Sensing of tryptophan by a non-toxic cobalt(II) complex

Abstract

A non-toxic, hemocompatible, fluorescent probe as cobalt(II) based complex [Co(PS)₄](ClO₄)₂ (1) (PS: N-pyridylsalicylaldimine) was successfully synthesized and characterised by single crystal X-ray diffraction studies. The structural analysis revealed that 1 exhibits intramolecular π–π interaction. The recognition ability of 1 towards various amino acids and proteins were studied by UV-vis and fluorescence spectroscopy. The titled complex selectively sense the Trp by reducing its internal fluorescence quenching. It also sense BSA with a detection limit of 56 nM. The binding constant of 1 was analysed by Hill's equation and it was found that it binds to Trp and BSA in the order of 10³ and 10⁴ respectively.

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Introduction

Amino acids with various functional groups in the side chain are essential building blocks for living organisms as they are the main components of proteins and peptides.^1a Tryptophan (Trp) is one such amino acid requisite by all forms of life for protein synthesis and other important metabolic functions.^1b Trp is recognized as a key to determine the activity, hydrophobicity and diversity in protein.² Hence, selective detection of Trp is vital to reveal its biochemical potentiality.

Proteins contain aromatic amino acids, such as, tryptophan (Trp), tyrosine (Tyr) and phenylalanine (Phen) as intrinsic flurophores. Among these, Trp is particularly useful because of its high quantum yield³ and fluorescent property modulated with respect to the local environment.⁴ The emission maxima of Trp in fluorescence spectrum depends strongly on the polarity of its microenvironment and ranging from 308 nm (azurin) to 355 nm (glucagen).⁴ The structural and dynamical aspects of native protein and its interactions with other molecules such as nucleic acids, proteins, ligands and compounds can be determined from the fluorescence behavior of Trp.⁵

Serum albumins (SAs) with its high abundance in blood plasma,⁶ are the globular protein, serve as a model protein for the study of protein–ligand⁷ and protein–solvent⁸ interactions. SAs facilitates the disposal and transport of various exogenous and endogenous molecules such as fatty acid, porphyrin, bilirubin, steroid etc. to the specific targets by hydrophobic, hydrophilic and ionic interactions.⁹ Bovine serum albumin (BSA) is one of such serum albumin which has two binding sites referred to as site I and II⁶ located in the hydrophobic cavities of subdomains IIA and IIIA, respectively. Therefore, detection of BSA is important to elucidate the protein function in living cells or tissues.

Metal complexes of Co(II) using chelating legends are of great interest in the areas of catalysis,^10a magnetism,^10b chemical sensors,^10c etc. However, an area of research has been focused on the interaction of biologically relevant systems with a large variety of Co(II) complexes. Cobalt(II) plays a crucial role in several biologically important processes, and is predominately found in the form of vitamin B₁₂ (cobalamin).¹¹ Several cobalt(II) compounds have been reported with therapeutically relevant antifungal, antibacterial, and antiprotozoal properties.¹²

We herein report a non toxic cobalt(II) complex as a luminescent sensor for Trp and BSA, for the first time. It is well known that the effect of compound on cell viability and proliferation forms the basis of toxicity. Thus, MTT assay was performed to measure the effect of the synthesized Co(II) complex on HeLa and MCF-7 cell proliferation. Although, the toxicity mechanisms are cell type-dependent, a simple hemolysis assay could be a good first line assessment for in vivo administering of any drug. To comply this we have also determined the hemocompatible dose of the synthesized Co(II) complex.

A thorough literature survey revealed that till date there is hardly any example of a complex capable of sensing Trp¹³ and BSA¹⁴ (Table 1 and 2). In this context the present endeavour towards the synthesis of a non-toxic Co(II) complex is clearly a new addition to this class over the state-of-the-art. It is also worthy to note that among the previously reported examples^13,14 the fluorescence intensity of the sensors were increased. However, for our case the fluorescence intensity of both Trp and BSA is increased.

Table 1 Comparisons of various Trp sensors

Sensor	Detection limit	Binding constant	Ref.
Ruthenium(II) complexes (I and II)	300 nM	3.0 × 10^4 (I), 2.1 × 10^3 (II)	16
8-(Alkoxy)quinoline-based fluorescent probe (1)	—	6.42 × 10^4 (1)	17
SQ	—	1.4 × 10^6	18
MMAPA	—	6.3 × 10^4	19
[Pt(bzimpy)Cl]^+ (1)	—	6.75 × 10^4	20
Amphiphilic fluorophore	—	3.47 × 10^5	21
Complex 1	56 nM	2.907 × 10^4

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Table 2 Comparisons of various BSA sensors

Sensor	Detection limit	Binding constant (M^−1)	Ref.
(R)-1	—	1.73 × 10^4	12
Calix[4]arene fluoroionophore	0.00826 nM	10.21 × 10^8	13
CuNPs	—		14
4-DPD-Ag NPs	20 μM		15
Complex 1	410 nM	1.77 × 10^3

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Results and discussion

The reaction of the ligand (PS) with a methanolic solution of Co(ClO₄)₂·6H₂O at room temperature produced deep pink crystals of the complex formulated as [Co(PS)₄](ClO₄)₂ (1). Single crystal X-ray analysis revealed that the central metal ion, Co(II), possess a distorted tetrahedral geometry coordinated through four pyridine nitrogen atoms from four different PS-ligand moieties (Fig. 1a).

Fig. 1 (a) The asymmetric unit of 1. This is the unique set of refined atoms; (b) the π–π interactions of 1. The angle between these two planes is 11.416°.

The complex displays equal Co–N bond distance with 2.080 Å (3) (Table S1†). The bond lengths and the bond angles are listed in Tables S1 and S2.† The interesting structural feature of the complex is that the four pyridine rings (of PS) lie parallel with the benzene rings (of the other PS) in the same complex with an inter-ring separation of 3.807 Å (Fig. 1b). Thus, the pyridine and benzene rings interact with each other through intramolecular π–π interaction.

The selectivity of the complex 1 with various amino acids was investigated by absorption and fluorescence titration experiments in phosphate buffer saline (PBS, pH = 7.4). In the absorption spectra, the complex 1 (1 μM) itself exhibits three absorption bands at 257, 290 and 352 nm (Fig. S2†). With the addition of Trp (40 μM), accompanying with the band at 352 nm three new bands were appeared at 271, 279 and 287 nm, however, two bands of the complex at 257 and 290 nm were abolished (Fig. 2). Again, the addition of other amino acids did not show any remarkable effect in the absorption band of 1. This study indicated that the complex was more sensitive towards Trp compared to other amino acids studied in our case (Fig. 3). Trp selectivity of 1 was evaluated through the changes in fluorescence emission intensity. The other amino acids did not produce a noticeable effect on the emission spectra of 1 compared to Trp except cysteine (Cys) which showed decreases in the fluorescence intensity at 350 nm (λ_ex = 290 nm) (Fig. S7†). The results indicated that the titled complex has excellent selectivity towards Trp. In order to determine the sensitivity of 1 towards Trp gradual addition of Trp (0–180 μM) into 1 (0.5 mM) showed a significant increase in the fluorescence intensity at 350 nm (λ_ex = 290 nm) (Fig. 4). The Job's plot analysis (Fig. S9†) of the fluorescence titration profile of 1 (5 μM) revealed a 1 : 4 stoichiometry between 1 and Trp with a binding constant of 1.77 × 10³ M⁻¹. The detection limit of 1 for Trp was determined from the fluorescence spectral changes and was found to be 410 nM.

Fig. 2 UV-vis spectra of 1 (1 μM) in the presence of increasing amount of Trp (0 to 40 μM) in PBS buffer (pH = 7.4).

Fig. 3 The absorption value of 1 (1 μM) at 287 nm in the presence of different amino acids (40 μM) in PBS buffer (pH 7.4).

Fig. 4 Fluorescence emission spectra of 1 (0.5 μM) upon addition of increasing concentrations of Trp (0–180 μM) in PBS buffer (pH 7.4). The arrow indicates the change in the emission intensity with the increased Trp concentration. λ_ex = 290 nm.

Tryptophan contains two nearby isoenergetic π–π* transitions, ¹L_a and ¹L_b which are approximately at a right angle to each other (Scheme 1a).¹⁵ The ¹L_a state is more sensitive to its environment than the ¹L_b state as the transition from ground to ¹L_a state involves a large change in permanent dipole moment (6D) arising from electron density shifting from the pyrrole ring to the benzene ring of the indole moiety.^16a In polar solvents the ¹L_a transition shifts to lower energy and the emission is obtained from ¹L_a state. Whereas, in a nonpolar environment, the ¹L_b state possess lower energy and dominates the emission.^16b The internal fluorescence quenching of Trp occurs significantly by both electrophilic protonation by the ammonium group (NH₃⁺) at the C-4 position of the excited indole moiety and by the charge–transfer interaction between the excited indole (electron donor) and the ammonium group (electron acceptor) of the side chain (Scheme 1b).¹⁷ During interaction with complex, the transition from ¹L_a to ground state increases, causes increment in the emission intensity of Trp without any spectral change (Fig. 5). This fluorescence enhancement is due to the interaction of NH₃⁺ group of Trp with the methoxy(–OMe) groups of 1 (Scheme 1c). Thus, the NH₃⁺ group cannot interact intramolecularly with the excited indole moiety resulting in the decrease of internal fluorescence quenching of Trp.¹⁷ The decrease in fluorescence intensity of 1 in presence of Cys is probably due to fluorescence resonance energy transfer (FRET) (Fig. 6).

Scheme 1 (a)Trp transition dipole; (b) Trp fluorescence quenching process; (c) proposed interaction mechanism of Trp with complex 1.

Fig. 5 Fluorescence spectra of 1 (0.5 μM) in both the presence and absence of Trp (180 μM), Trp (180 μM) in PBS buffer (pH = 7.4), (λ_ex = 290 nm) and difference spectra of [(1 + Trp) − Trp].

Fig. 6 UV-vis spectra of 1 (1 μM) in both the presence and absence of Cys (40 μM) and fluorescence spectra of 1 in PBS buffer (pH = 7.4). (λ_ex = 290 nm).

To establish the selectivity of the titled complex towards various proteins (containing varying numbers of Trp at different position) like bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lz) and hemoglobin (Hb), the fluorescence property of 1 was investigated. Fig. 7 showed the comparative fluorescence intensity of 1 (0.5 μM, PBS) with various proteins (10 μM). Results exhibited that the fluorescent intensity of BSA was higher compared to other tested proteins. The binding of 1 to HSA caused an increase in the emission intensity by a blue-shift of 2 nm (lesser than BSA-1 emission). The emission intensity of 1 was decreased on the addition of Hb, but the addition of Lz did not affect the emission property of 1 at all. This result established that the complex was more selective towards BSA. In order to evaluate the sensitivity of BSA the fluorescence titration experiment was done with gradual addition of BSA to 1 (0.5 μM, PBS). The emission intensity of BSA was increased with a blue-shift of 6 nm (from 350 nm to 344 nm) (Fig. 8). A separate study was performed in order to confirm the increment in fluorescence of BSA due to the interaction with 1. In this experiment the titled complex was excited in both the presence and absence of BSA at λ_ex = 290 nm. After subtracting the fluorescence intensity of 1 from BSA-1, it was clearly established a positive increment in fluorescence intensity (Fig. 9). Fig. S19† showed changes in fluorescence of BSA (at 350 nm) as a function of its concentration. The fluorescence value at 350 nm increased linearly with the concentration of BSA (0–50 μM) with a binding constant of 2.907 × 10⁴ M⁻¹ and the detection limit was found to be 56 nM.

Fig. 7 Fluorescence responses of 1 (0.5 μM) to different proteins (10 μM) in PBS buffer (pH 7.4).

Fig. 8 Fluorescence emission spectra of 1 (0.5 μM) upon addition of increasing concentrations of BSA (0, 1, 3, 5, 10, 20, 30, 40 and 50 μM) in PBS buffer (pH 7.4). The arrow indicates the change in the emission intensity with the increased BSA concentration. λ_ex = 290 nm.

Fig. 9 Fluorescence spectra of 1 (0.5 μM) in both the presence and absence of BSA (5 μM), BSA (5 μM) in PBS buffer (pH = 7.4), (λ_ex = 290 nm) and difference spectra of [(1 + BSA) − BSA].

BSA has two tryptophan (Trp) residues: one in subdomain IB (Trp-134), located on the surface (in the hydrophilic region of the protein) and another in subdomain IIA (Trp-212) (located within a hydrophobic binding pocket).⁶ Trp residues buried in the hydrophobic pocket of proteins were associated with shorter wavelength emission maximum around 340 nm while those on or near the surface of proteins were characterized with longer wavelength emission maximum. Thus, the fluorescence emission of BSA with maximum around 345 nm when excited at 295 nm came from the Trp residue at 212.¹⁸ The blue shifting of the fluorescence emission band with an increase in intensity indicated the interaction of the complex with the Trp-212 of BSA. HSA contains only one Trp at 214 in the subdomain IIA.¹⁹ Binding of 1 with Trp-214 of HSA was less compared to Trp-212 of BSA. This was might be due to the difference in the position of Trp residues in proteins. In Hb there were six Trp residues, three for each αβ dimer. α14Trp and β15Trp were located in inter-helical positions within their respective subunits, while the two β37Trp residues were situated in the inter-subunit (α1β2 and α2β1) interface.²⁰ The intrinsic fluorescence of Hb originates from the β-Trp37.²¹ In Hb three types (and in total six) of cysteine residues were there, one at each α subunit, α104Cys and two at each β subunit β93Cys and β112Cys. The b93Cys residue located at the α1β2 subunit interface, in the carboxyl terminal region of the β-chain, and in close to the proximal histidyl residue.^20a After addition of 1, 2, 3 mM of Hb, the emission intensity of complex 1 increased, decreased and again increased respectively (Fig. 10). Then further addition of Hb, the emission intensity of 1 was decreased consistently. As we have seen that binding of free Cys amino acid to 1 decreased emission intensity. Therefore, such irregular phenomena might be due to the competitive binding of 1 to β37Trp and b93Cys of Hb. Lz contained six Trp residues at positions 28, 62, 63, 108, 111, and 123. Three of which (Trp 62, 63, 108) were located in the region of the binding cleft of the enzyme.²² Studies revealed that the Trp residues at positions 62 and 108 contributed largely to the fluorescence emission of Lz, whereas the residues at positions 28 and 111 made only little contribution.²³ The interaction of 1 with Lz did not affect the emission behavior, suggested that Trp moiety of Lz could not interact with 1.

Fig. 10 Fluorescence spectra of 1 (0.5 μM) in the presence of increasing amount of Hb (0 to 7 μM) in PBS buffer (pH = 7.4) (λ_ex = 290 nm).

The MTT cell proliferation assay measured the cell proliferation rate and conversely, when metabolic events lead to apoptosis or necrosis, the reduction in cell viability. During our study, we found negligible changes in growth of cells in the presence of the complex. With increasing concentration of the complex there was no change in proliferation of cells in respect of DMF (diluent). On the basis of our result, as shown in Fig. 11a and b, we can predict that effect of 1 on both cell lines (MCF-7 and HeLa) are almost same. It gives strength to our finding that complex 1 is non toxic for cells and can be used against various tissues. From the hemolysis study it was noted that both 25 μM and 50 μM of 1 exhibited only 0.01% hemolysis (Fig. 12). It was reported earlier that materials with <5% hemolysis were regarded as hemocompatible.²⁴ Thus, both 25 μM and 50 μM concentration of the complex 1 were found to be hemocompatible. To study the fluorescence ability of 1 in an intracellular environment MCF-7 cell were treated with 12.5 mM of the complex and the fluorescence micrograph was obtained using LED based fluorescence microscope and the result was showed in Fig. 13.

Fig. 11 Effect of complex on cell viability in respect of diluent DMF. MTT assay was performed with MCF 7 (a), and HeLa (b) cells with increasing concentration of the complex. Percentages of cell viability were calculated in respect of untreated cells (100%) minus percentage inhibition by DMF or complex 1.

Fig. 12 Hemolysis assay visually and by taking absorption at 541 nm.

Fig. 13 Fluorescence property of complex 1: fluorescent image of MCF 7 cells were seen in the presence of complex under fluorescence microscope, 10× magnification. There were no fluorescence measured in untreated MCF 7 cells.

Conclusion

To the best of our knowledge this is the first report of a non-toxic Co(II) complex which can sense Trp and BSA by reducing internal fluorescence quenching of Trp in aqueous solution. The titled complex may become a convenient luminescent sensor for the recognition of Trp and BSA. Methods based on enhancement in Trp absorption and emission maxima should be invaluable in protein research. Moreover, we expect this new complex to be useful by virtue of its non-toxic, hemocompatible and excellent fluorescence behaviour.

Experimental section

Materials

All experiments were conducted in phosphate-buffered saline (PBS) buffer, pH 7.4. The pH was adjusted by addition of hydrochloric acid (HCl). Quartz distilled deionized water and analytical grade reagents were used throughout. All buffer solutions were passed through Millipore filters of 0.45 μm (Millipore India Pvt. Ltd., Bangalore, India) to remove any particulate matter. Proteins and amino acids solutions were prepared in phosphate buffer saline (PBS). Stock solution of complex 1 (1250 μM) was prepared in DMF.

2-Amino pyridine and 18 amino acids were purchased from SRL. 2,5-Dimethoxybenzaldehyde was purchased from Spectrochem. The reagents perchloric acid was purchased from Merck. All the chemicals and solvents were used without further purification.

Bovine serum albumin (BSA), human serum albumin (HSA), lysozyme (Lz) and hemoglobin (Hb) was obtained from Sigma-Aldrich Chemicals Co., (St. Louis, MO, USA) and used as received without further purification. The concentration of BSA, HSA, Lz and Hb were determined using molar extinction coefficients values 43 824 M⁻¹ cm⁻¹ at 280 nm for BSA, 36 600 M⁻¹ cm⁻¹ at 280 nm for HSA, 37 750 M⁻¹ cm⁻¹ at 280 nm for Lz and 179 000 M⁻¹ cm⁻¹ at 405 nm for Hb. No deviation from Beers law was observed in the concentration range employed in this study.

Caution! Perchlorate salt of metal complex with organic ligands is potentially explosive. Only a small amount of material should be prepared, and it should be handled with care.

Equipments and spectral measurements

Infrared spectra of the metal complex were recorded in the range of 3000–500 cm⁻¹ using a Perkin Elmer Spectrum two FT-IR spectrophotometer from KBr discs. LC-MS spectra were obtained using a Applied Biosystem, ATI-3000 mass spectrometer. A Shimadzu Pharmaspec 1601 unit (Shimadzu Corporation, Kyoto, Japan) was used for absorption spectral studies. Fluorescence spectral studies were performed on a Hitachi F7000 (Hitachi Ltd., Tokyo, Japan).

All spectral data were recorded at 25 °C. Fluorescence measurements were done using 1 cm quartz cell with 5 nm slit width for both excitation and emission experiments and the excitation wavelength was 290 nm (λ_ex = 290 nm).

Few drops of DMF solution were taken in methanol solution to record LC-MS.

Synthesis of N-pyridylsalicylaldimine (PS) and [Co(PS)₄](ClO₄)₂ complex (1)

2-Aminopyridine (0.9412 g, 10 mM) was dissolved in 20 ml methanol in a round-bottom flask. 10 mM of 2,5-dimethoxysalisaldehyde (1.6618 g) was added to this solution. After that the whole reaction mixture was refluxed for 2 h to get a greenish-yellow solution. The solution was cooled to room temperature and 4 ml solution was taken in a 100 ml beaker. To this solution, 10 ml methanolic solution of cobalt perchlorate (2 mM, 0.73 g) was added dropwise with continuous stirring. The resulting mixture was stirred for 2 h to get a transparent pink solution. The solution was kept in dark for crystallization. Diffraction quality deep pink coloured single crystals was obtained by slow evaporation of the solvent after seven days. Yield 70%. Anal. calcd. for C₅₆H₅₆Cl₂CoN₈O₁₆: C, 54.82; H, 4.60; N, 9.13%. Found: C, 54.81; H, 4.42; N, 9.08%. IR (KBr, cm⁻¹): 510 (w), 534 (m), 552.6 (w), 615 (m), 623 (s), 649 (m), 668 (m), 705.5 (s), 723 (w), 752.74 (m), 783.59 (m), 800 (m), 821 (m), 860.5 (m), 874 (w), 887.6 (m), 975 (w), 1038 (m), 1094 (s), 1151 (m), 1169 (m), 1182.7 (w), 1195.8 (w), 1223.3 (s), 1245 (m), 1271 (m), 1281 (m), 1294 (m), 1376 (s), 1421 (m), 1437 (m), 1474 (s), 1501 (s), 1563 (s), 1599 (m), 1619 (s), 1888 (w), 1973.6 (w), 2013 (w), 2157 (broad), 2313 (w), 2366 (w), 2840 (w), 2908 (w), 2941 (w), 2996 (w).

Crystallographic analysis

Single crystal X-ray diffraction intensity data of the title complexes were collected at 293(2) K using a Bruker APEX-II CCD diffractometer equipped with graphite monochromated MoK_α radiation (λ = 0.71073 Å). Data reduction was carried out using the program Bruker SAINT.²⁵ An absorption correction based on multi-scan method²⁶ was applied. The structures were solved by direct methods and refined by the full-matrix least-square technique on F² using the programs SHELXS97 and SHELXL97 (ref. 27) respectively. All calculations were carried out using WinGX system Ver-1.64 (ref. 28) and PLATON.²⁹ All hydrogen atoms were located from difference Fourier map and treated as riding. A summary of crystal data and relevant refinement parameters are given in Table 3. CCDC deposition no. 1419991 contains the supplementary crystallographic data for this paper.

Table 3 Crystal data and structure refinement parameters of co-complex

Structure	1
Formula	C56H56Cl2CoN8O16
Dcalc./g cm^−3	1.423
μ/mm^−1	0.470
Formula weight	1226.91
Colour	Pink
Shape	Irregular
Max size/mm	0.18
Mid size/mm	0.12
Min size/mm	0.10
T/K	120(2)
Crystal system	Tetragonal
Flack parameter	0.014(11)
Hooft parameter	0.012(11)
Space group	P4̄21c
a/Å	11.8148(3)
b/Å	11.8148(3)
c/Å	20.5068(8)
α/°	90
β/°	90
γ/°	90
V/Å	2862.55(18)
Z	2
Z′	0.25
θmin/°	1.986
θmax/°	26.492
Measured refl.	18 765
Independent refl.	2960
Reflections used	2638
Rint	0.0541
Parameters	190
Restraints	0
Largest peak	0.295
Deepest hole	−0.261
GooF	1.091
wR2 (all data)	0.0849
wR2	0.0820
R1 (all data)	0.0510
R1	0.0419

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Determination of binding constant

Scatchard equation. The binding constants between 1 and the tryptophan (Trp) or BSA have been determined from the fluorescence intensity data using Scatchard plot. The Scatchard equation is shown in eqn (1)^30,31

ν/C_F = nK_a − νK_a (1)

In eqn (1) ν = ratio of the concentration of bound ligand to total available binding sites. C_F = number of binding sites per Trp or BSA molecule. n = binding stoichiometry. The plot of the data ν/C_F vs. ν is non-linear which shows the cooperative binding.

C_F can be calculated from the equation C_F = (1 − a)D.

Where D = concentration of 1 and a = (I_f − I)/(I_f − I_b)

Again, I_f = fluorescence intensity of free complex 1

I = fluorescence intensity of after addition of tryptophan (Trp) or BSA

I_b = fluorescence intensity of 1 when fully bound to tryptophan (Trp) or BSA

Hill's equation. Since, the Scatchard plot is non-linear we have calculated the binding constant using Hills equation, eqn (2)³²

log[θ/(1 − θ)] = n log[Trp or BSA] − log K_d (2)

θ – number of binding sites (C_F in Scatchard equation), K_d – dissociation constant (the inverse of K_a), n – Hills coefficient of cooperativity which gives the nature of cooperativity. n > 1 positive cooperative binding, n < 1 negative cooperative binding and n = 0 non cooperative binding.

Determination of the detection limit

The detection limit (DL) of 1 for Trp or BSA was determined from the following equation:³³

DL = K × Sb1/S

where K = 2 or 3 (we take 2 in this case); Sb1 is the standard deviation of the blank solution; S is the slope of the calibration curve.

Cell culture

Human breast adenocarcinoma cell line (MCF 7), and human cervix adenocarcinoma cell line (HeLa) were procured from National Centre for Cell Science (NCCS), Pune, India. Cells were maintained in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS), 100 units per 1 ml penicillin, 100 mg ml⁻¹ streptomycin, 0.14% sodium bicarbonate and 0.1 mM sodium pyruvate. Cells were grown in CO₂ incubator (N-Biotech) at 37 °C in 5% CO₂ atmosphere with 95% humidity.

Cell viability study

Cells were seeded in 96-well plates at a density of 5 × 10³ cells per well in 200 μl culture medium. After 24 h, the cells were treated with DMF and complex dissolved in DMF with increasing concentration (6.25–50 μM). After 24 h, media was replaced with MTT solution (10 μl of 5 mg ml⁻¹ per well) prepared in PBS and incubated further for 3 h at 37 °C in a humidified incubator with 5% CO₂. Then 50 μl of isopropanol was added to the each well and plates were gently shaken for 1 min and absorbance was taken at 595 nm by micro titer plate reader (Bio-Rad). The percentage of cell viability was calculated as 100 − (Y − X/Y × 100), where Y is the mean optical density of control (untreated cells) and X is the mean optical density of treated cells with DMF or complex as described previously.³⁴ The all experiments were repeated three times independently. Results were presented as mean ± SD of triplicates from three independent experiments.

Fluorescence imaging

MCF 7 cells were seeded at 2 × 10⁵ cells on coverslips in 35 mm glass Petri plate culture dishes and left for 24 h at 37 °C with 5% CO₂. Complex 1 was added to the cells (to achieve a final concentration of 13 μM) and left to incubate for 5 h. Cells were washed with 1× PBS and imaged immediately, using a LED based fluorescence microscope, Magnus MLXi microscope. The cells were excited at 480 nm using LED cassettes and emission was collected using a long pass filter. Cells were observed immediately after PBS washing under 10× magnification and images were captured by digital SLR Olympus camera mounted on head for high resolution image.

Hemolysis assay

Hemocompatibility of 1 at 25 and 50 μM was determined in terms of percent hemolysis. Ethylenediamine tetraacetic acid (EDTA) stabilized human blood sample was freshly collected after obtaining informed consent from all human subjects. The experiment was performed in accordance with the guideline of Indian Council of Medical Research (ICMR), New Delhi, India and the study protocol were approved by the Ethical Committee of Vidyasagar University.³⁴ Whole blood was centrifuged at 1600 rpm for 5 min and the plasma, buffy coat, and the top layer of cells were discarded. The remaining packed red blood cells (RBCs) were washed four times with sterile isotonic PBS. After washing, 0.2 ml of packed RBCs was diluted to 4 ml with PBS (5% hematocrit).³⁵ The diluted suspension of RBCs (0.2 ml) was then mixed with varied concentrations of 1 in PBS (0.8 ml). Diluted suspension of RBCs mixed with 0.8 ml PBS and 0.8 ml double distilled water were used as negative and positive control, respectively. The mixture was gently vortexed and incubated at room temperature for 2 h. After centrifugation (1600 rpm, 5 min) of the incubated mixture, absorbance of the supernatant at 541 nm was measured by UV-vis spectrophotometer. Finally, hemocompatibility was evaluated in terms of percent hemolysis, where percent hemolysis = (A_S − A_N)/(A_P − A_N) × 100; A_S is the sample absorbance, A_N is the absorbance of the negative control and A_P is the absorbance of the positive control.

Acknowledgements

SKJ is thankful to UGC, New Delhi for providing Senior Research Fellowship. Departmental instrumental facilities from DST FIST and UGC SAP programs are gratefully acknowledged. We also acknowledge the USIC, Vidyasagar University for spectroscopic measurements.

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Footnote

† Electronic supplementary information (ESI) available. CCDC 1419991. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c6ra16086g

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