Cell (A549)Particle (Jasada Bhasma) Interactions >Cell (A549)Particle (Jasada Using Raman Spectroscopy Bhasma) Interactions Using Raman

Spectroscopy
G. Pyrgiotakis,1 T. K. Bhowmick,2 K. Finton,1 A. K. Suresh,2 S. G. Kane,2 J. R. Bellare,2 B. M. Moudgil1,3
1 2 3

Particle Engineering Research Center, University of Florida, Gainesvilla, Florida Department of Chemical Engineering, School of Biosciences and Bioengineering, IIT, Bombay 400 076, India Department of Materials Science and Engineering, University of Florida, Gainesville, Florida

Received 24 October 2007; revised 15 December 2007; accepted 5 January 2008 Published online 5 February 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bip.20947

ABSTRACT:
Current methods for the evaluation of cell interactions with particles are nonspecic, slow, and invasive to the cells. Raman spectroscopy is a noninvasive technique, and is used in the present study to investigate particlecell interactions. The main focus of the present study is to employ Raman spectroscopy for investigating the interaction of human lung adenocarcinoma cell line (A549) with the particulate system Jasada Bhasma, a traditional Indian medicine. Jasada Bhasma is a unique preparation of zinc and is traditionally used for the treatment of various diseases like diabetes, age-related eye diseases, and as a health promotional tonic. The Raman spectral analysis is executed by identifying the difference in intracellular DNA/RNA, and proteins and lipids concentration between particlestreated and untreated cells. Comparison between Bhasma-treated and -untreated cells indicates that vibrational peaks

corresponding to the DNA/RNA molecule show a signicant increase in cells treated with the Jasada Bhasma. Apart from the DNA molecule, several other vibrational peaks related to the protein molecules also show a signicant increase in A549 cells after treatment with Bhasma. These results indicate that Bhasma treatment of A549 possibly delays DNA degradation and enables retention of higher amount of protein molecules in the cells. # 2008 Wiley Periodicals, Inc. Biopolymers 89: 555564, 2008. Keywords: Raman spectroscopy; cellparticle interaction; A549; Bhasma; Bio-Raman; Micro Raman

This article was originally published online as an accepted preprint. The Published Online date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

Correspondence to: B. M. Moudgil; e-mail: bmoudgil@perc.u.edu Contract grant sponsor: Ministry of Human Resource Department (MHRD) Contract grant sponsor: Department of Biotechnology (DBT), Government of India Contract grant sponsor: Particle Engineering Research Centre (PERC), University of Florida, USA Contract grant sponsor: NSF-NIRT Contract grant number: EEC-94-02989 Contract grant sponsor: NIH P-20 Contract grant number: 1-P20-RR020654-01 C V 2008 Wiley Periodicals, Inc.

INTRODUCTION
he interaction of biological systems with particles depends on various factors that are not yet completely understood. Such factors depend on the nature of the particles (size, shape, composition, etc.) and the cell type and environment. Quantitative assessment and real-time monitoring of cellular response is one of the major focus of ongoing research today. Researchers have tried to evaluate the response of biological systems

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after particle uptake, using different cell and tissues as model systems.13 Such systems include microbial and animal cell lines (e.g., Yeast, HeLa, Macrophage, etc.).46 Most techniques for evaluation of the biological responses to particles rely on the standardized method, mainly microscopy and biological assays,7 which are often time-consuming, expensive, and destructive. Consequently, noninvasive, in situ, biocompatible instruments and techniques, especially to obtain a preliminary assessment, are critically needed. A wide variety of biochemical methods [e.g., (3-(4,5dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide) (MTT) assay, Neutral Red uptake assay, etc.] and nonbiochemical methods or techniques (e.g., uorescence microscopy, NMR, FTIR, etc.) are available for measuring biological responses from the cells. Raman microspectroscopy has recently emerged as a useful tool with many advantages for quantitative, in vivo, and real-time measurement of responses from biological systems.8 Use of the appropriate laser, 785 nm, source in the instrument prevents cellular degradation, even for long exposure times,9 allowing the noninvasive feature with low noise levels. Raman spectroscopy has therefore been recently examined for monitoring the behavior of cells, with the following advantages over conventional existing biological assays for studying live cells,10,11 (i) it is noninvasive, i.e., it can measure response from the cells without perturbing the cellular biochemistry, (ii) it has very low interference from water and carbon dioxide molecules compared to other vibrational spectroscopic techniques, (iii) no labeling is required, and (iv) it is relatively rapid (13 min).12 The current study involves investigating the interaction of a particulate material known as Bhasma, which belongs to the Indian traditional system of medicine. Jasada Bhasma is a special preparation of zinc and has been used by traditional medicine practitioners for the treatment of various diseases like diabetes and age-related eye diseases. Jasada Bhasma has been also used as a general tonic to increase energy, and to improve overall health and longevity.13,14 Clinical reports on Jasada Bhasma have served as an inspiration to undertake the present study.15,16 Another similar simultaneous study has indicated that Jasada Bhasma is able to protect yeast cells against free radical-mediated cellular damage.17 In the current work, an attempt has been made to quantify the cellular response of Jasada Bhasma to human lung adenocarcinoma (A549) cells. The response of this particular cell line (A549) with respect to several agents is well characterized using Raman spectroscopy.18 A549 cells have been used also as a model cell line for examining the effect of different materials including medicines.19,20 Notingher et al. have reported distinguishing the difference between live and dead A549 cell using Raman spectroscopy.21 They deter-

mined that several characteristic peaks in the Raman spectrum representing the DNA/RNA components were of lower intensity in dead A549 cells as compared to viable cells. The decrease in peak intensity indicated the degradation of both phosphodiester (O O) bonds and the base molecules of P DNA/RNA. They also showed a similar reduction in the intensity of the peak assigned to the vibration of phenylalanine molecule. In another study, Verrier et al. tested the toxic compound Triton X100 on A549 cells and showed the peak intensity reduction of the same bands.22 Raman spectroscopy was also employed for detecting the biochemical changes in brain tissue of mice because of radiation damage.23 A signicant reduction in the peak assigned to the C deformation C of unsaturated lipid was observed. Several other peaks assigned to the lipids and C ring stretch of phenylalanine C were also reduced due to radiation damage. The objective of the present study was to investigate the viability of the cells, identify the molecular changes occurring in the living cells after interaction with Jasada Bhasma using Raman spectroscopy, and to cross validate the results with MTT assays.

MATERIALS
Particulate System Synthesis
The method of preparation of Jasada Bhasma is tedious and timeconsuming and involves several steps. An authentic traditional medicine practitioner prepared Jasada Bhasma, whose family has been preparing the Bhasmas for a few generations. Preparation process of Jasada Bhasma is described in Ayurvedic texts,24 and the Bhasma employed in this study involved the following steps during preparation. Traditional names of each step have been mentioned in parentheses. In the rst step (Shodhan), raw zinc was melted and quenched into slaked lime water (ratio of 1:200) over 70 min. In the second step (Jarana), the product from the previous step was melted, while adding an equal weight of turmeric powder slowly. In third step (Dhavana), the product from the previous step was added to water in a container, allowed to settle overnight, and the supernatant was poured off. In the fourth step (Bhavana), the product from third step was mixed with turmeric quath (a decoction of turmeric in water) in a mortar and ground with pestle continuously. In the fth step (Puta), the ground material from the third step (Dhavana) was made into discs and placed inside an earthenware pot, which was covered with another earthenware pot. The entire assembly was placed in a pit with dry cow dung. The dung was set on re, and the material in the pot was heated for 45 h. Each fth (Puta) step was followed by a fourth (Bhavna) step, and this combination was repeated 13 times before completion of Jasada Bhasma preparation. Final Jasada Bhasma was collected in ne powder form after grinding the material obtained from the nal fth (Puta) step. Sources of other materials required for the present study are as follows. RPMI-1640 was purchased from Sigma-Aldrich. Fetal bovine serum (FBS) and antibiotic purchased from Gibco. Quartz powder was purchased from Min-U-Sil, West Virginia. The human adenocar-

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Table I Element O Na Mg Al Si S K Sn Ca Mn Fe Cu Zn
a

ICP Results for the Jasada Bhasma ICP (wt %) N/Aa 0.14 1.472 0.636 4.11 0.151 0.736 0.515 1.47 0.018 0.583 0.1 78.82 Error (wt %) N/A 0.06 0.161 0.106 0.694 0.038 0.042 0.28 0.248 0.001 0.063 0.023 0.547

media was removed and the cells were rinsed twice with PBS. New media with the Bhasma particles was added, and the cells were analyzed at specic time periods using Raman spectroscopy. Based on the fact that they are metal oxide particles, they are stable in water and are not expected to dissolve; they are endocytosed as whole by the cells. The mechanism of endocytosis has been extensively studied, and it is considered the major mechanism for particle uptake by cells.2628

Measurement of Mitochondrial Activity of A549 Cells With and Without Bhasma Treatment. The mitochondrial activity
of A549 cells was measured after treatment with different doses of Bhasma by the MTT assay. This test was performed on A549 cells after 24, 48, and 72 h of incubation, as described by Mosmann.29 Cells were grown in a 96-well plate in complete medium at 378C and 5% CO2. For each concentration of Jasada Bhasma used in the study (37.5, 75, 150, 300, 600, 1200, and 2400 ppm), eight different wells were used. Six hundred parts per million quartz particles were taken as positive control. MTT assays involved the following steps: the growth medium was removed from the culture wells and 10 ll of MTT solution added in each well of the plate. For the reduction of tetrazolium salt, plates were incubated at 378C for 4 h. The tetrazolium salt was reduced to formazan (blue) product within this time by the active mitochondria of the cells. In each well, 100 ll of isopropanol with 0.04N HCl was added for dissolving the reduced tetrazolium salt. The color of reduced salt was measured at 570 nm using microplate reader (Molecular Devices Corp.). The absorbance at 570 nm was taken as an index of activity of mitochondria. Based on the mitochondrial activity, the concentration of the Jasada Bhasma was selected for subsequent analysis.

Detectable with XPS, but not measurable with ICP.

cinoma cells (A549) were purchased from American Type Culture Collection (Manassas, VA). MTT assays were purchased by Chemicon International.

Chemical Composition and Size Characterization of Jasada Bhasma

Since detailed characterization of the particle has been discussed elsewhere,25 only a brief summary is presented in the paper. Compositionally, the particle was characterized with ICP and EDAX. The elemental content based on ICP analysis of Jasada Bhasma (Table I) shows that it contains 12 elements (Na, Mg, Al, Si, S, K, Ca, Mn, Fe, Cu, Zn, and Sn) with Zn in the form of ZnO as the major element (78.82 wt %). Jasada Bhasma has a wide distribution of size (15 nm to 3 lm); so for the characterization of the particle size, the powder was separated by ltration to fractioned (20- to 30-nm particles) and unfractioned powders ($1 lm). To determine the mean particle size, the Jasada Bhasma (in unfractioned and fractioned form) was analyzed by dynamic light scattering (DLS), supplied by the Brookhaven Zeta plus (Holtsville, NY). Particle size analysis results from DLS instrument show the mean size of the particles present in unfractionated part of Jasada Bhasma to be 1.2 lm (Figure 1a). Analysis of the fractionated part of Jasada Bhasma shows the presence of 30-nm size particles in the sample (Figure 1b). Particle size analysis of Jasada Bhasma was also done with TEM. It revealed that the unfractioned particles exhibit a wide size distribution, have irregular shape, and are strongly aggregated (Figure 1c). The TEM also revealed that the ne fraction of Jasada Bhasma is 1535 nm in size, and it exhibits a much narrower distribution of sizes with spherical shape (Figure 1d).

Raman SpectroscopySpectrum Collection, Baseline Correction, Deconvolution of Spectrum. The InVia system by
Renishaw, consisting of a Leica microscope connected to a Renishaw 2000 spectrometer, was used for the measurement of the spectra. The high power diode laser (115120 mW) produces laser light of 785 nm and does not cause any signicant damage to the cells even after 40-min exposure time.30 The laser spot size of the laser is 20 3 40 lm2, which is comparable to the size of the cell. For every measurement, the laser was placed, with the guide of a crosshair, above the nucleus and covered the entire cell. In that manner, the differences of the obtained spectra are exclusively due to the cell state and not the relevant position of the laser on the cells. The experimental approach for evaluating the effect of Jasada Bhasma medicine on A549 cells was similar to the one reported by Nortingher et al.21 In this study, we have collected spectra from 30 different cells adhered onto MgF2 plate. Each spectrum was collected for 120 s (similar results were obtained with 30-s exposure time) after focusing the 785-nm laser beam through 633 water immersion Leica objective. The obtained spectra were baseline-corrected and normalized in respect to the 1450 cm21 peak, representing the C deformation H in proteins and lipids, which is not signicantly affected by the intracellular changes. Data processing was performed using a custommade mathematical code written in Mathematica (Wolfram Research). The mathematical base of the normalization procedures, although elaborate, is simple algebraic calculations, where the only two effects are essentially the subtraction of the background and the multiplication of an appropriate factor to normalize the spectra.

Methods
Culture of A549 Cells With and Without Jasada Bhasma. A549 cells were grown in complete media (RPMI-1640, 5% FBS, and 1% antibiotic/antimycotic) in a cell culture ask. The cells were trypsinized and seeded at $5 3 103 on a 5- 3 5-mm MgF2 substrate (custom-made by Red-Optronics). The seeded MgF2 substrates were placed in 2 mL media in a six-well plate and incubated at 378C and 5% CO2 for 24 h. After reaching $80% conuency, the cell
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FIGURE 1 The particle size distribution of Bhasma particles. (a) Unfractioned particles size distribution. (b) TEM image of the unfractioned particles. (c) Fractioned particles size distribution. (d) TEM image of the fractioned particles. Reproduced from Ref. 25 with permission from Elsevier. within a span of four wave numbers (x0 6 2 cm21), and the FWHM of each peak was constrained at 30 wave numbers. The relative intensities were unconstrained. The prole of the peak was Gaussian, and the baseline was assumed linear (second and third degree polynomial was attempted with no signicant difference in the result). The curve t was carried out until local minimum or conversion was reached. The area under the curve was used as the intensity of the peak. The same process was repeated for the standard deviation spectrum, and the resultant areas used as the error of the peak intensity measurement.

Initially, the media spectrum was subtracted using nonlinear subtraction according to the method developed by Maquelin et al.31 For background subtraction, a spectrum was obtained by a cell-free region on the MgF2 plate while still focused on the cell focal plane. The spectra were subsequently normalized with respect to the 1450 cm21 peak originating from the CH2 stretching bond from all the cell components, which can be assumed to remain constant over the measurement time. A second normalization process was followed using the standard normal variate (SNV) method to further normalize the spectra and eliminate any intensity differences.32 The normalized spectra were treated with the algorithm developed by Lieber and Mahadevan-Jansen to remove the baseline.33 Although Lieber and Mahadevan-Jansen33 proved that the algorithm does not induce any changes, as a precaution, an extra normalization step was employed with respect to the 1450 cm21 peak and with the SNV method. The processed spectra were averaged, and the standard deviation was calculated. The average is a representative spectrum of the cells at a certain time point, and the standard deviation is the error of the measurement. The steps of the aforementioned algorithm were optimized to achieve minimum standard deviation. Assignment of the peaks was done according to Notingher et al.21 Deconvolution of spectra was carried out, and area under the individual bands computed using GRAMS software (ThermoGalactic). The peak position, x0, was xed according to Notingher

RESULTS
Mitochondrial Activity of A549 Cells Increased in Presence of Jasada Bhasma
Results presented in Figure 2 show that mitochondrial activity is maximum at 24-h incubation of A549 cells with Jasada Bhasma at (150300) ppm concentration. Mitochondrial activity of A549 cells is signicantly increased after 24-h incubation compared to control. The general decline in mitochondrial activity is an indication of cellular aging with time. The higher mitochondrial activity of
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FIGURE 2 Mitochondrial activity (MTT assay) of A549 cells after treatment with Jasada Bhasma at different time point of growth. Signicant difference observed after 24 h of treatment at 150 300 ppm concentration of Bhasma.

bases of DNA, and peak at 788 cm21 is responsible for phosphodiester (O O) stretching bond of DNA/RNA. The P result of best-tted peaks for Jasada Bhasma treated and untreated A549 cells are plotted in Figures 4a and 4b, respectively. In Bhasma-treated A549 cells, the area of the peak at 788 cm21 is $37% larger than the untreated A549 cells. In contrast, after treating cells with quartz particles, which are known for their toxicity, 788 cm21 peak decreased by 59%. No signicant difference has been observed in the peak area at 782 cm21. The decrease in the area of 788 cm21 peak in Bhasma-untreated A549 cells indicates less number of O O bonds, implying a greater fragmentation of DNA P in the control cell nucleus. Raman Spectrum of A549 Cells: 9951025 cm21 Region Information of Protein. This region is tted with two peaks at 1005 cm21 and 1013 cm21. Peak at 1005 cm21 is dominating in this region and assigned to ring stretching of phenylalanine amino acid molecule. Another peak at 1013 cm21 is a very small peak and corresponds to C stretching of deoxyO ribose sugar. Figures 5a and 5b show the tted curve for control and Jasada Bhasma-treated A549, cells respectively. Analysis of the result shows that peak at 1005 cm21 for Jasada Bhasma-treated A549 cells is about 36% larger than control A549 cells. The decrease in the area of the peak at 1005 cm21 of control A549 cells indicates that control cells contain less amount of phenylalanine, which indicates degradation as a result of cell aging. Raman Spectrum of A549 Cells: 10251150 cm21Information of DNA/RNA, Protein, Lipid, and Carbohydrate Molecules. This part of the spectrum is tted with six peaks. Peak at 1033 cm21 is originated by the in-plane vibration of

Bhasma-treated cells, as compared to control cells, is indicative of their higher viability.

Raman Spectrum from A549 Cells

Figure 3 shows the average Raman spectrum of quartztreated, Jasada Bhasma-treated, and untreated A549 cells, after background subtraction. Underneath each spectrum is shown the respective standard deviation of the spectrum that was used to calculate the error in the peak intensity. The conclusions regarding the state of the cells will be derived by comparing four particular regions (770803 cm21, 995 1020 cm21, 10251150 cm21, and 11501385 cm21) of the Bhasma-treated versus control (untreated) A549 cells, and the quartz-treated cells as positive control. These four regions of the spectrum contain information about several molecules such as DNA, protein, lipids, and carbohydrate. Tentative assignment of the peaks for A549 cells is shown in Table II. Peaks at 782, 788, and 1095 cm21 represent the vibrations of molecules related to DNA. Peaks at 1005, 1128, and 1209 cm21 are related to the vibrations of different protein molecules. Peaks at 1231, 1242, and 1284 cm21 represent different conformational structure of protein molecules (a helix b-sheet and random coils). Peaks at 1066, 1080, 1095, and 1128 cm21 are assigned to vibrations of C stretching of lipid moleC cules. The peak at 1450 cm21 corresponds to C H deformation vibration in all of the cellular components of the cells used for normalization.23,34,35 Raman Spectrum of A549 Cells: 770803 cm21 Region Information of DNA. This region of the spectra is tted with two peaks at 782 cm21 and 788 cm21. Peak at 782 cm21 is assigned to the ring stretching of cytosine and thymine
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FIGURE 3 The Raman spectrum from control, Jasada Bhasma, and quartz-treated A549 cells, at 24 h, with their respective errors, after the mathematical processing described in the text.

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Table II Assigned Raman Peaks for Different Molecules of the A549 Assigned Peaks for the Molecules Region of Spectrum A549 (cm21) 770803 9951020 10201150 Fitted Peaks for A549 (cm21) 782 788 1005 1013 1031 1049 1066 1080 1095 1128 1209 1231 1242 1258 1284 1301 1320 1342 1367 Carbo hydrate

DNA/RNA U, C, T ring br. O O P

Protein

Lipid

Ref. 3638 3638 38,39 21 40 41 42 21,39 38 39,42 42 43 43 43 42,43 42,43 21 38 44

Sym. ring br. Phe C deoxyribose O C in plane Phe H C str. N C str. N C str. N C 6H5, Phe, Trp C Amide III, random coil Amide III, b-sheet Amide III, b-sheet Amide III, a-helix C def. H C def. H Chain C str. C Chain C str. C Chain C str. C C str. O

PO2 str. 2

11901385

CH def. CH def. CH2 twist

G A, G

Sym. str. CH3

Abbreviations: O O phosphodiester; PO22, phosphodioxy; Phe, phenylalanine; Trp, tryptophan; A, adenine; G, guanine; C, cytosine; T, thymine; P , U, uracil; sym, symmetric; str, stretching; def, deformation.

C bond in phenylalanine amino acid of protein moleH cule. Peak at 1049 cm21 is assigned to the stretching of the C bond in carbohydrate. Peaks at 1066, 1080, and O 1128 cm21 are assigned for the C stretching of the lipids. C Peak at 1095 cm21 is assigned to the vibration of phosphodioxy (PO2) group of deoxyribose sugar. 2 Figures 6a and 6b show the results of the best tted peaks for Jasada Bhasma-untreated and treated A549 cells, respec-

tively. Jasada Bhasma-treated cells have $35% and $28% more peak area at 1033 and 1080 cm21, respectively as compared to untreated A549 cells. This indicates higher amount of intracellular protein as well as phenylalanine amino acid molecules in the treated cells as compared to the untreated cells. Another peak at 1049 cm21 also increased ($28%) after treatment with Jasada Bhasma, indicating higher amount of carbohydrate content in Bhasma-treated cells.

FIGURE 4 Peak tting of the 770803 cm21 region corresponding to the (a) control A549 cells and (b) Jasada Bhasma-treated A549 cells.

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FIGURE 5 Peak tting of the 9951025 cm21 region corresponding to the (a) control A549 cells and (b) Jasada Bhasma-treated A549 cells.

At the same time, exposure of A549 cells to quartz particles signicantly decreased peak intensity as compared to the control cells. Raman Spectrum of A549 Cells: 11901385 cm21Information of DNA/RNA, Protein, Lipid, and Carbohydrate Molecules. This region of the spectra is crowded with several vibrational frequencies corresponding to proteins, nucleic acids, and lipids. This part of the spectra from A549 cells is tted with nine peaks. Peak at 1209 cm21 corresponds to the C 6H5 bond stretching of phenylalanine. Peak at C 1258 cm21 is related with the in-plane deformation of CH bond of lipid molecules. Peak at 1231 cm21 is related to the amide III random coil structure of protein. Peaks at 1242 and 1258 cm21 correspond to b-sheet structure, and peak at 1284 cm21 is assigned to the amide III a helix vibration of protein molecule. CH2 twisting of phospholipids is assigned at 1301 cm21 peak. Peaks at 1342 cm21 and 1320 cm21 are assigned to the vibration of adenine, guanine,

carbohydrates, and C deformation of protein molecules. H Symmetric CH3 stretching behavior of phospholipids molecule is assigned to the peak at 1367 cm21. The high number of peaks (nine peaks with maximum FWHM 30 wave numbers in a span of 200 wave numbers) can be a source of error, and so the results of those peaks were cross examined with the peaks of other regions. Figures 7a and 7b show the result of the best-t peaks in the spectrum from Jasada Bhasma treated and untreated A549 cells, respectively. After comparing all nine peaks from Jasada Bhasmatreated and untreated A549 cells, a signicant difference is observed in the peak at 1242 cm21. In Jasada Bhasma-treated A549 cells, area of the peak at 1242 cm21 increased by $75%, which indicate that cellular proteins tend to conserve the b-sheet structure after treatment with Jasada Bhasma. This result shows a similar trend, with peaks at 1005 cm21 assigned to the vibration of phenylalanine amino acid. At the same time, peak area at 1242 cm21 is reduced by $20% in quartz-treated A549 cells.

FIGURE 6 Peak tting of the 10201150 cm21 region corresponding to the (a) control A549 cells and (b) Jasada Bhasma-treated A549 cells.

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FIGURE 7 Peak tting of the 11901385 cm21 region corresponding to the (a) control A549 cells and (b) Jasada Bhasma-treated A549 cells.

DISCUSSION
Growth and Morphology
The objective of this study was to probe the effect of Bhasma particles on biological systems at the molecular level using Raman spectroscopy. The cellular aging is manifested by the damages of nucleic acids, protein, and lipids molecules, which are caused by the oxygen-containing free radicals such as superoxide, nitric oxide, and hydroxyl radicals.36 Reactive oxygen species (ROS) are generated inside the cells through several metabolic reactions. Accumulation of the free radicals inside the cells leads toward the fragmentation of the DNA molecule, conversion of proteins into small chain polypeptides or amino acids, and change of peroxidation of lipid molecules resulting in the death of the cells.37 According to another theory of aging, mitochondria plays a signicant role in aging, and aging processes are the cumulative result of the ROSa normal byproduct of the aerobic cells. MTT assay shows that Bhasma treatment increased the mitochondrial activity of A549 cells, perhaps due to ROS scavenging in the Bhasma-treated cells.38

bond of nucleic acids in A549 cells, suggests that after treatment with Jasada Bhasma, cells are able to prevent the degradation of nucleic acid molecules. The changes in the Raman peaks (1005, 1080, and 1242 cm21) that are related to the vibration of phenylalanine and several conformational structure of the protein molecules indicate that Jasada Bhasmatreated A549 cells are able to maintain higher amount of intracellular protein compared to the untreated A549 cells. Peak at 1049 cm21 indicates the presence of higher amount of carbohydrate molecules in Bhasma-treated A549 cells. In the case of quartz (positive control), all the major peaks were signicantly reduced in intensity as compared to both the control and Bhasma-treated A549 cells.

CONCLUSION
Interaction of the particles with biological systems including living cells is one of the most intriguing areas of basic and applied research at the interface of biology and particulate

Raman Spectra, Peaks, and Molecular Information

After examining the four specic regions of the average spectra from A549 cells, signicant changes in the several signature peaks were observed. These signature peaks are the markers of the cellular responses after interaction with particulate materials. Peak-tting analysis resulted in higher area of certain peaks in the spectra of Bhasma-treated cells as compared to the control cells. Figure 8 summarizes the changes in the signature peaks area in the spectra of A549 cells. The most signicant enhancement occurred in the peak area at 788, 1005, 1033, 1049, 1080, and 1242 cm21 of Jasada Bhasma-treated A549 cells. The changes in the Raman peaks (788 cm21), corresponding to the vibrations of O O P

FIGURE 8 Bar chart demonstrating the spectral differences between Jasada Bhasma-treated and untreated cells.

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materials. In this study, Raman spectroscopy was used to monitor cellular response through molecular markers. The positive effect of the traditional Indian medicine Bhasma indicated by Raman spectroscopy was supported by MTT assay that showed the improvement of mitochondrial activity after treating A549 cells with Bhasma particles. Signicant peak area increase (788, 1005, 1031, 1049, 1080, and 1242 cm21) was observed in the spectra of Jasada Bhasma-treated A549 cells. The higher peak area at 788 cm21 corresponding to the vibration of O O bond of nucleic P acid may be attributed to slower degradation of DNA in the presence of Bhasma particles. Higher peak areas at 1005, 1031, 1034, and 1080 cm21 that are assigned to the different protein molecules show higher amount of protein content in Bhasma-treated cells. Higher peak area at 1242 cm21 indicates that the ordered structure of protein molecules is better persevered after treatment with Jasada Bhasma. Altogether, these results indicate that Jasada Bhasma treatment of A549 cells at a particular dose results in enhanced cellular viability by maintaining better intracellular molecular structure. To be more specic, the Jasada Bhasma treatment seems to prevent the degradation of the intracellular DNA/RNA and proteins. The DNA being protected from oxidative stress, the stability of the bond O O is indicated by the higher intensity, P for the case of Bhasma-treated cells. In the case of proteins it is different, since the proteins are not only protected, but the production of proteins appears to be higher in relation to the untreated cells, since the ribosomes (the main source of protein within the cells) are protected from oxidative stress. Although the reason of this is not yet known, it is believed that it is due to the prevention of the oxidative stress, as has been noticed for other metal oxide particles such as CeO2,39,40 Y2O3,41 and other Bhasma powders.38,42 Furthermore, establishing these spectral signatures of particulate matter (Bhasma) interaction with the cells is expected to lead to reliable monitoring of cellular response by Raman spectroscopy.
We acknowledge Vaidya Ajit Joshi for providing samples. They are also grateful to the Sophisticated Analytical Instrumentation Facility (SAIF), Mumbai, India for providing instrumental facilities. They also acknowledge the Elsevier B.V. Publications for granting permission to reproduce Figure 1.

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