Ag(I) and Au(III) Mercaptobenzothiazole complexes induced apoptotic cell death

2-Mercaptobenzothiazole (MBT) complexes of Ag(I) and Au(III) were synthesized by wet chemical method. The structural, optical, 1HNMR, ICP – MS and electrochemical studies of the complexes were carried out. The TUNEL assay studies of Ag(I)MBT and Au(III)MBT complexes on A549 cell line indicated induced apoptosis in the cells. TUNEL assay showed 60% cell viability for Ag(I)MBT whereas 80% for Au(III)MBT. Thus Ag(I)MBT can induce cell apoptosis in cells at a higher rate than Au(III)MBT. Therefore these complexes studied here can be a viable option as anti – proliferating agent.


Synthesis of Tetra Chloro Auric(III) acid (HAuCl 4 ). The tetrachloroauric acid (HAuCl 4 ) solution
(0.01 M) was synthesized by dissolving 2.5 gm of Au granules in 100 ml of aqua regia by continuous stirring and allowed to evaporate until dry. The residue obtained was washed with Conc. HCl and evaporated until dry. This process was repeated 3-4 times. Distilled water was added to the residue and evaporated until dry. This process was repeated twice to obtain crystals 5 of HAuCl 4 (Fig. 1).
Synthesis of Au(III)MBT complex. 1 mmol commercial MBT (0.167 gm) was dissolved in 10 ml of ethanol.
5 ml of HAuCl 4 (0.01M) was added to 5 ml of acetonitrile. This solution was then added to the ethanolic MBT and refluxed for 3 hours at 80 °C. After cooling the solution was filtered and the filtrate was kept aside for slow evaporation 3     were used with no further purification. XRD spectrum was recorded for Ag(I)MBT and Au(III)MBT complexes using BRUKER D8 Advance X-ray diffractometer with Cu Kα source (λ = 1.5406 A°). The UV-Vis absorbance spectrum was obtained by measuring the absorbance using Specord/210, analyticjena UV-Vis spectrophotometer. FTIR spectra were recorded using SHIMADZU, IRAffinity1 spectrometer.

Results and Discussion
Structural Analysis. Figure 6 represents the XRD spectra of Ag(I)MBT and Au(III)MBT complexes. The observed XRD spectra is in satisfactory agreement with the literature 5,15-17 . In Fig. 6a Fig. 7. In Fig. 6b, the characteristic peaks for Au(III) is seen at 38°, 44°. The broadening of the XRD around 20° to 50° is caused due to the presence of ion pair 3 (Calculated lattice constants for Au(III)MBT (a = 7.9939A°; b = 5.9315A°; c = 15.0911A°) agree satisfactorily with the literature for monoclinic lattice. The d value is calculated as 5 nm and the space group is 'p21/n' ref. 3 ).
Optical Analysis. The optical studies of Ag(I)MBT and Au(III)MBT were carried out using UV-Visible and FTIR spectroscopy to understand their optical properties.
Electronic spectroscopy analysis. The absorption spectra of MBT, Ag(I)MBT and Au(III)MBT was recorded in chloroform (Fig. 8). MBT showed an absorption spectrum at 239 and 329 nm. The peak at 239 nm (5.18 eV)     Vibrational spectroscopy analysis. Figure 9 shows the FTIR spectrum of MBT, Ag(I)MBT and Au(III)MBT.
The stretching vibration at 3072 cm −1 for MBT is attributed to aromatic C-H stretching which is red shifted to 3061 cm −1 in Au(III)MBT and completely absent in Ag(I)MBT. The selection criteria for IR active species state that "vibrations involving dipole moments that are perpendicular to the surface only get excited" 18 . As seen from section 3.1, due to the presence of aromatic C-H bond perpendicular to Au(III) ion, the complex Au(III)MBT obeys the selection rule and a C-H stretching peak is seen at 3061 cm −1 , whereas in the case of Ag(I)MBT, the aromatic C-H stretching is parallel or in plane to the Ag(I) ion, therefore peak at 3061 cm −1 is not observed for Ag(I) MBT. Thus it can be concluded that MBT complexes with Ag(I) ion is in planar geometry and with Au(III) is in near perpendicular geometry. At lower wavenumber region, the in-plane C-C stretching at 1593 cm −1 for MBT is red shifted to 1514 cm   The 1 HNMR spectra of both the complexes exhibit 1H peaks related to four aromatic protons present in the benzene ring of the mercaptobenzothiazole (Fig. 11). A closer inspection of the 1 HNMR data revealed that in case of Ag(I)MBT all the aromatic protons are shifted to high frequency (downfield) compared to that of the Au(III) MBT complex. There is a pair of deshieled doublet pattern observed in both the complexes corresponding to protons attached to C4 and C7. In case of Ag(I)MBT there are two triplets at 7.48 and 7.57 ppm while the triplets have merged together and observed as a single multiplet pattern at 7.34 ppm in case of Au(III)MBT complex. Further, the -SH proton peak is missing in case of Ag(I)MBT which is clearly observed in case of Au(III)MBT as a broad singlet at 12.39 ppm. This observation can be attributed to the fact that in case of Au(III)MBT complex sulphur has not coordinated to the Au(III) ion and remains as -SH whereas in case of Ag(I)MBT, the -SH proton is involved in the complex formation via coordinating with the Ag(I) ion. Such coordination has also brought an overall deshielding effect for all the aromatic protons of Ag(I)MBT. On the basis of NMR analysis, we tried to support that complexation of silver with MBT has happened through -SH group, while in case of gold the -SH group remains intact. Further, the aromatic protons of MBT experienced a greater downfield shift in case of silver complex compared to that of the gold complex. We must clarify that we have not tried to determine the complex structure using NMR data, rather we have used NMR to identify the event of complexation and the different mode of complexation in case of Au(III)MBT and in case of Ag(I)MBT. Thus the structure shown in Fig. 7 of section 3.1 is supported by UV-Visible, FTIR and 1 HNMR studies. Electrochemical studies. Electrochemical studies were carried out using cyclic voltammetric technique employing Zahner Zennium electrochemical workstation. In a standard three-electrode electrochemical cell, the working electrode was a gold electrode (surface 0.02 cm 2 ), whose potential was controlled against the saturated calomel reference electrode (SCE). Platinum coil served as a counter electrode. Figure 12  peak corresponds to the redox reaction Au 3+ + 2e → Au + , the second peak accounts for AuCl 4 − + 3e → Au and the third peak indicates the formation of Au 3+ + 3e → Au. This demonstrates the presence of AuCl 4 − anion and free Au 3+ ions in the solution. Anodic peaks were noticed at 0.6 and -0.6 V. This is due to the oxidation of Au + ions released in the solution back to Au 3+ ions, as Au 3+ is the most stable oxidation state for Au. Thus in addition to UV-Visible, FTIR and 1 HNMR studies, electrochemical analysis of Ag(I)MBT also support the formation of S coordinated Ag(I) ions and ion pair complexes in Au(III).

Inductively coupled plasma mass spectroscopy (ICP-MS) analysis. ICP-MS
Cell Culture and TUNEL Analysis. During apoptosis, DNA cleaves to generate myriad fragments with double stranded and single stranded nick in the nucleus 21,22 . The fragmentation of DNA during apoptosis are labeled in-situ by attaching fluorescent-tagged nucleotides into partially degraded DNA using terminal deoxynucleotidyl transferase (TdT) and DNA polymerase 23,24 . The tail labeling reaction was done using TdT [25][26][27][28][29] . This assay is commonly known as TUNEL 'TDT-mediated dUTP-biotin nick-end labelling' 30 . Different variations of this assay have been developed 29 . Amongst all variants, the assay based on incorporation of BrdUTP seems to be more promising in terms of simplicity and sensitivity 28 . In this assay FITC-conjugated anti-BrdU attaches to poly BrdU found at the site of DSBs 31 . DNA denaturation is not required for the attachment of antibody to poly BrdU in DSB but it is required to detect the monomer incorporation during the process of DNA replication 31,32 . To perform this assay prefixation of cells using formaldehyde was done to retain the oligomeric fragments inside the cell. Labelling of nicks with FITC-tagged anti-BrdU antibody can be combined with red fluroscence staining of DNA. These cell subpopulations were analyzed under multiparameter cytometer to differentiate the cell which undergo apoptosis process from non-apoptotic subpopulations 25,26 . A549 cells were platted into six well tissue culture plates for various treatment experiments and on subsequent day at 70-80% confluence. From the TUNEL 14 assay analysis of Ag(I)MBT and Au(III)MBT treated cells, we observed a new crucial insight into the mechanism. The probability of induced apoptosis in A549 cells upon treatment with both Ag(I)MBT and Au(III) MBT had been noticed (cf section 3.8). A549 cells were seeded into six wells tissue culture plates and twenty wells were treated with or without (DMSO) as control experiments. Then the cells were treated with Ag(I)MBT (Fig. 13a) and Au(III)MBT (Fig. 13b) complexes at different concentrations ranging from 0 to 40 μM for Ag(I) MBT and 0 to 80 μM for Au(III)MBT. After 24 hrs of the treatment, post-treated cells were processed for detection of apoptosis in the terminal via deoxynucleotidyl transferased UTP nick-end labeling (TUNEL) analysis 14 . Figure 13 depicts the bar chart of the TUNEL analysis with mean ± SD of three independent experiments which were performed in triplicates and *p < 0.05 as compared to control cells. The TUNEL assay studies in conjunction  with the Fluorescence microscope images of A549cell line treated with Ag(I)MBT and Au(III)MBT complexes indicated induced apoptosis in the cells (Fig. 14). TUNEL assay showed 60% cell viability for Ag(I)MBT at concentration of 30 μM whereas 80% for Au(III)MBT at 60 μM. Thus Ag(I)MBT can induce cell apoptosis in A549 cell line at a higher rate and lower concentration than Au(III)MBT. The cell viability was achieved for Ag(I)MBT at half the concentration of Au(III)MBT. Therefore, these complexes studied here can be a viable option as antiproliferative agent.
To understand cell viability effects of Ag(I)MBT and Au(III)MBT complexes, we have performed MTT assay. The viable cells can change MTT to colored formazan crystals which are imaged by bright field image microscopy. Cells were platted into 96 well plates and next day they were treated with appropriate doses of Ag(I)MBT and Au(III)MBT complexes as shown in Supporting Information. MTT solution was used by diluting 5 mg of MTT reagent in 1 ml of PBS and incubated to cell samples for 4 hours at 37 °C in dark. One hour incubation at 37 °C in dark was given after addition of acidic isopropanol and proper mixing to dissolve formazan crystals. Absorbance were monitored at 570 nm wavelength with the help of microplate reader. As there is no additional data to support the proposed mechanism, we have performed the detailed bright field image microscopy and counted the cell numbers manually. The data clearly represent the overall good health and fitness of the cells. We have observed the difference between the control (non-treated), Ag(I)MBT and Au(III)MBT complexes treated samples in terms of cellular proliferation (cf. Supporting Information).

Plausible mechanistic pathway for Ag(I)MBT and Au(III)MBT based apoptosis of cell. Ag(I)
MBT or Au(III)MBT get attached to cell surface receptor and get engulfed as a vesicle carrying the complex, which forms an early endosome. Once the early endosome gets attached to lysosome, its pH reduces 20 to form late endosome (ca. pH = 5.5). As a consequence of reduced pH, Ag or Au gets released from the complex. MBT metabolizes to benzothiazole and H 3 S + which makes the lysosomal environment even more acidic. This strong acidic environment facilitates the release of Ag or Au and benzothiazole out of late endosome. The escaped Ag or Au and benzothiazole get into the nucleus and damages the DNA which leads to the induction of apoptosis 20,33,34 (cf. Fig. 15). Analogous to the proposed mechanism, Ag(I)MBT is reduced to Ag in acidic pH leading to degradation of Ag(I)MBT complex when electrochemically perturbed. This unique observation in the cyclic voltametric analysis of Ag(I)MBT and Au(III)MBT implicitly support the formation of free Ag and Au in cells at acidic pH of 5.5.

Perspectives and Summary
Ag(I)MBT and Au(III)MBT synthesized via wet chemical method is characterized for optical, structural, electrochemical properties. The structural and optical studies confirmed the formation of linear complex for Ag(I) MBT and near perpendicular complex for Au(III)MBT. 1 HNMR studies also supported the linear and perpendicular structure of Ag(I)MBT and Au(III)MBT complexes. The electrochemical analysis at acidic pH of 5 showed release of Ag + ions at cellular pH. This released Ag + is reduced to metallic silver in the cathodic scan, whereas Au 3+ reduces to Au + . This unique observation from electrochemical analysis supported the plausible mechanism of apoptosis in cells by Ag(I)MBT complex. The TUNEL and MTT assay on A549 cells and control cells revealed induced apoptosis and cellular anti-proliferation. Thus the complexes studied in the present investigation can be a viable option as anti-proliferative agent.