Supporting Information

Water soluble luminescent cyclometalated platinum(II) complex - A suitable probe for bio-imaging applications

Sheik Saleem Pashaa#, Pradip Dasb#, Nigam P. Rathc, Debashree Bandyopadhyayd, Nikhil R. Janab*, Inamur Rahaman Laskar*a

aDepartment of Chemistry, Birla Institute of Technology and Science, Pilani Campus, Pilani, Rajasthan, 333 031 Indiair_laskar@pilani.bits-pilani.ac.in; bCentre for Advanced Materials, Indian Association for the Cultivation of Science, Jadavpur, Kolkata 32, India camnrj@iacs.res.in; cDepartment of Chemistry and Biochemistry, University of Missouri-St Louis, 1 University Blvd, St Louis, MO 63121, USA rathn@umsl.edu; dDepartment of Biology, Birla Institute of Technolgy and Science, Hyderabad Campus, Hyderabad, Telangana, 500078, India banerjee.debi@hyderabad.bits-pilani.ac.in

#Both authors have the same contribution to this work

Materials

Potassium tetrachloroplatinate(II), 2-phenylpyridine, 2-ethoxyethanol and ethylenediamine were

purchased from Sigma Aldrich Chemical Company Ltd. The solvents were procured from

Merck. Dulbecco’s modified eagle medium (DMEM), penicillin/streptomycin, Triton X-100 and

propidium iodide were purchased from Sigma-Aldrich. Fetal bovine serum (FBS) was purchased

from Invitrogen. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was

purchased from Himedia.

Characterization

1H NMR and 13C NMR spectra were recorded on a 400 MHz Brucker NMR. UV-Vis absorption

spectra were recorded on a Shimadzu Spectrophotometer (model UV-1800 and 2550). Steady-

state Photoluminescence (PL) spectra were recorded on Horiba Jobin Yvon Spectrofluorometer

(FluoroMax-4). Differential interference contrast and fluorescence images of the cells were

performed by using an Olympus IX81 microscope with image-pro plus (version 7.0) software.

Synthesis

Synthesis of [C^NPtNCl] complex 1

It was synthesized by following the procedure as stated in the literature [1].

Synthesis of [Pt(ppy)(en)]Cl complex 2

It was synthesized by modifying the methodology as reported earlier [2]. Ethylenediamine (5.5

mmol) was added at room temperature to a stirred solution of complex 1 (1.85 mmol) in DCM

(10 ml). After 5 minutes of stirring, the solvent was evaporated under reduced pressure and the

crude product was purified by column chromatography (60-120 mess of silica gel) which

resulted a yellow solid powder (yield 72-82%). 1H NMR spectra of 2, [1H NMR (400 MHz,

DMSO-d6) 8.57 – 8.55 (m, 1H), 8.03 (dd, J = 11.1, 4.5 Hz, 2H), 8.03 (dd, J = 11.1, 4.5 Hz, 2H),

7.66 – 7.58 (m, 2H), 7.26 (dd, J = 7.4, 5.9 Hz, 4H), 7.17 – 7.08 (m, 3H), 2.86 (s, 3H); 13C NMR:

(101 MHz, DMSO-d6) δ 150.35, 145.02, 142.78, 139.31, 132.63, 129.58, 123.61, 123.45, 122.70,

118.85, 43.64.

Fabrication of thin-film of complex 2 on substrate for PL measurement

A 10-3 M solution of complex 2, was prepared in methanol. Two drops of the methanol solution

were placed on a thin glass substrate (2 x 2 cm2) and the solvent was allowed to evaporate

slowly.

Single crystal X-ray structure

A single crystal X-ray diffraction data set was collected on a Bruker AXS Kappa Apex II

diffractometer equipped with an Oxford Cryosystem 700 Plus liquid nitrogen based cooling

device. The data set was recorded at 100K using a combination of φ and ω scans to obtain a data

set complete up to 78.4 in 2. Data reduction and corrections were done using APEX II [3]

software suite (Bruker AXS). The crystal structure was solved using direct methods

(SHELXS97) [4] available in the Shelxtl [5] suite and the structure was refined by full matrix

least squares refinement process using SHELXL97 [4].

Computational calculations using Density Functional Theory (DFT):

The crystal structure of the 2 was optimized at ground state level using Density Functional

Theory (DFT). B3LYP hybrid functional was used in DFT. Double-Zeta basis set (LANL2DZ)

and effective core potential were approximated for platinum atom. The four atoms co-ordinated

to platinum and the counter-ion chloride was treated separately from the remaining atoms in the

complex. The basis set applied on chlorine atom, 6-311++G (3df, 4pd), contains additional

diffusion function to represent its anionic nature. Three nitrogen atoms and one carbon atom co-

ordinated to platinum were assigned as 6-31G** basis set. Remaining carbon and hydrogen

atoms were assigned with 6-31G* and 3-21G basis sets, respectively. Time-dependent DFT (TD-

DFT) calculations were performed on the lowest singlet ground state to probe the absorption and

emission properties, using the same functional and basis sets. As the UV-visible absorption and

photoluminescence experiments were recorded in methanol solvent, all the computational

calculations were performed in methanol solution (ε=30) using polarizable continuum model.

Ten lowest singlet and triplet roots of the non-Hermitian eigenvalue equations were obtained to

determine the vertical excitation energies. Oscillator strengths were deduced from the dipole

transition matrix elements for singlet states only. All the calculations were performed using

GAMESS-US software. Canonical Molecular Orbital analysis (CMO) has been performed to

analyze the composition and bonding nature of molecular orbitals (MO), using NBO 5.0

software. CMO analysis gives effective information on i) bonding characters of MO, like HOMO

− x, LUMO + y etc; ii) the energies of individual MOs. The partial charge transfer (CT) has been

characterized for HOMO − x to LUMO + y transition (Equation 1).

CT(M) = [%(M)HOMO−x]‒[%(M)LUMO+y] ...Equation 1

In Equation 1, “%(M)HOMO - x” and “%(M)LUMO + y” are percentage of metal character

obtained from CMO analysis. When the contributions to an excited state comes from the

multiple single-electron excitations, the metal CT character is described by the Equation 2

CTi (M) =∑ [Ci ( i - j )]2 ((M)i -(M)j ).......Equation 2,

where Ci (i-j) are co-efficients expressed as the excitation amplitudes corresponding to

transitions between i to j states.

Cell Imaging

For cell imaging studies of the complex 2 three cancer cells, namely, HeLa (human cervical

cancer cell), U87MG (human glioblastoma cell) and Nuro2a (mouse neuroblastoma cell) and

normal cells namely 3T3-L1 (mouse fibroblast cell) were used. All the cells were purchased

from National Center for Cell Science, Pune (India). Cells were cultured in Dulbecco’s modified

eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1%

penicillin/streptomycin at 37ºC and 5% CO2. Next, cells were seeded into 24-well tissue culture

plate in presence of 500 µL DMEM medium and grown overnight. Next, aqueous solution of

platinum(II) complex was added at a final of concentration of 170 µM to each well and

incubated for different times. Next, cells were washed with PBS buffer solution, supplanted with

500 µL DMEM medium and used for imaging study.

Co-localization Study

For nucleus co-localization studies HeLa cells were seeded into a 4-well chamber slide in 500

µL supplemented DMEM medium and allowed to adhere for overnight. Then cells were

incubated with an aqueous solution of the complex 2 (final concentration, 170 µM) for 4 hrs.

Next, cells were washed with PBS buffer solution and fixed with 4% paraformaldehyde for 20

min. Then cells were permeabilized by adding 500 µL 0.3% Triton X-100 in PBS solution for 20

min. Then, cell nucleus was stained with aqueous solution of propidium iodide. Next, fixed cells

were mounted with 50% glycerol and imaged under fluorescence microscope.

Cytotoxicity Assays

In vitro cytotoxicity of the complex 2 was measured using standard MTT assay. The cells were

seeded in presence of 500 µL of supplemented DMEM medium into a 24-well plate and allowed

to stand overnight at 37 ºC and 5% CO2. After overnight growth, different amounts (10-500 µL)

of 1 mM aqueous complex 2 were added to each well and incubated for 24 hrs. After incubation,

the cells were washed with PBS buffer solution and fresh 500 µL supplemented DMEM medium

was added. The cells were incubated for 4 hrs after adding 50 µL of aqueous solution of MTT (5

mg/mL) to each well. Next, violet formazan was dissolved in sodium dodecyl sulfate solution in

a water/DMF mixture and absorbance was measured at 570 nm. The cell viability was measured

assuming 100% viability for control cells exclusive of any platinum complex.

Fig. S1. Complex 2 : 1HNMR (400 MHz, DMSO-D6) δ 8.66 (t, J = 5.3 Hz, 1H), 8.08 (dt, J = 18.5, 8.0 Hz, 2H), 7.75 – 7.67 (m, 1H), 7.40 – 7.23 (m, 2H), 7.11 (dd, J = 5.2, 3.5 Hz, 2H), 6.12 (s, 2H), 5.38 (s, 2H), 2.67 (s, 4H).

Fig. S2. 13C NMR : (101 MHz, DMSO-D6) δ 166.88 (s), 151.61 (s), 145.55 (s), 144.85 (s), 140.28 (s), 134.17 (s), 129.97 (s), 124.42 (s), 124.00 (s), 123.71 (s), 119.81 (s), 48.30 (s), 43.95 (s).

Fig. S3. Differential interference contrast (a) and luminescence image (b) of complex 2 labeled 3T3-L1 cells. Cells are incubated with it for 4 hrs (final concentration of 170 µM) and imaged under UV light.

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Fig. S4. Differential interference contrast image (a, c, e, g, i, k) and luminescence images (b, d, f, h, j, l) of live HeLa cells after different times incubation (15 min to 24 hrs) with complex 2 at final concentration of 170 µM. The luminescence images are captured under UV excitation.

Fig. S5. Differential interference contrast image (a, c, e, g, i, k) and luminescence images (b, d, f, h, j, l) of live Neuro-2a cells after different times incubation (15 min to 24 hrs) with complex 2 at final concentration of 170 µM. The luminescence images are captured under UV excitation.

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Propidium Iodide Platinum Complex

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Fig. S6. a) High magnification luminescence merged image of complex 2 and propidium iodide labeled HeLa cells and b) luminescence intensity profile across the line shown in image a. Green and red line corresponding to complex 2 and propidium iodide, respectively.

Fig. S7. The stability of complex 2 in pure fetal bovine serum (FBS). (a) Digital images of complex 2 in pure FBS after different times. Top and bottom row shows the images of solution under ordinary light and UV light. (b) The photoluminescence spectra of corresponding solution of complex 2 in pure FBS under 375 nm excitation. The result of the study shows that complex 2 has high colloidal stability in FBS and also intact its photoluminescence property.

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Fig. S8. A series of luminescence images of complex 2 labeled HeLa cells using at different Z planes from bottom to top with successive Z-axis slices of 6 µm each, demonstrating that the complex 2 is located in nucleus of cell as well as cell cytoplasm.

Table S1: Excited state electronic properties of complex 2 in methanol solvent.

Transition from So to

λcal(nm) E(ev) F (oscillator strength)

Assignments MLCT character (%)

S1 371.70 3.34 0.035 HOMO→LUMO 96.04% 31.69S2 346.08 3.58 0.010 HOMO-1→LUMO

98.01%84.97

S3 306.70 4.04 0.092 HOMO-2→LUMO 76.09%

10.34

T1 483.24 2.57 0.0 HOMO→LUMO 67.86% 22.39T2 412.51 3.01 0.0 HOMO-1→LUMO

34.30%HOMO-2→LUMO 33.39%HOMO→LUMO 8.47%

29.744.542.79

References

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Choudhury and I. R. Laskar, RSC Advances. 4, 2014, 50549 .

[2] P. I. Kvam and J. Songstad, Acta Chemica scandnavica. 49, 1995, 313.

[3] M. Nardelli. J. Appl. Crystallogr. 28, 1995, 569.

[4] APEX2, SADABS and SAINT; Bruker AXS Inc. Madison, Wisconsin, USA, 2008.

[5] G. M. Sheldrick, Acta Crystallogr. A64 (2008) 112.