum from thepurified solution (curve 2). The TEM analysis inthe following section will reveal that the trian-gles synthesized by this recipe are small in size,and thus it is very difficult to separate them com-

pletely from the spherical particles by the use ofcentrifugation based purification.

The as-synthesized and purified gold nano-triangle solutions were analyzed by TEM imag-ing. Figure 2A shows the TEM micrograph of theas-synthesized gold nanotriangles, which clearlyshows a large population of spherical gold nano-triangles along with the triangles. However, themost important observation to be made here isthat the triangles synthesized by this method (in-creasing the amount of reducing agent) are muchsmaller in size as compared to those formedby the standard method described elsewhere[31].Figure 2B shows the representative TEM micro-graph obtained for the purified gold nanotrian-gles. The image clearly indicates that the pop-ulation of gold spherical particles has reducedconsiderably in the solution after the purifica-tion step and the relative population of triangleshas improved significantly. Figure 2C shows theparticles size distribution plot for the gold nan-otriangle synthesized by the modified protocol.It is observed that nearly 60% population of thegold nanotriangles is in the size range 80–120nm compared to the average size of 440 nmobserved for the gold nanotriangles synthesisof standard method. This observation corrobo-rates well with the optical absorption spectra ofthe solution, which showed that the NIR peakwas centered at approximately 950 nm (Figure1B, curve 2). Thus, TEM imaging and particlesize distribution analysis clearly indicates thatthe gold nanotriangles, which have absorbance

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Biogenic Gold Nanotriangles in Animal Cell Lines 257

FIGURE 3. (A) MTT assay showing cell viabilityduring exposure of RAW264.7 cells to varyingconcentration of gold nanotriangles for 24 h. Thewhite and gray bars correspond to exposure ofcells to gold salt precursor and gold nanotrian-gles respectively. (B) MTT assay showing cellviability during exposure of gold nanotriangle tocancerous MCF-7 (�), non-cancerous NIH 3T3(•), and exposure of gold salt precursor to non-cancerous NIH 3T3 (�) for 24 h.

maxima at 950 nm, are predominantly those thathave an edge length ranging between 80–120nm.

The cytotoxicity of gold nanotriangles underin vitro conditions in RAW264.7 macrophagecells was examined in terms of the effect ofgold nanoparticles on cell proliferation by theMTT assay. Figure 3A shows the histogram plotof MTT assay results for cell viability studiesperformed on RAW264.7 macrophage cell lineafter exposure to varying concentrations of goldnanotriangles. The white bars correspond to thegold precursor (aqueous solution of HauCl4)while the gray bars correspond to the% cellviability at the given concentration of goldnanotriangles. The plot clearly shows 75% cellviability for the cells that were treated with 800µM concentration of gold nanotriangles for 24h. On the contrary, the LD50 (lethal dose 50, i.e.,the concentration of the material in questionwhich causes death of 50% of cell population) isachieved in the case of gold precursor at a con-centration as low as 50 µM. Thus, it can be easilyconcluded from this result that the gold salt pre-cursor (AuCl4− ions) is highly toxic to the cellswhile the biogenic gold nanotriangles do notshow acute toxicity even at high concentrations.This result becomes even more important whenit is compared with the result of the same exper-

iment using chemically synthesized sphericalgold nanoparticles[43]. The borohydride-reducedgold nanoparticles showed nearly 80% cellviability at the exposure to 100 µM concen-tration for 24 h. Thus, it shows that biogenicgold nanotriangles are more biocompatiblethan chemically synthesized borohydride goldnanoparticles and can be looked upon as promis-ing candidates for biomedical applications.

Figure 3B shows the MTT assay results forcell viability studies performed on cancerousMCF-7 (�) and non-cancerous NIH 3T3 (•) cellsafter 24 h exposure to gold nanotriangles. Simul-taneously, NIH 3T3 cells were also exposed togold salt precursors (�) and checked for cell vi-ability after 24 h. The percentage viability of thecells has been calculated considering the respec-tive untreated cells to have 100% viability. TheMCF-7 cells show almost 100% viability afterexposure to 400 µM concentration of gold nano-triangles for 24 h. It is also seen that the viabilityat lower concentrations is nearly 110%, whichsuggests that the gold nanotriangles have somegrowth proliferating effects on the MCF-7 cells.When the gold nanotriangles were exposed tothe non-cancerous NIH 3T3 fibroblast cells, itwas observed that the cells show 83% viabilityfor 400 µM concentration of the gold nanotri-angles after exposure for 24 h. When these non-cancerous cells were exposed to the gold saltprecursor, the LD50 was attained at the concen-tration of 50 µM and at 400 µM concentration,almost all the cells were found dead. Thus, theseresults suggest that the gold nanotriangles do notshow any cellular toxicity to non-phagocytoticcancerous as well as non-cancerous cells whilethe gold salt precursor showed acute toxicity tothe cells.

The RAW264.7 cells were seeded at the celldensity of 1 × 106 cells/well, in the 6-well tis-sue culture plates and grown overnight. The cellswere treated with 250 µM concentration of goldnanotriangles for 24 h. Simultaneously, the cellstreated with 5 µM concentration of bacteriallipopolysacchride (LPS) were taken as the pos-itive control for this experiment while the un-treated cells served as the standard control. Theculture supernatants in all the cases were as-sayed for the pro-inflammatory cytokines TNF-α (tumor necrosis factor-α) by enzyme-linked

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immunosorbent assay (ELISA) using the Opt-EIA kits (BD Biosciences). The ELISA ex-periments were performed as per the instruc-tions given by the manufacturer. The RAW264.7macrophage cells were chosen for this exper-iment due to the fact that macrophages areprimary immune effector cells and thus, therewould be maximum probability of particle inter-nalization by them through active phagocytosis.Therefore, they are the suitable candidates forstudy of stress induction.

Macrophages are one of the primary im-mune effector cells and are known to be activelyphagocytotic in nature. Thus, in addition to thecytotoxicity studies on exposure to the gold nan-otriangles, the immunological response of theRAW264.7 macrophages cells was analyzed byenzyme linked immunosorbent assay. Here, thepro-inflammatory cytokine, TNF-α productionwas analyzed at the protein level to check thestress induction in the cells on exposure to goldnanotriangles. Figure 4 shows the histogram ofthe results from the ELISA analysis for the un-treated control cells, bacterial LPS treated pos-itive control, and the cells treated with 250 µMconcentration of gold nanotriangles. It can beclearly seen that the untreated control cells didnot show any sign of stress and the produc-tion of TNF-α cytokine was negligible. How-ever, the macrophage cells, which were exposedto 250 µM concentration of gold nanotriangles,

FIGURE 4. The ELISA analysis usingRAW264.7 macrophage cells showing theexpression of TNF-α in control, lipopolysac-charide (LPS) treated and gold nanotriangles(Au-TNP) treated cells.

show 35 pg/mL concentration of the cytokine.Thus, it can be concluded that exposure of thegold nanotriangles does cause some stress to themacrophages, even though the cells are viableafter 24 hours of incubation. The cells that wereincubated with bacterial LPS showed a signifi-cant amount of TNF-α production, which is ex-pected as bacterial LPS in known to cause stressto the cells. Thus, the ELISA analysis suggeststhat the exposure of gold nanotriangles to themacrophage cells indeed induces stress on thecells and elicits immunological response in theform of production of the pro-inflammatory cy-tokine TNF-α. However, the cytokine producedwas much smaller in concentration comparedto the positive LPS treated control even at highconcentration.

The NIH 3T3 fibroblast cells were treatedwith 50, 100, and 250 µM concentrations of goldnanotriangles and incubated for 24 h in a humidi-fied atmosphere of 5% CO2 and 95% air at 37◦C.Thereafter, the cells were washed thoroughly inphosphate buffer saline (PBS, pH 7.4) to removeany surface bound or uncoordinated gold nan-otriangles. These cells were then checked forchange in their cellular granularity using flow cy-tometry. Additionally, the cells were also imagedunder an inverted phase contrast microscope toconfirm that the gold nanotriangles are internal-ized inside the cells. The advantage of using goldnanotriangles is that they are much bigger in sizewhen compared with spherical nanoparticles orquantum dots, which have been used earlier forsuch studies. Thus, they can be easily viewed un-der a simple inverted phase contrast microscopeat 60X magnification. The control untreated andthe cells treated with 100 µM concentration ofgold nanotriangles were also analyzed by atomicforce microscopy (AFM) to observe any changein the surface texture of the cells on exposure.

The NIH 3T3 fibroblast cells were analyzedusing a flow cytometer to find any change in thecellular granularity after exposure to the goldnanotriangles. In principle, if the gold nanotri-angles are internalized by the cells, the gran-ularity inside the cells should increase, whichcould be picked up by analyzing the side scatterplot. Figure 5 shows the forward and side scatter(FSC/SSC) plots obtained for the untreated con-trol cells (Figure 5A) and the cells treated with

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FIGURE 5. The flow cytometry analysis of (A)untreated control NIH 3T3 fibroblast cells and (B)fibroblast cells treated with 50 µM concentrationof gold nanotriangles for 24 h.

50 µM concentration of gold nanotriangles (Fig-ure 5B). Thus, Figure 5A reveals the granularityof the untreated control cells, which shows onlyone type of cells predominantly. However, whenthe FSC/SSC plot for the cells treated with 50µM concentration of gold nanotriangles is ob-served (Figure 5B), we see that the majority ofthe cells show an increased side scatter profileas compared to the control (Figure 5A). Thus, inthe FSC/SSC plot of the treated cells, we havetwo types of cell populations: one, which showsthe FSC/SSC profile corresponding to that of thecontrol cells, and the other, which shows an in-creased side scatter plot. The population of cellsof the former types is the one that does not showuptake of gold nanotriangles, and, thus, the gran-ularity of the cells do not change when comparedwith the control cells (Figure 5A). This may bedue to the fact that at a given time, not all cellsin the population will have phagocytotic activityand so not all cells are able to take up the goldnanotriangles in the vicinity.

It is known that the increase in the side scat-ter profile of a cell is a direct measure of theincreased granularity inside that cell. Thus, theFSC/SSC plot of the treated cells clearly indi-cates that the cellular granularity inside a ma-jority of cells increases after the treatment withgold nanotriangles. This gives an indirect indica-tion that the gold nanotriangles are internalizedinside the fibroblast cells and are localized in thecytoplasmic space, thereby increasing the cellu-lar granularity. It is important to realize that thereis a streak of dots, which are diverging from the

original cell population compared to the controland show a varying degree of side scatter. Thisshows that different cells show different degreeof uptake of the gold nanotriangles and, thus, agraded variation in the side scatter is obtained.This can be accounted for based on the fact thatall cells in a population do not show same de-gree of phagocytotic activity and, thus, the cel-lular uptake of gold nanotriangles will not beuniform in all the cells. However, to further con-firm this observation, the untreated controls aswell as the experimental treated fibroblast cellswere observed under an inverted phase contrastmicroscope, for direct visual evidence.

The non-cancerous NIH 3T3 cells weretreated with 100 and 250 µM concentration ofgold nanotriangles for 24 h and imaged underan inverted phase contrast microscope at 60Xmagnification. The untreated cells were taken ascontrol for this experiment. Figure 6A shows thecontrol fibroblast cell. It can be seen that the cellsare either attached to the surface of the cultureplate and can be seen in their elongated and flatmorphology or are detached from the substrateand dispersed in the culture medium, showinga spherical morphology. Figure 6B shows thefibroblast cells treated with 100 µM concentra-tion of gold nanotriangles. It can be seen that thecells show a lot of particulate matter within thecytoplasm while the round nucleus of the cellsdo not show them. This proves that the goldnanotriangles do get internalized inside the cellsand compartmentalize in the cytoplasmic space.This also confirms that the gold nanotriangles donot enter the nucleus of the cells. The arrows inthe image show the cells with the gold nanotri-angles showing the contrast in the cytoplasmicregion while the nuclear region does not showany contrast.

Figure 6C shows the fibroblast cells treatedwith a 250 µM concentration of gold nanotrian-gles. The image shows a highly dense contrastin the cytoplasmic region of the cells indicatingthat the cellular uptake of gold nanotriangles ismuch higher. Thus, it can be concluded that theamount of uptake of the gold nanotriangles bythe cells is concentration dependent. Also, at thisconcentration, the nuclear region of low contrastis seen prominently and confirms the observa-tion that the gold nanotriangles are confined in

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FIGURE 6. (A) Control untreated NIH 3T3 fibroblast cells. (B) Fibroblast cells treated with 100 µMconcentration of gold nanotriangles. (C) Fibroblast cells treated with 250 µM concentration of goldnanotriangles.

the cell cytoplasmic space and do not enter thenucleus. Another important observation that canbe made from the images in Figure 6 A and B isthat the density of the particles in the differentcells in not uniform; some cells show high up-take of gold nanotriangles while several othersshow lower concentration of gold nanotriangles.This observation confirms the graded side scat-ter population of the cells obtained in the flowcytometry analysis (Figure 5B), which is pri-marily due to non-uniform density of the goldnanotriangles in the cytoplasm of the fibroblastcells.

Figure 7A shows the atomic force microscopyimage of the untreated NIH 3T3 fibroblast cellsshowing their elongated and flat morphology onattachment to the substrate. The height of thecell was calculated across the line drawn at the

edge of the cell and it was found to be 245 nm(Figure 7B). The 3D profile of the untreated fi-broblast cell has been shown in the Figure 7C,which shows a smooth and uniform surface withno deformity. However, the fibroblast cells thatwere treated with 100 µM concentration of goldnanotriangles showed a very rough surface tex-ture (Figure 8A). The AFM micrograph revealsthe presence of pits on their surface, which havebeen indicated in the image by white arrows. The3D profile of the same image has been shown inthe Figure 8B where the surface pits can be ob-served much more prominently correspondingto the surface features indicated in the 2D image(Figure 8A). The size of pits was found to be300 to 400 nm, which might have formed due toentry of the gold nanotriangles into the cells atthese sites.

FIGURE 7. (A) AFM micrograph of untreated NIH 3T3 fibroblast cell. (B) Line profile showing theheight of the cell in A. The height of the cell was found to be 245 nm. (C) 3D profile of the untreatedfibroblast cells showing a smooth topology (color figure available online).

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FIGURE 8. (A) The height mode AFM image of the NIH 3T3 fibroblast cells treated with 100 µMconcentration of gold nanotriangles. The arrows indicate the surface deformities of the cells. (B)3D profile of the 2D image shown in A, showing the pits at the surface of the fibroblast cells (colorfigure available online).

The observations made in the aforementionedsection clearly indicate that exposure of the fi-broblast cells to the gold nanotriangles renderstheir surface highly rough and irregular and the3D profile shows the presence of pits on the sur-face. The conclusions drawn by phase contrastmicroscopy clearly prove that the fibroblast cellsinternalize the gold nanotriangles and compart-mentalize them in the cytoplasmic space. Thus,the pits that are seen at the surface of the fibrob-last cell in Figure 8 could be the sites of the entryof the gold nanotriangles into the cell. It is verydifficult to predict whether the entry of the goldnanotriangles is activated or it is a passive modeof entry, where the sharp triangles cut throughthe surface of the cells. However, the latter ar-gument seems unlikely due to the fact that thecytotoxicity studies show 80% viability of theNIH 3T3 fibroblast cells on treatment with 100µM concentration of gold nanotriangles (Fig-ure 3B). Thus, such high cell viability will bedifficult to explain if the gold nanotriangles arecutting through to enter the cells, which willcause damage to the cellular integrity. The otherpossibility could be the entry of the gold nano-triangles by pinocytosis (cell drinking), which isalso unlikely because the size of the gold nan-otriangles is too large. It has been reported inliterature that the particles that are smaller than100 nm in size enter in the cell by pinocytosiswhile the particles bigger than that enter throughphagocytosis (cell eating).[46] Thus, the entry ofthe gold nanotriangles might be due to phago-cytosis. However, this possibility could only beconfirmed by further experimentation.

CONCLUSIONS

The work presented in this paper discusses thecytotoxicity and biocompatibility issues of thebiogenic gold nanotriangles synthesized by us-ing lemon grass extract. Biogenic nanostructureshave been considered to have better biocompat-ibility than the chemically or physically synthe-sized nanostructures. However, there is a dearthof scientific research to prove this assumption.An attempt has been made to address these is-sues in this work. Another highlight of this workis that the anisotropic gold nanotriangles withhighly unusual optical properties have been usedto carry out the work, which may find futureapplication in hyperthermic treatment of cancercells. The results of the cytotoxicity studies sug-gest that the gold nanotriangles do not cause anyharm to the cancerous (RAW264.7 and MCF-7)as well as non-cancerous (NIH 3T3) cells up to400 µM concentration after 24 h of exposure.The possibility of induction of stress by thesegold nanotriangles has been studied by ELISAanalysis, which suggests that they cause somestress-induced production of TNF-α cytokines.The flow cytometry analysis and phase contrastmicroscopy imaging suggests that the gold nano-triangles are internalized inside the cells and arecompartmentalized in the cytoplasmic space butdo not enter inside the nucleus of the cells. Thecomparative AFM analysis of the control un-treated fibroblast cells and fibroblast cells treatedwith gold nanotriangles reveals that the surfaceof the treated cells is highly irregular. The sur-face of the control untreated cells was found to

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be very smooth and regular as opposed to thetreated ones, which showed pits of the sizes of300 to 400 nm. It is speculated that these surfacepits are the sites of entry of the gold nanotrian-gles into the cells. The exact mechanism of entryof the gold nanotriangles inside the cells has notbeen fully understood. However, phagocytosismight be the process operative in this case. Theaverage size of the gold nanotriangles has beencalculated to be 113 nm, and thus they are toobig to be taken inside by pinocytosis. Phago-cytosis has been reported to be the process bywhich particles bigger than 100 nm enter insidethe cells.

In conclusion, it has been shown in this pa-per that the biogenic gold nanotriangles showbiocompatibility better than the chemically syn-thesized nanoparticles. The nanotriangles havebeen found to be non-toxic to the animal cellsat high concentrations, although they do elicitsome immunological response. They have beenshown to be internalized by the cells and areconfined in the cytoplasmic space but do not en-ter into the nucleus. Thus, these biogenic goldnanotriangles could be an excellent scaffold fordelivery of drugs, genes, or growth factors insidethe cells. Additionally, since these nanoparticlesshow a strong and tunable NIR absorption, theycan serve as excellent candidates for hyperther-mic treatment of cancer cells.

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