Ru(ii)arene(N^N bpy/phen)-based RAPTA complexes for in vitro anti-tumour activity in human glioblastoma cancer cell lines and in vivo toxicity studies in a zebrafish model

Herein, we have introduced a series of half-sandwich Ru(ii)arene(N^N bpy/phen)-based RAPTA complexes for brain cancer therapy. Among all the synthesized complexes, [(η6-p-cymene)RuII(κ2-N,N-4,7dimethyl phenanthroline)(PTA)]·2PF6 (4c) and [(η6-p-cymene)RuII(κ2-N,N-4,7diphenyl phenanthroline)(PTA)]·2PF6 (4d) showed outstanding potency against the T98G, LN229 and U87MG cancer cells. The antiproliferative activity of these complexes was reinforced by neurosphere, DNA intercalation, agarose gel electrophoresis, cell cycle analysis and time-dependent ROS detection assays. The real-time reverse transcription (RT)-polymerase chain reaction (PCR) study showed that complex 4c inhibited the TNF-α-induced NF-κB phosphorylation in glioma cells. Moreover, the in vivo biodistribution of complex 4c in different organs and the morphological patterns of widely used zebrafish embryos due to toxic effects have been evaluated.


DNA binding study
The binding capability of the complexes with calf-thymus DNA (Ct-DNA) was evaluated using electronic absorption spectroscopy, and the competitive binding assay was studied using ethidium bromide (EtBr) as a quencher by fluorescence spectroscopy.

UV-visible studies 1
The DNA binding experiment was performed in aqueous medium using RAPTA complex 4c in Tris-HCl buffer (5 mM Tris-HCl in water, pH 7.4). The concentration of Ct-DNA was determined using its absorbance at 260 nm and a known molar absorption coefficient of 6600 M -1 .cm -1 . In cuvettes, an equal amount of DNA was introduced to both the sample and the reference. The concentration of CT-DNA was increased as the titration progressed. The sample was equilibrated with CT-DNA for around 5 minutes before each measurement, and then the complex's absorbance was measured. The intrinsic DNA binding constant (K b ) was calculated using the equation (i):

UV and Fluorescence study
All of these RAPTA complexes were studied in a 10% DMSO solution using UV and fluorescence. The fluorescence quantum yields (Ф) were then estimated using the comparative William's approach, which entails utilising a well-characterized standard with a known quantum yield value and a 10% DMSO solution. 2 Quinine sulphate was employed as a standard. Quantum yield was calculated according to the equation (ii): Where, φ = quantum yield, I = peak area, OD = absorbance at λmax, = refractive index of solvent (s) and reference (R). Here, we have used quinine sulphate as a standard for calculating the quantum yield.

Ethidium bromide displacement assay
To demonstrate the manner of binding between the complexes and DNA, the ethidium bromide (EtBr) displacement experiment was used.

Protein binding studies 5
The main component is serum albumin proteins in drug transport and metabolism, as we all know. The interaction of the complex with human serum albumin (BSA) was examined using a tryptophan emission quenching experiment. The association of the RAPTA complexes 4c and 4d with the protein BSA was detected using a tryptophan emission quenching assay.  The conductivity of the RAPTA complexes was measured using a conductivity-TDS meter-307 (Systronics, India) and a cell constant of 1.0 cm -1 to verify the interaction of the complexes with DMSO and aqueous DMSO.

n-Octanol-water partition coefficient (log P o/w ) 8
Using the previously reported shake flask approach, the log P o/w of the RAPTA complexes followed the previously described procedure. An orbital shaker was used to shake a known amount of each complexes in water (pre-saturated with n-octanol). The solution was centrifuged at 3000 rpm for 10 minutes to allow phase separation. Different ratios (0.5:1, 1:1, and 2:1) of saturated solutions were shaken for 20 minutes on an orbital shaker with presaturated n-octanol to get the partition coefficient. The absorbance of aliquots of the aqueous and octanol layers were measured with a UV-Vis spectrophotometer after adequate dilution.
The concentrations of the complexes in each layer were estimated using the corresponding molar extinction coefficients, and the partition coefficient (log P o/w ) values were computed from the ratio.

Viscosity measurement 9
A hydrodynamic method such as a viscosity was performed using an Ostwald Viscometer to determine the binding manner of complexes using compound 4c, 4d, and EtBr treated DNA with respect to cisplatin.