Synthesis, characterisation, cellular uptake and cytotoxicity of functionalised magnetic ruthenium (II) polypyridine complex core-shell nanocomposite

Accepted Manuscript Synthesis, characterisation, cellular uptake and cytotoxicity of functionalised magnetic ruthenium (II) polypyridine complex core-shell nanocomposite

Kandibanda Srinivasa Rao, Gundeboina Narasihmha, Sourav Das, Sunkara V. Manorama PII: DOI: Reference:

S1011-1344(17)31020-5 doi:10.1016/j.jphotobiol.2017.10.037 JPB 11040

To appear in:

Journal of Photochemistry & Photobiology, B: Biology

Received date: Revised date: Accepted date:

8 August 2017 17 September 2017 30 October 2017

Please cite this article as: Kandibanda Srinivasa Rao, Gundeboina Narasihmha, Sourav Das, Sunkara V. Manorama , Synthesis, characterisation, cellular uptake and cytotoxicity of functionalised magnetic ruthenium (II) polypyridine complex core-shell nanocomposite. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Jpb(2017), doi:10.1016/j.jphotobiol.2017.10.037

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ACCEPTED MANUSCRIPT Synthesis, characterisation, cellular uptake and cytotoxicity of functionalised magnetic ruthenium (II) polypyridine complex core-shell nanocomposite

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Kandibanda Srinivasa Rao[a]*, Gundeboina Narasihmha[a], Sourav Das[b], Sunkara V.

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Manorama[a]*

[a] Nanomaterials Laboratory, Inorganic and Physical Chemistry Division, CSIR - Indian

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Institute of Chemical Technology, Hyderabad, India, 500007.

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[b] Biomaterials Group, CSIR-Indian Institute of Chemical Technology, Uppal Road, Tarnaka, Hyderabad - 500007, India.

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Corresponding authors:

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* Tel: 91-9849149508, E-Mail: [email protected],

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* Tel: 91-9866061347, E-mail: [email protected], [email protected]

ACCEPTED MANUSCRIPT Abstract The development of multifunctional nanoparticles comprising of a magnetic core in conjunction with appropriate molecules with capabilities to impart functionalities like luminescent, specific binding sites to facilitate attachment of moieties. This has attracted

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increasing attention and enable identification of promising candidates using for applications such as diagnostics and cure through early detection and localized delivery. Many studies

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have been performed on the synthesis and cellular interactions of core− shell nanoparticles, in

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which a functional inorganic core is coated with a biocompatible polymer layer that should reduce nonspecific uptake and cytotoxicity Here we report the synthesis and characterisation multifunctional

core-shell

magnetic,

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of

luminescent

nanocomposite

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(Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2). Fe3O4 as core and a luminescent ruthenium(II) complex encapsulated with silica shell, and then it is functionalized by an amine group by

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APTMS. The magnetic, luminescent, and biological activity of this multifunctional

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nanocomposite have also been studied to prove the nanocomposite is biocompatible, cellular uptake. The synthesized nanocomposite was completely characterised by X-ray diffraction (XRD), Fourier-transform infrared (FTIR) spectroscopy, transmission electron microscopy

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(TEM), vibrating sample magnetometer (VSM), and emission spectroscopy. MTT assay and

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cellular uptake by flow cytometry results proved that magnetic ruthenium(II) polypyridine complex – core shell nanocomposite has biocompatablity, minimum cytotoxicity and internalised inside B16F10 cells and confirms the potential biomedical applications. Keywords: Fe3O4. bifunctional • core-shell nanocomposites • ruthenium(II) polypyridine • MTT assay. cellular uptake.

ACCEPTED MANUSCRIPT 1.0 Introduction Magnetic nanoparticles, specially Fe3O4 nanoparticles are of great interest for the study of magnetic properties and for practical applications such as magnetic resonance imaging (MRI) contrast enhancement[1,2], magnetic immobilization,[3] bioelectrocatalysis[4],

therapeutic applications[7-9]. Magnetic NPs need to be

uniform size with high magnetic

to obtain high application sensitivity or

coupled with other functional

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moments[10],

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and drug targeting[5,6], and they have also been studied for biological imaging, sensing and

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components so that multifunctionality can be present within one nanostructure for simultaneous detections[11]. Reactivity of iron oxide nanoparticles is high as the particle

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dimensions are reduced, and particles with relatively small sizes may undergo rapid

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degradation when they are directly exposed to certain environments[12], consequently, a suitable coating is essential to overcome such limitations. With the properties of stability,

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biocompatibility, and easy functionality, silica acts as an optimum alternative to

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encapsulating these magnetic nanoparticles[13-15].

These days much attention is being directed towards the synthesis and application of

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dye-encapsulated silica nanoparticles [16-20].

On one hand, the encapsulation of

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luminescent dye in nanoparticles often increases their photostability and emission quantum yield due to their isolation from possible quenchers such as molecular oxygen and water, and added to that silica is relatively easy to functionalize and conjugate to bioactive molecules, which shows great potential in bioanalysis. luminescent dye encapsulated silica nanoparticles were prepared by the reverse microemulsion method, because of the possibility of size control and further miniaturization[21], among many dyes encapsulated, ruthenium(II) complexes prove to be suitable due to its good stability and high quantum yield [22]. Besides, its strong electrostatic interaction with silica makes its leaching almost negligible.

ACCEPTED MANUSCRIPT Consequently, ruthenium(II) complexes encapsulated silica nanoparticles can be extensively applied in bioanalysis and biodetection[23,24].

we have synthesized a new kind of

biocompatible nanoarchitecture containing a magnetic Fe3O4 core and a luminescent ruthenium(II) complexes encapsulated silica shell and it is functionalised by amine group. The cell viability and uptake assays reveal good biocompatibility of these hybrid

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nanoparticles. Hence, the composite nanoparticles are of potential to be further explored as

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therapeutic vector in biomedical field. Another important aspect is that silica is highly

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biocompatible and its surface can be easily modified with amines, thiols or carboxyl groups,

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which enables covalent modification of the particle surface for biological applications [25].

The Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2 nanocomposite was characterized by spectroscopy,

transmission

electron

microscopy

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FTIR

(TEM),

vibrating

sample

magnetometer (VSM), and emission spectroscopy. Here we report the synthesis and

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characterisation of functionalised core-shell magnetic, luminescent nanocomposite. Fe3O4 as

functionalized

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core and a luminescent ruthenium(II) complex encapsulated with silica shell, and then it is by an amine group by 3-aminopropyl trimthoxy silane (APTMS).

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Biocompatablity, minimum cytotoxicity of this nanocomposite was confirmed by MTT assay with B16F10 and Chinese hamster ovary (CHO) cell lines and internalisation of this

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functionalised nanocomposite inside B16F10 cells has been quantified by flow cytometry. 2.0 Materials and Methods Fe(acac)3, 1,2 Hexanediol, Oleic acid, Oleylamine, TritonX-100, Cyclohexane, Tetraethyl orthosilicate (TEOS),

ruthenium(III) chloride hydrate, 1,10-Phenanthroline,

APTMS, All the antibiotics ( penicillin, streptomycin, kanamycin) were obtained from Sigma-Aldrich, Diphenylether, 1-Hexanol, NH4OH from SD fine chemicals, MTT (3-(4, 5dimethylthiazol- 2-yl)-2, 5-diphenyl tetrazolium bromide) reagent was obtained from

ACCEPTED MANUSCRIPT Calbiochem. All cell lines B16 F10, CHO were purchased from the American Type Culture Collection (ATCC), USA. All reagents were used as received without further purification. After each step of the synthesis (as shown in scheme 2) the materials have been characterized to confirm the product and the reaction continued until the bifunctional nanocomposite, and further functionalized by amine has been synthesised. FT-IR spectra were recorded at room

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temperature(RT) over the range of 4000–400 cm-1 on an Alpha-Bruker FTIR spectrometer

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with a wave number resolution of 4 cm-1. Powder X-ray diffraction (XRD) patterns were recorded in reflection mode on a Seimens (Cheshire, UK) D5000 X-ray diffractometer over a

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2θ range from 10-650 using CuKα (=1.5406Å ) monochromatic radiation source to identify

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the crystal phase. Transmission electron microscope (TEM; Philips Tecnai FEI F20, operating at 200 kV) was used to investigate the morphology and size of the particles.

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The UV-Visible spectra were recorded on a Shimadzu (Model UV-3600) spectrophotometer. The fluorescence measurements were performed on a Fluorolog-3 spectrofluorometer (Spex

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model, JobinYvon). The concentration of the solutions was maintained constantly at

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OD~0.05 for all the compounds. Magnetic studies were carried on with a Micro-sense vibrating sample magnetometer (VSM) in the applied magnetic field sweeping from -5 to 5

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KOe.

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2.1 Preparation of Fe3O4

Fe3O4(magnetite) nanoparticles were synthesised by thermal decomposition method by adopting a reported procedure [26]. 1.76g (5 mmoles) of Fe(acac)3 was dissolved in 30ml of diphenyl ether, 3.0g (25 mmoles) of 1,2 hexanediol was added to the solution. 4.7ml (15 mmoles) of oleic acid and 4.9ml (15 mmoles) of oleylamine were added respectively under nitrogen while stirring and continued the reaction at RT for 1 hour to get the homogeneous mixture and then this reaction mixture heated at 320oC for 30mins. Black precipitation was

ACCEPTED MANUSCRIPT obtained at the bottom. Particles were separated by centrifugation and followed by washings several times with Ethanol. Finally washed with Acetone and dried under vacuum. 2.2 Preparation of Fe3O4@SiO2: Silica was encapsulated onto the synthesized Fe3O4 nanoparticles by reverse

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microemulsion method. 7.2ml (12.3 mmoles) of TritonX-100 and 7.1ml (56.5 mmoles) of 1Hexanol were dissolved in 30ml of Cyclohexane respectively and the mixture was stirred.

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115mg (0.5mmoles) of Fe3O4 dispersed in 2ml of deionised Millipore water was added to the

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above solution while stirring. 400µl of TEOS was added slowly (drop-wise) to the reaction mixture followed by addition of 250µl of NH4OH after 20 minutes at room temperature to

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initiate catalysis and stirring continued for further 24 hours at room temperature. Acetone was

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added to break the microemulsion and the particles were collected at the bottom and separated by centrifugation. The final product i.e. Fe3O4@SiO2 was washed with water and

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ethanol several times and finally with acetone and vacuum dried.

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2.3 Preparation of Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2: Fe3O4@SiO2 was coated with [Ru(Phen)3]2+by using reverse microemulsion method.

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7.2ml of TritonX-100 (12.3 mmoles), 7.1ml (56.5 mmoles) of 1-Hexanol and 30ml of Cyclohexane were taken in 100 ml RB flask. 145mg of Fe3O4@SiO2 (0.5 mmoles) and

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320mg (0.5 mmoles) of the synthesized [Ru(Phen)3]2+was dissolved in millipore water and added to the above solution. 400µl of TEOS was added slowly (drop-wise) to the reaction mixture and 250µl of NH4OH was added after 20 minutes at room temperature to initiate catalysis and stirring were continued for 24 hours at room temperature. The microemulsion was broken by adding acetone and separating the particles by centrifugation. The final product i.e. Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 was washed with water and ethanol several times and finally with acetone and vacuum dried.

ACCEPTED MANUSCRIPT 2.4 Preparation of Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2: Amine functionalization of the Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 was accomplished by in a typical reaction 50mg (0.045mmols) of Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 and 1.7ml (12.0mmoles) of APTMS were added to 35ml of CH3CN and the reaction mixture was

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stirred at room temperature for 24h. This was followed by solvent decantation and the product was washed first with CH3CN followed by ethanol and acetone and the product was

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vacuum dried.

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2.5 Cell culture experiments

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cancer cell line (B16F10: mouse melanoma cell line) and normal cell line (CHO: chinese hamster ovarian cell line) were cultured in DMEM (Dulbecco’s Modified Eagle Medium)

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media supplemented with 10% fetal bovine serum (FBS), 1% antibiotics (penicillinstreptomycin-kanamycin) and in a humidified 5% CO2 incubator at 37 ºC for all in vitro

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experiments. The cells were incubated with different concentrations of Fe3O4 (S-1),

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Fe3O4@SiO2 (S-2), Fe3O4@SiO2@[Ru(Phen)32+]@SiO2@NH2 (S-3) for certain time point. 2.6 Cell viability test using MTT reagent

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It is a colorimetric assay to check the cytotoxicity of any compound [27]. Briefly, cancer cell (B16F10) and normal cell (CHO) were seeded in 96 well tissue culture plate at a density of

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10 × 103 cells per well for 24 hours. After 80 % confluency of the cells, they were incubated with S-1, S-2, S-3 with different (10 µg/mL, 50 µg/mL, 100 µg/mL) concentrations for another 24 hours. The cells were washed with PBS and MTT solution (0.5 mg mL−1 in DMEM) was added in each well and incubated at 37 °C for 4 h. 100 µL of DMSO:MeOH [1:1 (v/v) solution] was added in each well after replacing the MTT solution to solubilize the insoluble formazan dye to violet colour solution. Finally, the absorbance of the solution was measured at 570 nm using a BioTek multimode plate reader [28].

ACCEPTED MANUSCRIPT 2.7 Uptake study by ICP-OES: The ICP-OES method was performed to determine the cellular uptake efficiency of the nanomaterials. 2 × 106 cells were seeded in each T 25 flask. After 80% confluency, the cells were incubated at 37 °C for 24 h with 100 μg/mL of each nanomaterial (S-1, S-2, S-3). After

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the incubation period, cells were washed thoroughly with Dulbecco's phosphate-buffered saline (DPBS) and trypsinized. The total numbers of cells were counted using the

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haemocytometer and the samples were digested with 1 mL of 70% nitric acid and kept in

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water bath for overnight at 55 ºC. The digested solutions were diluted to 10 mL with Milli-Q H2O and used for ICP-OES analysis. The total iron (Fe) concentration was determined using

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the ICP-OES method.

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2.8 Uptake study by Fluorescence Assisted Cell Sorting (FACS) :

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B16F10 cell line was used to investigate the uptake of nonomaterials (S-2, S-3). 2 × 25

flask one day before treatment. All the nonomaterials

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106 cells were seeded in each T

(Dose: 100 µg/mL) were incubated with cells for 4 hours at (37°C, 5% CO2). After

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incubation, the cells were washed thoroughly with DPBS and trypsinized. Cells were then centrifuged to collect the pellet. The cell pellet was re-suspended in DPBS. The cellular

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uptake of the nonomaterials was analyzed by flow cytometry (FACS Canto II, Becton Dickinson, San Jose, CA, U.S) using the red fluorescence of [Ru(Phen)3]2+ , and their interactions were studied using confocal laser scanning microscopy. 3.0 Results and discussion At each step during the synthesis of the nanocomposite the product was confirmed by the characterization by different analytical techniques to ascertain the formation of the

ACCEPTED MANUSCRIPT targeted material and confirm

the physical characteristics like

size, morphology,

composition etc. The crystal structure of the as-synthesized nanocomposite was characterized by X-ray diffraction (XRD), and the XRD patterns are illustrated in figure 1(a-d). The diffraction peaks

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have been indexed and assigned to the cubic structure of Fe3O4 (figure 1a), which is in good agreement with the reported values (JCPDS card: 82-1533). The pattern of the weakening intensity

with

no

change

in

the

position

of

peaks

from

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peak

Fe3O4

to

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Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2NH2 (1a to 1c) can be observed in figure 1. The peaks of Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 (figure 1c) are much weaker than those of the Fe3O4, and

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this can be attributed to the formation of core-shell structure thereby increased screening the

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magnetic nanoparticles by the silica and [Ru(Phen)3]+2 in the final nanocomposites [29]. The

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intensity of peaks of the major diffractions for each sample is given in Table 1.

ACCEPTED MANUSCRIPT Figure 1. XRD Pattern of (a) Fe3O4 (b) Fe3O4@SiO2 (c) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 (d) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

FTIR spectra were recorded to identify the functional groups on the nanocomposite at different stages of the synthesis. The significant peak at 590cm-1 (Fe-O) (figure 2a) indicates

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the formation Fe3O4 and Silica coating over Fe3O4 was confirmed by observing the characteristic peak of (Si-O-Si) at 1090 (figure 2b). The peaks corresponding to phenyl group were observed and it indicates the presence of [Ru(Phen)3]+2 over

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at 1400-1600 cm-1

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Fe3O4@SiO2 (figure 2c). Functionalization of NH2 on dye encapsulated Fe3O4@SiO2 is

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confirmed by the evidence of a peak at 2928cm-1(figure 2d). These characteristic IR peaks

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prove the sequential coatings in order.

Pyridyl

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(c)

(a)

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(b)

Si-O

Si-O-Si

Si-O-Fe

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Transmittance (%)

(d)

Fe-O

4000

3500

3000

2500

2000

1500

1000

500

-1

Wavenumber (cm ) Figure 2.FTIR spectra of (a) Fe3O4 (b) Fe3O4@SiO2 (c) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 (d) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

ACCEPTED MANUSCRIPT The simple reverse microemulsion method was used to prepare the silica– encapsulated Fe3O4 nanocomposite, which was coated with dye and subsequently functionalized with an amine. Silica layer thickness is thin and it was controled by varying the volume of tetraethyl orthosilicate (TEOS). TEM images of bifunctional luminescent magnetic nanocomposite are shown in figure 3(a-d). The size of the particles observed from

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the micrographs reveal the diameter ranging from 10 nm to 110 nm. The Fe3O4@SiO2 core-

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shell structure of the nanocomposites, as revealed from the TEM images, shows a particle

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diameter of 15 nm and a silica shell thickness of ca. 5 nm. The main advantages of encapsulating Fe3O4 nanocomposite within silica is the compatibility rendered in biological

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systems [30]. Since the monolayer of the ruthenium (II) complex is thin to be observed by TEM, it is difficult to differentiate the inner silica layer and the outer protective silica layer

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from the TEM image. The size of dye encapsulated silica coated Fe3O4 and functionalised

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particles are 80 nm and 110 nm respectively.

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3.

TEM

images

of

(a)

Fe3O4

(b)

Fe3O4@SiO2

(c)

Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 (d) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

Free ruthenium (II) complex, Fe3O4, Fe3O4@SiO2, and Functionalized dye nanocomposite

Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

i,e

were

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encapsulated

characterised by UV-Vis absorption spectra that were recorded in aqueous solution as shown figure

4(a-d).

The

spectra

of

the

Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

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in

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nanocomposite and the ruthenium (II) complex were compared in aqueous solution. The UV-

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Vis absorption bands at ca. 260 and 400-500 nm of the Ru(Phen)3+2 are assigned as the Intra ligand (IL) and the metal-to-ligand charge transfer (MLCT) transitions of the ruthenium (II)

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complex, respectively, confirming the successful immobilization of the ruthenium (II) complex to the magnetic nanocomposite. The intensity of the peak at 260 nm decreased in

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dye encapsulated Fe3O4@SiO2 NPs (figure 4b) compared with free Ru(Phen)3+2, similarly the

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intensity of functionalized dye encapsulated Fe3O4@SiO2 nanocomposite peak (figure 4c) still decreased, this is because of increasing thickness of silica layer. The overall decrease in

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intensity from figure 4a-4c at 400-500 nm could also be attributed to the screening due to

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silica layer. As anticipated no excitation was observed in the case of Fe3O4@SiO2 (figure 4d).

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(b) (c)

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400

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(d)

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Absorbance

(a)

600

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800

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Wavelength(nm)

Figure 4.UV-Vis spectra of (a)[Ru(Phen)3]2+(100µM) (b)Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2

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(100µM) (c)Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2 (100µM) (d) Fe3O4@SiO2(100µM)

Emission spectra of the as-prepared Fe3O4@SiO2 nanoparticles, the ruthenium (II)

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complex coated on Fe3O4@SiO2 nanocomposite, functionalized dye encapsulated

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Fe3O4@SiO2 nanocomposite in aqueous solution were recorded at an excitation wavelength of 460 nm and compared as shown in figure.5(a-d). The silica-coated iron oxide nanoparticles were found to be nonemissive upon light excitation figure 5a. The fluorescent excited state of [Ru(Phen)3]2+ is assigned to the metal to ligand charge transfer (MLCT) state. Figure 5, shows that the bifunctional nanocomposite exhibits (figure 5b) an emission peak at a position slightly red-shifted from that of the free ruthenium complex (figure 5d), which is due to the screening effect of silica layer between ruthenium complex and functionalised nanocomposite. Reduction of intensity was also observed (figure 5d - 5b) from free

ACCEPTED MANUSCRIPT [Ru(Phen)3]2+ to silica coated particles and functionalized dye magnetic nanocomposite, this is due to the thin coatings over [Ru(Phen)3]2+.

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Intensity (a.u.)

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(d)

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(c)

(a)

600

650

700

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550

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(b)

750

800

850

5.

Fluorescence

spectra

of

(a)

Fe3O4@SiO2

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Figure

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Wavelength (nm)

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(b)Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2 (c) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2 (d) [Ru(Phen)3]2+

The magnetic property of Fe3O4, silica-coated Fe3O4 nanoparticles and bifunctional nanocomposite are studied on powder samples at room temperature by vibrating sample magnetometer. Figure 6 reveals the typically low coercivity and remanence of all synthesised nanocomposites, which indicates the

superparamagnetic character of the as-prepared

magnetic nanoparticles. The Fe3O4, silica-coated Fe3O4, bifunctional nanocomposite and its amine functionalized nanocomposite saturation magnetizations are 65.5, 19.1, 8.9 and 6.2

ACCEPTED MANUSCRIPT emu/g, respectively. The lower saturation magnetization of silica coated Fe3O4 and these

40

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(a) (b) (c) (d)

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20

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-20 -40 -60 -2000

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4000

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-4000

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Saturation Magnetisaion(emu)

functional nanocomposite could be attributed to the presence of the outer shell of silica.

Applied Magnetic Field(Oe)

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Figure 6. VSM spectra of (a) Fe3O4 (b) Fe3O4@SiO2 (c) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2

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(d) Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2

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The dispersed nanocomposite in solution was inductive to the external magnetic field. The nanocomposite was attracted to the magnet very quickly and accumulated near it

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within minutes when an external magnet was placed close to the glass sample tube, leaving the bulk solution clear and transparent. Absorption and emission spectra were recorded for the clear transparent liquid, that did not show any characteristics implying that there no traces of material. After removal of the external magnet and on vigorous shaking, the nanocomposite could be rapidly redispersed.

ACCEPTED MANUSCRIPT 3.1 Cell Viability Test by MTT Analysis Cytotoxicity is major limitation for biological applications, it is caused by leaching of dye in composite, and this problem was adequately taken care of by the SiO 2 coating on the ruthenium complex. This was experimentally validated by the cytotoxicity studies that were undertaken on the Fe3O4@SiO2@[Ru(Phen)3]2+@SiO2@NH2, which showed a minimum

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cytotoxicity. It is one of the basic assay to determine the cell viability against any cytotoxic

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material. We carried out MTT assay in B16F10 and CHO cell lines. Result revealed that in CHO cell line as well as in B16F10 cell line for all the three compounds, Fe3O4 (S-1),

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Fe3O4@SiO2 (S-2), Fe3O4@SiO2@[Ru(Phen)32+]@SiO2@NH2 (S-3). The cells were viable

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for more than ~70% [figure 7(a-b)]. This result confirmed the biocompatible nature of all the

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B16F10

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% Cell Viability

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three compounds [(S-1), (S-2), (S-3)].

Concentration

24 Hrs

ACCEPTED MANUSCRIPT Figure 7a. Cell viability assay using MTT reagent. Biocompatibility of S-1, S-2 and S-3

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were tested in B16F10 cells in a dose-dependent manner.

24 Hrs

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% Cell Viability

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CHO

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Concentration

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Figure 7b. Cell viability assay using MTT reagent. Biocompatibility of S-1, S-2 and S-3

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were tested in CHO cells in a dose-dependent manner.

3.2 Cellular uptake of nanomaterials using ICP-OES: We carried out cellular uptake studies of S-1, S-2, S-3 in the B16F10 cell line to confirm their internalization through the ICP-OES technique using the iron (Fe) content per cell (figure 8). We calculated the Fe concentration in picogram (pg) per unit of cell in B16F10 for each nanomaterials treated cells [(S-1: 1.53 pg), (S-2: 1.55 pg), (S-3: 1.93 pg)] (figure 8). The result exhibited that all the three compounds (S-1 to S-3) internalized inside B16F10 cells

ACCEPTED MANUSCRIPT after an incubation period of 24 hours. For the UT cell (without treated with nanoparticles)

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no uptake of iron has observed.

Figure 8. Cellular uptakes of nanomaterials (S-1, S-2 and S-3) in B16F10 cell line through

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ICP-OES analysis using the iron content per cell. This figure indicates the internalization of S-1, S-2 and S-3 compounds in B16F10 cells after 24 hours of incubation.

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3.3 Quantification of cellular uptake by flow cytometry: The uptake of the nanomaterials [S-3, S-2 (taken as negative control)] were further carried out by FACS using the red fluorescence property of the [Ru(Phen)3]2+ in B16F10 cells (figure 9 a-c). Untreated cells (figure 9a) and negative control S-2 (figure 9 b) did not show any shift of red fluorescence in the PE channel as S-2 has no [Ru(Phen)3]2+ moiety in its structure. On the other hand, the cells treated with S-3 exhibited shift in red fluorescence indicating a cellular uptake of that nanomaterial (figure 9c). The quantification result showed

ACCEPTED MANUSCRIPT the cellular uptake of the S-3 (33 %) is greater than S-2 (5 %) at same dose (figure 9d). This result indicates that S-3 nanomaterial having the [Ru(Phen)3]2+ moeity is internalized in the

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cell which is also confirmed by ICP-OES analysis.

Figure 9. Flow cytometry analysis for the cellular uptake of S-2, S-3 nanomaterials in

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B16F10 cells. The red fluorescence property of [Ru(Phen)3]2+ was utilized to study the

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cellular entry of S-3 nanomaterial. a: untreated B16F10 cells, b: control cells [treated with S2 (100 μg/mL)], c: cells treated with S-3 (100 μg/mL). d: % of the red shift (channel) represented in histogram. 4.0 Conclusion The objective is to synthesis and charecterisation of

new kind of biocompatible

nanoarchitecture containing a magnetic Fe3O4 core and a luminescent ruthenium(II) complex functionalized by amine group. MTT assay and cellular uptake by flow cytometry results

ACCEPTED MANUSCRIPT confirm that functionalised magnetic ruthenium(II) polypyridine complex – core shell nanocomposite has biocompatablity, minimum cytotoxicity and internalised inside B16F10 cells.

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Acknowledgements

This work was supported by the Department of Science and Technology (SERB),

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INDIA. KSR is thankful to DST for providing financial assistance under Fast track young acknowledges

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scientist scheme (SR/FT/CS-80/2011) and GN and Chitta Ranjan Patra

CSIR-India for financial support from 12th FYP project(ADD: CS0302), and SD

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acknowledges UGC for their Junior Research Fellowships. The authors thank Dr.Giribabu,

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Dr. Ravibabu for assistance in UV-visible and Fluorescence measurements. Dr. L.Satyanarayana for his assistance in carrying out the VSM measurements and Dr.Patra for

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valuable help in biological studies.

ACCEPTED MANUSCRIPT References

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ACCEPTED MANUSCRIPT Table1. The intensity of X-ray diffraction peaks before and after coating

Diffraction Fe3O4

Fe3O4@SiO2

Fe3O4@SiO2@

Fe3O4@SiO2@

[Ru(Phen)3]2+@SiO2

[Ru(Phen)3]2+@SiO2@NH2

70

50

20

15

311

210

150

60

50

400

40

20

20

15

333

60

30

25

20

440

60

30

25

20

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220

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peak

Intensity (counts approximately )

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Scheme:1

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Graphical abstract

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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT Highlights  Synthesis of multifunctional nanocomposite  Surface functionalised by amine group

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 Proved biocompatibility of new kind of nanocomposite