l-Cysteine-Conjugated Ruthenium Hydrous Oxide Nanomaterials

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L-Cysteine Conjugated Ruthenium Hydrous Oxide Nanomaterials with Anti-cancer Active Application Bichitra Nandi Ganguly, Buddhadeb Maity, Tapan Kumar Maity, Joydeb Manna, Madhusudan Roy, Manabendra Mukherjee, Sushanta Debnath, Partha Saha, Nagaraju Shilpa, and Rohit Kumar Rana Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b01408 • Publication Date (Web): 27 Dec 2017 Downloaded from http://pubs.acs.org on December 28, 2017

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L-Cysteine Conjugated Ruthenium Hydrous Oxide Nanomaterials with Anti-cancer Active Application

Bichitra Nandi Ganguly1*, Buddhadeb Maity2, Tapan Kumar Maity2, Joydeb Manna2, Modhusudan Roy1 Manabendra Mukherjee1 Sushanta Debnath1, Partha Saha1,3, Nagaraju Shilpa4, Rohit Kumar Rana4 1

Saha Institute of Nuclear Physics, Kolkata-700064, India. Department of Chemistry, Mahishadal Raj College, Mahishadal, East Midnapur, West Bengal721628, India. 3 Homi Bhaba National Institute, Mumbai-700094, India. 4 Nanomaterials Laboratory, I & PC Division, CSIR-Indian Institute of Chemical Technology, Hyderabad 500007, India.

2

*1

Corresponding author’s email: [email protected]

ACS Paragon Plus Environment

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L-Cysteine Conjugated Ruthenium Hydrous Oxide Nanomaterials with Anti-cancer Active Applications Abstract Bio-active nanomaterials, namely: ruthenium hydrous oxide (or ruthenium oxy-hydroxide), RuOx(OH)y and also a surface-conjugated novel material of the same within the template of an amino acid molecule: L-cysteine have been studied. These compounds have been prepared through a simple wet chemical route, under physiological condition, such that they could be suitably used in anti-cancer applications. Several physical methods were used for the nanomaterial characterization, e.g.: thermal analysis of the as prepared ruthenium hydrous oxide by differential scanning calorimetry (DSC) followed by thermal gravimetric analysis (TGA). This confirms the material to be a precursor for anhydrous nano-crystalline ruthenium oxide (RuO2), as is affirmed by powder X-ray diffraction pattern. Also, optical spectroscopic absorption (UVVis and FT-IR) study of these nano-particles(NPs) to ascertain their surface conjugation with Lcysteine have been performed. Besides these, surface morphology of the NPs were studied by field emission scanning electron microscopy (FE-SEM) along with their elemental purity check through energy dispersive X-ray analysis (EDX). Their surface chemical micro-environments were examined by X-ray photo electron spectroscopy (XPS). The hydrodynamic size of the prepared NPs were measured through dynamic light scattering (DLS) studies. Further, biological consequences of these NPs on cancerous HeLa cells and their cytotoxicity effects have been reported with MTT assay, such an application has not been reported so far.

KEYWORDS: Ruthenium hydrous oxide / RuOx(OH)y; Surface conjugation with L-cysteine ; bio-active nanomaterial; Surface chemical analysis, DLS studies, in-vitro cellular studies.


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1. Introduction : It is the beneficial interaction of nano-materials and biological systems that has emerged as the forefront scaffolds in the design of bio-active materials1,2 for fruitful applications. In such a nascent field, design of new materials involving inorganic nanoparticles2 coroneted by functional surface active groups3-6 like proteins7-9, sugar molecules10, or co-polymers11 play important roles in bio-active spheres. Nanoparticles typically possess a larger percentage of atoms at the material’s surface, which can lead to increased surface reactivity12, can maximize their ability to be loaded with therapeutic agents to deliver them to target cells. By appropriate chemical design, these nanomaterials can acquire the ability to selectively target particular types of cells and cause anti-proliferative effects. Metal oxide nanoparticles: including zinc oxide13,14, hydrated gallium oxide15, silver oxide16,, have been versatile platforms for biomedical applications and therapeutic intervention. There is an urgent need to develop new class of anticancer agents, and recent studies demonstrate that transition metal oxides/compounds17-20, exhibiting variable valency states, under physiological conditions hold considerable promise.

It is well known that iron either in Fe (II or III) oxidation state is very important to sustain life as it binds not only to heme protein but also to other vital macromolecules and proteins. It is through their redox reactions (or electron transfer reactions), the energy is produced or cellular metabolism propagates. One of these molecules is transferrin, a protein that helps cells to get iron in soluble form and is useful for the metabolic processes where iron plays a role, particularly during cell growth and division. So it is supposedly easy to connect the fact that tumour cells require more iron than healthy cells because they have a cyclic activity faster than their healthy counterparts and therefore express a larger amount of transferrin receptors to get this essential element. The chemical similarity of ruthenium21,22 to that of iron (Table-1), being the member of the same group in the periodic table makes it obvious to compare the possibilities of redox reactions. Moreover, it is plausible that Ru (III) transfoms to active Ru(II) state, under reducing environment, in the prevailing physiological condition and selectively binds to cancerous cells17 . Transferrin is present in plasma and in other fluids in the body, can be easily accessible to ruthenium ionic species when the same enters blood circulation. Therefore, it is pertinent here to


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exploit simple mixed multiple valent oxidation states of ruthenium23, in the form of its hydrous oxide nano-particle species (NPs) for their cytotoxic effects, concomitantly studying their surface chemical properties and consequent detailed material characteristics of this nano-oxide material. It is noteworthy to mention that ruthenium complexes have attracted much attention as building blocks for new transition-metal-based antitumor agents. Although the pharmacological target of antitumor ruthenium complexes has not been unequivocally identified, but it is generally accepted that their cytotoxicity is related to their ability to bind DNA24,25. Ruthenium compounds have been shown to inhibit DNA replication, possess mutagenic activity, induce SOS repair, bind to nuclear DNA and reduce RNA synthesis, which are all consistent with DNA binding of these compounds in vivo. The electronic nature of ruthenium renders it very susceptible to nitrogen and sulphur donor molecules, such as an amino-acid like L-cysteine (see Figure 1a). Therefore the same amino acid has been employed here to surface conjugate with ruthenium hydrous oxide NPs to evolve a novel material that would make the ruthenium hydrous oxide suspension soluble at physiological pH (7-7.5) and thus would be biologically more versatile for application. In this context, it is pertinent to develop a systematic study of cysteine(CS) templated ruthenium hydrous oxide material for application as anti-cancer agent. Also, it is worthwhile to mention that during natural cell proliferation, ruthenium (ionic species) if present in the system, could bind to CS molecule during protein isoprenylation26 and inhibit the process. Thus in this case, the surface conjugated nano-material has been attempted to mingle with the natural cellular processes to arrest cell multiplication. This kind of attempt with ruthenium hydrous oxide-CS surface conjugation has not been reported earlier. Also, it is envisaged that such a nano-material suspension involving ruthenium could be radio-chemically tagged with a cyclotron produced Ru-95 radioisotope27, a short lived positron emitter (half-life 1.643hrs), since chemical properties of an element are invariant with the change of the isotope of that element. The same nano-particles/or compounds bearing Ru ions could be suitably labeled in aqueous phase and could be an important application for developing a radio-isotopically labeled drug molecule for diagnostic studies involving positron emission tomography.


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Table 1. Some important chemical parameters for Fe and Ru as transition metals (reference 20, 21) Parameter

Fe

Ru

Electronic Configuration :

[Ar] 3d 4s

[Kr] 4d7 5s1

Oxidation states

0 to VI, II and III are most probable

0 to VIII, II, III and VI are most probable

ionic radii (Coordination VI)

Fe+2= 0.076 nm, Fe+3= 0.064 nm

Ru+3=0.072 nm Ru+4= 0.067 nm

Ionization Potential

Ist = 1.265 x10-18J IInd= 2.592 x10-18J IIIrd= 4.909 x10-18J

Ist = 1.179 x10-18J IInd= 2.685 x 10-18J IIIrd= 4.559 x10-18J

Electronegetivity (Pauling)

1.81

2.2

6

2

2. Experimental 2.1.Chemical Method : RuCl3.3H2O and L-Cysteine (M.F.C3H7NO2S) were procured from Aldrich and Ammonia from Merck, India. All these chemicals were used as purchased without further purification. For all experiments, millipore water has been used.

a) Chemical synthesis of pure ruthenium hydrous oxide (ruthenium oxy – hydroxide): Rucl3.3H2O has been dissolved in water (concentration:0.5M) and then ammonia solution (1:1 volume) was added to this solution drop by drop, maintaining pH ≈7- 7.5, initially ruthenium oxy-hydroxide precipitate has been collected and re-dispersed into water and then centrifuged repeatedly for removing of chloride ion. Finally the precipitate was recollected and dried at 353K for 6 hours to get pure ruthenium hydrous oxide (ruthenium oxy-hydroxide). The dry precipitate was calcined at four different temperatures 473, 573, 623 and 673 K for 6 hours. b) Preparation of Cysteine conjugated ruthenium hydrous oxide:


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To the initial aliquot portion of aqueous solution of RuCl3 .3H2O, 2% L-Cysteine solution was added (see Figure 1a), maintaining pH at 7-7.5, i.e. at physiological condition, this precursor solution was kept over a sand bath at slightly elevated temperature (313K) with in vacuum chamber for a week, when the fine grains of the L-Cysteine conjugated ruthenium hydrous oxide compound appeared, (see the flow chart at Figure 1b) they were isolated, washed and dried, these grains were soluble under the same pH conditions.

2.2. Physical Instrumental Methods : a) Thermal analysis : Differential Scanning Calorimetry(DSC) and Thermogravimetry(TG): Calorimetric measurements has been performed using a differential scanning calorimeter (DSC: Model No. 204 F1, NETZSCH, Germany) in the range of 270 – 440 K , purging with 99.999 % pure nitrogen gas. Figure 2 a. represents the DSC scan of the sample in the range of 283-773K at the rate of 5K/minute. The TG measurements have been taken using a thermo-gravimetric analyzer (TGA) of Netzsch, Germany (model: STA 449C) using 99.999% percent pure nitrogen as purge. The analysed results are given in Figure 2 b. b) X-ray Diffraction measurements: The ruthenium oxide powder sample has been subjected to X-ray diffraction analysis using Seifert XDAL 3000 diffractometer with CuKα radiation (wavelength of radiation, λ= 0.154 nm). The data have been collected in the range (2θ) ~ 10o – 90o with a step size of 0.02o at room temperature. Si has been used as external standard. The XRD pattern has been shown in Figure 3. The grain size of the powder sample has been calculated using Scherrer formula28: Dhkl = Kλ/βP cosθ , where, Dhkl is the average grain size, K the shape factor (taken as 0.9), λ is the Xray wavelength, βP is the full width at half maximum (FWHM) intensity after correction of instrumental resolution and 2θ is the Bragg angle chosen at ~ 28º.

c) Field Emission Scanning Electron Microscopy (FE-SEM) and Energy Dispersive analysis X-ray (EDAX) measurements :


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Field emission scanning electron microscopy (FE-SEM) was carried out on a JEOL JSM7610F instrument (Tokyo, Japan), and energy dispersive X-ray spectroscopy (EDX) was performed with an OXFORD Inca detector interfaced at 15 kV without sample sputtering. The results are shown in Figure 4.a and b.

d) X-ray Photo electron Spectroscopy (XPS) measurements : XPS core-level spectra were taken with an Omicron Multiprobe (Omicron NanoTechnology GmbH., UK) spectrometer fitted with an EA125 hemispherical analyzer. A mono-chromated Al Kα X-ray source operated at 150W was used for the experiments. The analyzer pass energy was kept fixed at 6.40x10-18J for all the scans. As the samples were treated with a low energy electron gun (SL1000, Omicron) with a large spot size that was used to neutralize the samples. The potential of the electron gun was kept fixed at - 4.80x10-19 J for all the samples with respect to the ground. The binding energy of the peaks were corrected by shifting the peak positions by an equal amount that was required to shift the main peak of the corresponding C1s spectrum to 4.57 x10-17J. The results are shown in Figure 5.

2.3. Spectroscopic methods of characterization a) UV-Vis spectroscopic measurement: UV−Vis diffuse reflectance spectra were recorded on a Cary-5000 UV−Vis spectrophotometer in the region of 200−800 nm, after dispersing the samples in KBr (moisture free), results shown in Figure 6.

b) Fourier Transform Infrared (FT-IR) spectra: Fourier transform infrared (FT-IR) spectra of the powder (as pellets in KBr, without moisture) were recorded using Perking Elmer FT-IR system, spectrum 100 in the range of 450 – 4000 cm-1 with a resolution of 0.2 cm-1 as shown in Figure 7.

2.4. Dynamic light scatting measurements(DLS): Both the samples hydrated Ru-oxide and Ru-Cs conjugated NPs were dispersed in aqueous solutions and homogenized, the pH 7-7.5 was maintained in all the cases. The DLS measurements were performed by using Malveen Zetasizer for the aliquot portions of these solutions. The results have been shown in Figure 8.


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2.5. Biological Experiments MTT assay for cell viability : Cytotoxicity of hydrous ruthenium oxide and the CS conjugated nano-particles on HeLa cells have been determined by conventional MTT [MTT= (3-(4, 5-Dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide, a yellow tetrazole] assay29. Exponentially growing HeLa cells were seeded in 96-well flat-bottom culture plates at a density of 6 x 104 cells per well in 100 µl DMEM medium (Himedia, India) containing 10% FBS (Gibco, USA). The cells were allowed to grow for 24h at 310K in a CO2 incubator (New Brunswick Scientific, U.S.A.), and then the medium was replaced with 100 µl fresh medium containing various concentrations of hydrous ruthenium oxide(0 to 1 µg/ml) and also Ru hydrous oxide-CS separately (same concentration) . The assay was performed in quadruplet for each concentration. The cells were then incubated for 24h, after which the culture medium was removed aseptically, and 100 µl of 3 mg/ml MTT reagent in DMEM (without FBS) was added to each well. Thereafter, it was incubated for 4h; during which active mitochondria of viable cells reduce MTT to purple formazan. Unreduced MTT were then discarded and “MTT solubilization solution” [10% Triton-X-100 in acidic (0.1N HCl) iso-propanol] (100 µl) was added into each well to dissolve the formazan precipitate, which was then measured spectrophotometrically using a microplate reader at 570 nm. The cytotoxic effect of each treatment was expressed as percentage of cell viability relative to the untreated control cells. The simplest estimate of IC50 is a plot of concentration of hydrous Ru oxide-CS (X) Or bare Ru-hydrous oxide against percentage of cell viability (Y), the data has been fitted with a straight line (linear regression). IC50 value is then estimated using the fitted line, i.e., Y = a * X + b, and IC50 = (0.5 - b)/a. (see Figure 9)

3. Result and Discussion DSC and TG analysis of RuOx(OH)y:


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The sample obtained after precipitation, was initially dried under vacuum oven for two days under 353K which

has been designated as hydrous ruthenium oxide30,31 (RuO2.xH2O or

ruthenium oxy-hydroxide : RuOx(OH)y). The correlation between the structure and properties of this material is not well understood, but the picture that emerges23 is: the nano-particles of this hydrous oxide consists of disordered arrangements of RuO6 chains, capped by OH groups. Their surface chemistry much relies on interfacial electron transfer between the nano-particles and control of their microstructure. The material needs to be well characterized due to its structural disorder and variable valency state, especially with respect to the oxidation state of Ru and its structural water or -OH content, whereas the crystalline Ru(IV) oxide, RuO2, has been thoroughly characterized and can be used as a reference material for the hydrous samples. In order to elucidate the water content of the substance and to understand the structural parameters32,33 at different stages of drying processes, further thermal analysis was carried out through DSC and TG measurements and the results are shown in Figure 2.a and b. An endothermic peak was observed in the range 400–472 K (Figure 2a) with a calculated small enthalpy change of +128.4J/g, but ruthenium oxy–hydroxide has been still in amorphous state which corroborate with the XRD data(as shown in the following results). The first endothermic peak was observed in the range 556–610 K, is due to partial transformation of amorphous to crystalline structure transition corresponding to ∆H ~- 302.7J/g. The second sharp peak has been observed in the range 610–638 K, due to an amorphous to crystalline structure phase transition of already known rutile structure of RuO2 with a ∆H ~-568.6J/g , which could be confirmed by XRD as shown in the following section. Such clear enthalpy changes were not stated in earlier studies31. In TG analysis as shown in Figure 2b, the weight loss was found to occur in stages, the initial stage could be assigned to physical dehydration viz; desorption of water and also for entrapped water (in the range 323– 538 K) and the second one was attributed to chemical dehydration (in the range 538–623 K). Total weight losses of 51 wt% were observed after treatment at 773K. Thus the hydrated oxide is the precursor material which after stepwise dehydration procedure yields RuO2.

X-ray Diffraction (XRD) study of RuOx(OH)y: XRD results give us the characteristic diffraction pattern of the crystalline form, through characteristic peaks at the Bragg angles. Figure 3 shows the XRD patterns of the ruthenium oxy-


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hydroxide powder samples as prepared after drying in vacuum at 353K and after calcination at 473, 573, 623 and 673 K. For samples calcined at 573, 623 and 673K, the diffracted peaks corresponds to (110), (101), (200), (211), (220), (002), (310), (112), (301), (202) planes. But with the samples heated at 353 and 473 K, no characteristic diffraction peak has been found. It has been confirmed23,30,31 that, crystallinity occurs at a temperature above 573K. These results corroborate with the DSC and TG analyzed data, which are in concurrence to the various effects of endothermic transitions around temperatures ≥ 573K. Further, the average grain sizes of the ruthenium oxide samples were estimated from Xray line broadening using Debye-Scherrer’s equation28. The particles sizes have been found to be 7.3, 7.6, and 8.5 nm at 573, 623 and 673 K respectively and as per the general trend, size of nano-particle has been found to increase with increasing temperature13 in most of the cases. The sample after calcining to >623K, XRD peaks were assigned to RuO2 in a tetragonal crystallographic structure30. No presence of Ru in a metallic form was found. The crystalline Ru(IV) oxide, RuO2, has been thoroughly characterized and can be used as a reference material for the hydrous ruthenium oxide where the material has not been well characterized due to its structural disorder and variable chemistry. Further, since the RuO2. xH2O materials are amorphous or poorly crystalline, their structure can be best elucidated from the extended analysis of their electronic charge and the complicated hydrous shell as well as their surface structure through other spectroscopic methods23. Here so far we only confirm that the hydrated ruthenium oxide is the precursor material of the tetravalent Ru (IV) in the crystalline anhydrous rutile RuO2. Just as the as prepared sample shows poor crystallinity as the structure may not be in order or the hydrated (encapsulated, water sphere) micro-structure could obstruct the diffraction pattern (as seen from the Figure 3), so also the nano-sized particles synthesized within the template of cysteine –amino acid and dried in a vacuum oven at ~313 K do not show crystallinity.

FE-SEM and EDAX analysis : Surface morphology of the powdered samples have been examined under scanning electron microscope, the results are shown in Figure 4. The as prepared sample of hydrous ruthenium oxide at 353K showed the swollen nano-structure granules of the substance under FE-SEM


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(Figure 4a, inset). The nano-material appears to be aggregated, due to evaporation process under vacuum. The elemental compositions as found from the EDAX analysis in the same figure confirm the presence of Ru and O which attributes to a critical check on the chemical composition of the nano-material and thus the purity of the same, immediately after sample preparation and drying process. However, the morphology of the L-cysteine templated samples show a remarkable change in shape and structure. The nano-sized particles now appear as elongated narrow-tubular structure. Figure 4 b. is a representative SEM image (inset) where such shape changes can be seen. This effect also confirms the observations of an earlier study by Nazar33 in developing mesoporous ordered structures of ruthenium hydrous oxide. Dipole-Dipole interaction of polar amino acid moiety acting as surfactant molecule with the redox active charged surfaces of the nascent nanoparticle32 and a slow process of growth along the surface of the macromolecule could be responsible for such a distinct change of the structure for the surface conjugated NPs although the detailed physico-chemical mechanism remains to be explored. It is worthwhile to report that surface coroneted nano-particle clusters have been shown to present a different morphological effect when a certain concentration level of the surface active molecule has been attained through aqueous sol-gel route, as shown in case of the synthesis of ZnO NPs13, GaO(OH)15 etc. It is possible that the surface polar –SH, :N and :O groups of the amino acid-CS moiety links with ruthenium hydrous oxide (with its redox charge centers)23 from all side and serves as a template, therefore the growth of the nano-particles are oriented according to the surface area and attractive potential provided by the CS moiety (as is explained by the spectroscopic observation in the following paragraphs). In the slow process of charge-dipole interaction, shape of the nano grains could have been changed due to the conjugating ligand, as is evidenced by the FE-SEM figures. The chemical composition vis-a-vis distribution of element constituents of the L-cysteine capped RuOx(OH)y NPs were also determined by EDAX analysis, confirming the presence of C, Ru, N, O and S, which has been shown in Figure 4 b.

XPS analysis The X-ray photoelectron spectroscopy of Ru 3d region for RuOx(OH)y exhibits two broad peaks at ~4.49 x10-17 and ~4.57 x 10-17J (Figure. 5a), the intensity arising from the 3d5/2 and 3d3/2


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electrons. Several studies have reported the XPS of different forms of hydrated ruthenium oxide 34,35, 23, 36–40

. There is general consensus that the signal appears from a mixture of Ru (III) and

Ru(IV), which causes the broad bands. In addition, the hydroxyl and oxygen functionalities surrounding the Ru also lead to broadening; There is also contribution from the C 1s in this binding energy range. The peak around 4.47x 10-17 - 4.49x 10-17 J could be due to Ru (III) state and those around 4.55 x10-17 and 4.57x 10-17J could be due to Ru(IV) state. XPS data of RuOx(OH)y in O 1s and Ru 3d region is shown in Figure. 5b. around 8.49x10-178.51x 10-17J with a more or less broad peak due to oxygen 1s region (Figure. 5a), indicating the presence of multiple oxygen species. Based on the literature35, this broad region could be assigned to a variety of oxy-hydroxy group (or hydrated sphere) in the order of increasing binding energy to Ru–O–Ru, Ru–OH and Ru–H2O, respectively. XPS study (Figure 5) of ruthenium oxy-hydroxy NPs conjugated to L- cystein –amino acid show broad and modified peaks Figure 5c for Ru 3d binding energy, N (1S electron binding energy peaks Figure 5d), O (1s electron binding energy peak, Figure 5 e) and the presence of S (2p electron binding energy peak, Figure 5f) which are evidently attached to the hydrated ruthenium oxide by coordination through the lone pair of electrons, viz N: , O: and also S: as electron donor groups to ruthenium at variable valency states as transition metal ion within the nano-tubular NPs. Thus these evidently confer that the hydrated ruthenium oxide NPs are conjugated to the amino acid L-cysteine.

UV-Vis reflectance spectra analysis The reflectance spectra41 of pure ruthenium hydrous oxide NPs, L–Cysteine and ruthenium oxy-hydroxide nanoparticles conjugated to cysteine template have been shown in Figure 6. For pure ruthenium hydrous oxide (oxy-hydroxide) with the mixed valence state, the reflectance starts enhancing over a broad region from 400nm onwards to UV region and maximized to almost saturation, which is characteristic of d-d transition. In fact this black or the dark brown substance covers a wide region of wavelength for absorbance. As for pure Lcysteine, the reflectance starts decreasing from ~500nm onwards shows several minima around 450 to 250nm and grows strongly towards the maxima in UV region. Most importantly, for RuOx(OH)y nanoparticles in presence of cysteine template, the reflectance drops from a maxima at λ359.9 nm to a minima at λ283 nm and then exhibits a sharp drop/peak at λ234nm, where the same


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is discernible from the continuously growing reflectance intensity towards UV region. It means that the material has electronic energy levels that are separated by an energy in the UV region(the absorption maxima are blue shifted, as compared to pure cysteine) which absorb light energy of characteristic wavelength, to move electrons from an occupied energy level into its exited level. This causes a relative decrease in the amount of light at that particular energy, relative to a reference source, eventually the percentage reflectance decreases. This kind of observation has been related to charge transfer complex formation42. From these results, it is evident that surface conjugated ruthenium hydrous oxide species have typical structural sites where L-cysteine amino acid has coordinated possibly through :N, :O and :S moieties, as the observed results in case of pure ruthenium hydrous oxide NPs and that of L-Cysteine as powdered sample are very different.

FT- IR Investigation The structural analysis43,44 of ruthenium oxy-hydroxide nanoparticles in presence of L-cysteine template have been further supported through FT-IR investigation, shown in Figure 7. The FT-IR spectra of ruthenium oxy- hydroxide at 353 and 673 K have been shown in Figure 7a. The FTIR spectra show absorption bands corresponding to the residual functional groups of RuOx(OH)y as shown in Table-2. It is important to note that only one absorption band corresponding to peroxo group (1110 cm-1) occurs after heating to 673°K and the absorption around 3600 cm-1 is almost vanished. The structural analysis of ruthenium oxy-hydroxide nanoparticle in the presence of cysteine template was further supported through FT-IR investigation. In the spectra Figure 7b, peaks appeared at 3456 cm-1 and 3313 cm-1 which are assigned to N-H stretching vibration of pure Lcysteine and cysteine conjugated RuOx(OH)y respectively. The S-H stretching appeared in pure cysteine at

2400 cm-1 is shifted to 2225 cm-1 and broadened in cysteine templated ruthenium

hydrous oxide compound. The broad and prominent peak observed at 1787 cm-1 indicates the C=O stretching of carbonyl group, that is also responsible in ruthenium hydrous oxide conjugation as it suffers a strong shift from its position originally at 1864 cm-1 in pure cysteine.

Table 2. Important Infrared absorption frequency criteria of the ruthenium hydrous oxide nanomaterial after heating at 353K and at 673K.


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Wave number (cm-1)

Characteristic group and modes of vibration

483, 650

Vibrational stretching mode of RuO2

1111, 890

Stretching vibration of per-oxo group

1526, 1728

Bending vibration of hydroxyl group in water molecule only in as prepared ruthenium oxide hydrated non-crystalline sample

3600

Stretching vibration of OH group in the hydrated sample as stated above.

Ru-O stretching vibration is also observed at~ 650 cm-1, in the presence of cysteine templated compound although it appears submerged due to several other low frequency bands of C-H bending in cysteine. From these observations, it is concluded that S-H functional group, N-H and C=O are all affected, they are directly involved in RuOx(OH)y co-ordination through S:, N: and O: in L-cysteine molecule which prominently suffer notable frequency shifts (see Table 3).

Table 3. The characteristic group frequencies in L-cysteine and its conjugated compound of ruthenium hydrous oxide depicting the changes in the functional group frequencies.

Peak

position Characteristic group and

(frequency cm-1)

mode of vibration

3456

N-H stretching

pure cysteine

3313

N-H stretching

Ru-oxide conjugated cysteine

2400

S-H stretching

Pure cysteine

2225 broadened

S-H stretching


1864

C=O stretching

Pure cysteine

1787

C=O stretching


Compound


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DLS Studies: The hydrodynamic size of the hydrated Ru-oxide and cysteine conjugated Ru-oxide nano particles were measured and reported in the Figure 8., which clearly shows that conjugated RuNPs are far smaller than the bare hydrated Ru-oxide nano particles, although the FE-SEM investigation shows a different picture. The same could be due to the drying and agglomeration processes involved while data taking. The NPs dispersions in solutions were found to be stable for repeated observations. This experiments were performed to judge if the aqueous dispersion of the NPs would be suitable for biological cellular uptake. Thus taking the cue from Figure 8, one could judge the ease of entry of Ru-CS conjugated NPs in the biological system as compared to their bare oxide form.

Biological studies: Biological treatment of Ruthenium hydrous oxide NPs and CS templated NPs with the HeLa cells were studied on the basis of the experiments reported17, on rapidly growing HeLa cell line (in-vitro). Since the surface of the HeLa cells contain the high affinity transferrin receptors, which are the principal vehicles by which iron is brought in to the cells17,45, it could also play the same role for ruthenium17. In this case transferrin (associated to its receptor molecule at the cell surface) instead of binding to iron, it is also capable of binding to hydrated Ru (III) in the nano – particles which could then be internalized46. For the rapidly growing cells, in physiological medium, under reducing conditions (due to insufficient supply of oxygen, appropriate environment exists to reduce Ru+3 to Ru+2) the active species of Ru+2 could be thus rendered possible17 to bind with appropriate DNA sites or proteins molecules to produce cytotoxic effects 47

.

It has been observed that the CS wrapped nano-particles (as seen from Figure 8), have an advantage over that of bare ruthenium hydrated oxide particles as they are already surface coronated by CS amino acid molecules, which could get easy access through the membranes of the HeLa cells rather than the pristine surface of the NPs. It is also worth mentioning here that the hydrodynamic size of Ru-CS coated NPs are much smaller as compared to Ru-hydrous oxide as shown by DLS studies in Figure 8. and this could be another reason, for ease of intake by the cells, beside being surface conjugated by CS .


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A systematic dose dependent cell viability assay following a standard protocol48 was performed on the HeLa cell line through standard MTT assay27 which shows (Figure 9) results for IC50 value at a dose of ~30.33 µg ml−1 for CS templated ruthenium hydrous oxide. Whereas, when the same method has been followed for the bare ruthenium-hydrous oxide nano-particle, it shows the cell viability IC-50 value at ~46.52µg ml−1. This is however an average effect on the cell line.

4. Conclusion From the experimental evidences, we conclude that ruthenium hydrous oxide nano-structures as prepared under physiological conditions are amorphous nano-materials wrapped with hydrated spheres, which is basically the precursor material for RuO2 NPs (nano crystallites) with rutile structure. These nano-particles were successfully surface conjugated with amino acid molecule like L-cysteine, as a drug carrier, where in their structural morphology changes to elongated nano -tubular structures after drying process as suggested by FE-SEM studies. We have made a detailed spectroscopic study of these NPs on material aspects, where changes due to surface chemical conjugation were studied. Further, for application in biological cell line, the hydrodynamic size of the NPs were assessed by DLS studies wherein the size of the Ru-CS conjugated particles were found to be much smaller as compared to bare Ru-hydrous oxide NPs. Our initial investigation and application of the surface conjugated nanoparticles when injected in cancerous HeLa cell line (in-vitro), showed that those were taken up by the malignant cells and MTT assay showed fairly good cytotoxic effects as compared to bare ruthenium hydrous oxide particle. Thus these nano materials could probably also lead to a new therapeutic route. However, the mechanistic processes of cellular uptake is beyond the scope our discussion. For a detailed biological aspect, more investigation towards apoptosis through the ability of NPs to bind to DNA or necrosis by adhering to the cell surface would be necessary, which could be a separate issue for further biological investigation. Also, it is envisaged that these NPs hold potential in nuclear medicine, if the same could be radio-chemically tagged with a short lived radiotracer Ru95-a positron emitter for future investigation in medical diagnostic processes.

Acknowledgements


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1

BNG, 2BM and 2TKM acknowledge the technical help received from Soma Roy, Anish Karmahapatra, and the research fellows for the operation of the facilities like high temperature furnace and FT-IR spectroscopy.

5. References 1. Juan J. Giner-Casares, Malou Henriksen-Lacey, Marc Coronado-Puchau, Luis M. LizMarzán, Inorganic nanoparticles for biomedicine: where materials scientists meet medical research, Materials Today 2016,19, 19–28. 2. Kristof Zarschler, Louise Rocks, Nadia Licciardello, Luca Boselli, Ester Polo, Karina Pombo Garcia, Luisa De Cola, Holger Stephan, Ultra-small inorganic nanoparticles: State-of-the-art and perspectives for biomedical applications, Nanomedicine: Nanotechnology, Biology and Medicine, 2016,12, 1663–1701. 3.Singh B, Singh S, Singh J, Saini GS, Mehta DS, Singh G, Tripathi SK, Kaura A. Understanding the adsorption behavior of surface active molecules on ZnO nanostructures by experimental and first-principles calculations, Phys Chem Chem Phys. 2015, 17, 30450-30460. 4. Chandraboss VL, Karthikeyan B, Senthilvelan S., Experimental and first-principles study of guanine adsorption on ZnO clusters, Phys Chem Chem Phys. 2014, 16, 23461-23475. 5. Saha S, Sarkar P., Understanding the interaction of DNA-RNA nucleobases with different ZnO nanomaterials, Phys Chem Chem Phys. 2014, 16, 15355-15366. 6. Sperling R. A. and Parak W. J., Surface modification, functionalization and bioconjugation of colloidal inorganic nanoparticles, Phil. Trans. R. Soc. A 2010, 368, 1333–1383. 7. Mu Q., Kievit F., Kant R., Lin G., Jeon M. Zhang M., Anti-HER2/neu peptide-conjugated iron oxide nanoparticles for targeted delivery of paclitaxel to breast cancer cells, Nanoscale 2015. 7, 18010-18014. 8. Song C., Zhong Y., Jiang X., Peng F., Lu Y., Ji X., et al., Peptide-conjugated fluorescent silicon nanoparticles enabling simultaneous tracking and specific destruction of cancer cells, Anal. Chem. 2015. 87, 6718-6723. 9. Meziani M.J., Sun Y.P., Protein-conjugated nanoparticles from rapid expansion of supercritical fluid solution into aqueous solution, J. Am. Chem. Soc. 2003, 125, 8015–8018. 10. Lemarchand C., Gref R., Couvreur P., Polysaccharide-decorated nanoparticles, Eur. J. Pharm. Biopharm. 2004, 58, 327–341.


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2006,311, 622–627. 13. Dutta Sreetama and Ganguly Bichitra N, Characterization of ZnO nano particles grown in presence of Folic Acid template, J. Nanobiotechnology, 2012, 10, 29-38. 14. Rasmussen John W., Martinez Ezequiel, Louka Panagiota, and Wingett Denise G., Zinc Oxide Nanoparticles for Selective Destruction of Tumor Cells and Potential for Drug Delivery Applications, Expert Opin Drug Deliv. 2010, 7, 1063–1077. 15. Ganguly Bichitra Nandi, Verma Vivek, Chatterjee Debanuj, Satpati Biswarup, Debnath Sushanta and Saha Partha, Study of Gallium Oxide Nanoparticles Conjugated with β-cyclodextrin An Application to Combat Cancer, ACS Materials and Interfaces : 2016, 8, 17127- 17137.

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23. Adeyemo Adedunni, Hunter Gary, Dutta Prabir K., Interaction of CO with hydrous ruthenium oxide and development of a chemoresistive ambient CO sensor, Sensors and Actuators B. 2011, 152, 307–315. 24. Frühauf S, Zeller WJ., New platinum, titanium, and ruthenium complexes with different patterns of DNA damage in rat ovarian tumor cells. Cancer Res. 1991, 51, 2943-2948. 25. Gallori, E., Vettori, C., Alessio, E., Vilchez, F.G., Vilaplana, R., Orioli,P., Casini, A., Messori, L., DNA as a possible target for antitumor ruthenium(III) complexes a spectroscopic and molecular biology study of the interactions of two representative antineoplastic ruthenium(III) complexes with DNA. Arch. Biochem. Biophys. 2000, 376, 156–162. 26. Noda T, Iwakiri R, Fujimoto K, Rhoads CA, Aw TY, Exogenous cysteine and cystine promote cell proliferation in CaCo-2 cells, Cell Prolif. 2002, 35, 117-129. 27. https://en.wikipedia.org/wiki/Isotopes_of_ruthenium 28.CullityB.D, Stock S.R. Elements of X-ray Difrraction, 2001, Prentice Hall, Englewood Cliffs, NJ. 29. Rubinstein L. V., Shoemaker R. H., Paull K. D., . Simon R. M, Tosini S., Skehan P., Scudiero D. A., Monks A. and Boyd M. R., Isolation, Structural Identification and Cytotoxic Activity of Hexanic Extract, Cyperenoic acid, and Jatrophone Terpenes from Jatropha ribifolia Roots. J. Natl. Cancer Inst. 1990, 8, 1113-1117. 30. David A. McKeown, Patrick L. Hagans, Linda P. L. Carette, Andrea E. Russell, Karen E. Swider, and Debra R. Rolison, Structure of Hydrous Ruthenium Oxides: Implications for Charge Storage, J. Phys. Chem. B. 1999, 103, 4825-4832. 31. Cruz J. C., Baglio V., Siracusano S., Antonucci V., Aricò A. S. , Ornelas R., Ortiz-Frade L., Osorio-Monreal G., Durón-Torres S. M., Arriaga L.G., Preparation and Characterization of RuO2 Catalysts for Oxygen Evolution in a Solid Polymer Electrolyte, Int. J. Electrochem. Sci. 2011, 6, 6607 – 6619. 32. Zang L., Kisch H., Room temperature oxidation of carbon monoxide catalyzed by hydrous ruthenium dioxide, Angew.Chem. 2000, 39, 3921-3922. 33. Oh Si Hyoung and Nazar Linda F., Direct synthesis of electroactive mesoporous hydrous crystalline RuO2 templated by a cationic surfactant, J. Mater. Chem. 2010, 20, 3834–3839.

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40. Kim K.S. and Winogard N., X-ray Photoelectron Spectroscopic Studies of Ruthenium – Oxygen Surfaces, J of Catalysis. 1974, 35, 66-72. 41. Audrey L. Companion, Theory and Applications of Diffuse Reflectance Spectroscopy, 1965, Springer US. 42. Yasmina Nesrine Tchenar, Abderrahim Choukchou-Braham and Redouane Bachir, RuO2 supported on V2O5–Al2O3 material as heterogeneous catalyst for cyclohexane oxidation reaction, Bull. Mater. Sci., 2012, 35, 673–681. 43. Bellamy, L.J. The Infrared Spectra of Complex Molecules; Methuen: London, 1959. 44. Svehla, G. Wilson & Wilson’s Comprehensive Analytical Chemistry, Vol.VI, Analytical Infrared Spectroscopy, Elsevier: Amsterdam, 1976. 45. Lamb, J. E.; Ray, F.; Ward, J. H.; Kushner, J. P.; Kaplan, J.Internalization and Subcellular Localization of Transferrin and Transferrin Receptors in HeLa cells. J. Biol. Chem. 1983, 258, 8751−8758. 46. Som P., Oster Z. H., Matsui K., Guglielmi G., Persson B. R., Pellettieri M. L., Srivastava S. C., Richards P., Atkins H. L. and Brill A. B., Eur. J. Nucl. Med. Mol. Imaging. 1983, 8, 491494.


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47. Gatenby R. A. and Gillies R. J., Why do cancers have high aerobic glycolysis?, Nat. Rev. Cancer, 2004, 4, 891-899. 48. MTT Cell Proliferation Assay. http://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx.


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Figures:

Figure 1a : Molecular structure of L-Cysteine used as conjugating system in solution, red : oxygen atoms, black : carbon atoms, yellow: sulphur atoms, white: hydrogen atoms (a model structure).


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RuCl3.3H2O

2% Cysteine solution

Precursor solution at pH~7.5

Slow evaporation process

Cysteine templated ruthenium oxy-hydroxide Grains (NPs)

Figure 1b. Flow chart of chemical preparation of RuOx(OH)y /ruthenium hydrated oxide nanoparticles in presence of L-cysteine template.


Langmuir

Energy / mWmg

-1

0

430.3K 587 K

(a)

-10

-20

613.6 K

-30 400

600

800

Temperature (K)

100

Weight loss %

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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(b) 80

60

200

400

600

800

Temperature (K)

Figure 2. : Results of thermal analysis of the as prepared ruthenium hydrous oxide sample a) DSC analysis, showing the transition points and b)TGA analysis showing the weight loss around the transition point.


220 002 310 112 301 202

211

101 200

Intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

110

Page 25 of 35

673 K 623K 573K

473 K 353K

20

40

60

80

2-Theta Figure 3. Powder XRD patterns of as-synthesized and calcined ruthenium hydrous oxide or Ruoxy hydroxide at different temperature. Crystallinity gains prominence from 573K onwards.


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4a. EDAX investigation showing the elemental composition: Ru and O in the sample and the corresponding FE-SEM images of hydrated Ruthenium oxide nano particles (inset)


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Figure 4 b. EDAX of Cysteine conjugated hydrated RuO2 sample showing the elemental constituents: Ru , S, O, N, C etc. and the inset is the FE-SEM image of L-cysteine conjugated nano tubular structure of ruthenium hydrated oxide material.


Langmuir

455.33

15000

RuO(OH)xRu3d ,

451.33 Intensity

457.90 10000

(a)

5000

0 440

445

450

455

460

465

Binding energy (10

-19

470

475

x J)

20000

8 5 0 .3 0 Ru O (O H )X , O 1 s (b )

15000

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 28 of 35

10000

5000 840

850

860

B in d in g ene rg y x 10

-19

870

J

Figure 5 : XPS study of freshly prepared Ru-oxy -hydroxy compound treated at 80°C, binding energies (J) of a) Ru 3d and b) O 1s bound to Ru has been shown.


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454.24

9000

Ru 3d

(c)

Intensity

457.95

6000

3000

0 440

450

460

Binding energy x 10

470 -19

J

15000 639.87

Ru-CS, N 1s (d)

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

10000

5000

635

640 645 Binding Energy x10-19 J

650


Langmuir

848.04

15000

Ru-CS, O1s

Intensity

(e)

10000

5000

0 840

850 Binding energy x10-19 J

860

259.39

10000

Ru-CS , S 2p (f) Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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5000

0 255

260

265

270

Binding energy x 10-19 J

Figure 5 : XPS of Ru oxy-hydroxy compound doped with 2% cysteine showing the characteristic binding energy (eV) peaks for c) Ru -3d, d) N 1s, e) O 1s and f)S 2p


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1068nm

100 80

% Reflectance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

234nm

60 Hydrated Ru-oxide Cysteine Cysteine-Hydrated Ru -oxide

40 20 0

min at 425.84nm

200

300

400

500

600

700

800

Wave length (nm) Figure 6. UV-Visible reflectance spectra of hydrated Ruthenium oxide (oxy-hydroxy compound RuOx(OH)y) NPs(black), pure cysteine(red) and cysteine conjugated RuOx(OH)y NPs.(blue)


Langmuir

a) 1.9 (ii)

1.8

Intensity (a. u.)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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-1

650 cm

1.7

-1

510cm

-1

1111cm

1.6

(i) -1

1520-1700cm

1.5

-1

3300-3700cm -1

890cm

1000

2000

3000

Wave number (cm-1)

4000

Figure 7a. FT IR spectroscopic studies of i) as prepared ruthenium hydrated oxide dried at~80°C(in black), ii) ruthenium oxide heated at 400°C (in red).


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b)

(i) Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

2225 cm 650cm

-1

1787cm

-1

3313cm

-1

1 8 6 4 cm

-1

2400cm

2000

(ii)

-1

3456cm

1000

-1

3000

-1

W a ve n u m b e r (c m -1 )

4000

Figure 7b. FT IR spectroscopic studies of i) L-cysteine conjugated with ruthenium hydrous oxide (in red)NPs as compared to ii) L-Cysteine (in black)


Langmuir

75.08nm

15000

Ru-oxy- hyroxide

Number density

a) 10000

5000

0 -100

0

100

200

300

400

500

Diameter (nm)

65.64nm

3000

Ru-cysteine nanoparticles

b) Number density

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

2000

1000

0 -100

0

100

200

300

400

500

Diameter (nm)

Figure 8. Dynamic light scattering study of hydrodynamic size of the nanoparticles a) hydrated Ru-oxide , b) CS conjugated Ru oxide , depicting the size distribution in the dispersed media.


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100

Ru(OH)x Ru(OH)x -Cys Percentage of viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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50

0 0

20

40

60

Conc (µ µ g/mL) Figure 9. Results for dose dependent cell viability as determined through MTT assay, showing cytotoxic effect on HeLa cells using, cysteine templated ruthenium hydrous NPs (in red) and bare ruthenium hydrous oxide(blue).


l-Cysteine-Conjugated Ruthenium Hydrous Oxide Nanomaterials

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