Biophysical insights into the binding capability of Cu(II) schiff base complex with BSA protein and cytotoxicity studies against SiHa

Herein, we have explored the effects of chlorinated mononuclear Cu(II) complex upon binding with BSA protein (bovine serum albumin) and its in vitro anti-proliferative potentiality against SiHa cell. The complex was synthesized involving a Schiff base ligand having N,N,O donor centers and characterized by several spectroscopic studies. Structure, DFT studies and Hirshfeld surface (HS) analyses were identified using crystallographic computational studies. The binding interaction with BSA depicts the efficacy of the complex towards promising binding of it with BSA. Further, the complex shows a moderate cytotoxicity against SiHa cancer cell signifying its potentiality as an anti-proliferative agent for human cervix uteri carcinoma.


Introduction
In recent times, there is a considerable demand for the synthesis of effective anticancer drugs as millions of deaths occurring each year globally are due to cancer [1,2,3]. In this regard, the booming discovery of platinum based cisplatin and its analogues as effective anticancer drugs is undoubtedly remarkable. But to avoid the serious remedial drawbacks of platinum based complexes, chemists are prompted to develop non-platinum metal complexes [4,5]. Thereafter a lot of complexes other than platinum metal are synthesized which are vastly utilized in the field of biology along with anticancer [6], antiseptic [7] and opposed to fungal [8] applications.
But to explain the effective metabolism and transferring process of any medicine, it is very much important to inspect the binding interaction studies of that medicine with serum albumins. These proteins are studied expansively because of their easy accessibility, high stability, low-priced and extraordinary binding capability with ligand. Besides, albumin plays an important function during the transfer process [29]. So, interaction of drugs with albumin protein might be observed as a replica to get elementary idea about these interactions. Because of structural homology of more than 76% with HSA (Human Serum Albumin), interaction study with BSA was carried out extensively in the past years [30,31].
The current paper reported the synthesis of a Cu(II) Schiff base complex containing ligand (L) resulting from 5-chloro salicylaldehyde and ethylenediamine. BSA interaction with the complex has been studied by UV-spectrophotometry, fluorescence spectroscopy, circular dichroism spectroscopy and the binding mode is discussed. The focus is also centered in the investigation of cytotoxicity of the said complex on the SiHa cancer cell line.

Materials and methods used
Materials employed in various experiments of current studies are used without further purifications as they are all of pure reagent grade. The reagents like 5-chloro salicylaldehyde, ethylenediamine, methanol, CuCl 2 .2H 2 O, and Et 3 N etc. were procured from Merck. BSA purchased from Sigma-Aldrich Chemicals (USA) was prepared from phosphate buffer (PB) of 10 mM concentration to maintain pH 7.4. 5 μM of BSA was utilized for lifetime and steady-state studies whereas 2 μM concentration of BSA was used during CD study.

Preparation of L
It was made from the reaction of 5-chloro salicylaldehyde (1mmol) and ethylenediamine (1mmol) with stirring for 2 h in methanol solvent. Yellow colour solution was developed and was employed straightforward for complex preparation (Scheme 1).

Preparation of complex
CuCl 2 .2H 2 O (1 mmol) and ligand L were reacted in methanol to prepare the copper complex under the present study under refluxing condition for about 3 h (Scheme 1). A deep green colour solution was observed. Single crystals of green colour were achieved after few days from slow evaporation technique. These single crystals are finally used for X-ray diffraction.

Spectroscopic techniques
FTIR study was done in a IR spectrometer (Shimadzu). UV-Vis studies were carried out in a double beam spectrophotometer purchased from Systronic. ESI-MS study for complex was done by mass spectrometer (XEVO G2-XS QTOF).

Single crystal X-ray diffraction studies
Crystal data collection was performed at 150 K by a graphite monochromated X-ray diffractometer (Bruker Kappa) with Mo-Kα radiation of λ ¼ 0.71073 Å. APEX-II (v2.0-2) program (Bruker) was employed for data processing using φ and ω-scan techniques. For data correction towards Lorentz and polarization effects, SADABS program [32] was used. SHELXT 2014/5 was utilized for solving the structure by direct method and subsequent refinement was performed with the help of F 2 by means of SHELXL2018/3 [33]. Refinement of non-hydrogen atoms was done including parameters related to anisotropic displacement. Thermal parameters used for refinement of hydrogen atoms are 1.2 or 1.5 times greater than subsequent mother atoms. Table 1 shows data and parameters related to crystallographic studies of the complex. The CCDC number, 2165565 is gained from CCDC Centre (Cambridge).

Hirshfeld surface (HS) analyses
In the solid-state, the complex shows various types of important noncovalent interactions to build the lattice. This knowledge of non-covalent interactions of the complex molecule in solid-state helped to understand their interaction capability and types of interactions with other molecules like protein or small organic molecules. To understand the aforesaid weak intermolecular forces in the crystal in a deeper way, the Hirshfeld surfaces analysis is used as a theoretical tool. With this study, all types of atom-atom short contacts locations can be investigated quantitatively. The Crystal Explorer 3.1 software [34] has been used for quantitative analysis of intermolecular interactions in the crystal structure by HS analysis. The HS analysis involves the partition of electron density (ED) of a molecule into atomic fragments such that a molecule within a crystal is given by a weighting function where ρ ðrÞ is a spherically averaged Hartree-Fock atomic ED function of i th nucleus. The cutoff of the weight function is 0.5 Å.
Further, different interactions in the crystal structure have been visualized using different functions such as d e , d i ; d norm , shape index and curvedness mapped on the HS of the molecules. The normalized contact distance (d norm Þ in the HS analysis is given by r vdw e d e denotes HS distance from nearest nucleus situated outside surface, d i is the HS distance from nearest nucleus situated inside surface and r vdw corresponds to van der Waals radius. The d norm parameter on the HS of the molecule is visualized in a red-white-blue color code. The corresponding intermolecular contacts which are less than their vdW radii are indicated by red regions on the HS. Whereas the blue regions show intermolecular contacts of distance longer than their vdW radii. White regions signify that the contact distance is identical to the summation of vdW radii. Complementary nature of intermolecular interactions during the packing of molecules is identified by shape index. The curvedness is also useful parameter to understand the interactions such as π…π interactions. The flat regions of a surface are highlighted by a low value of curvedness. Similarly, the 2D fingerprint plot associated with the HS give quantitative information on the percentage contribution of individual interactions to the supramolecular assembly.

DFT computation calculations
With the help of Gaussian 09 DFT study of the complex was done. Hay double-ζ basis set and effective core potential (ECP) are employed to signify the inner electrons of Cu. Besides, elemental atoms (C, H, N, Cl) were illustrated by 6-31G (d,p) basis sets. For all computations, CPCM (conductor-like polarizable continuum model) was employed. The atomic coordinates of crystallographic structures optimize closed-shell geometry.

Steady-state experiments
The steady-state measurements were performed by UV-vis spectrophotometer (Cary 100) and Fluorolog 3-111 (Horiba JobinYvon). The fluorescence spectral intensities were modified to avoid the inner filter effect coming from significant absorption of complex below 300 nm (along with the absorption maximum at 370 nm) by using Eq. (1) [35]:

CD spectroscopic study
The CD spectra were taken in a spectropolarimeter (JASCO J-815) after correcting baseline at 298 K and using a 0.1 cm cuvette of quartz. The spectra were recorded within the wavelength range of 200-260 nm with a scanning rate of 100 nm min -1, and each spectrum was as an average of three scans. The obtained ellipticity (θ) was expressed in MRE (Molar Residual Elipticity) and shown in Eq. (4) [36]: Here, M and a denote molecular weight of protein and the number of amino acid residues, respectively. cis the concentration in g L À1 , and l is the path length.

Docking studies
AutoDock 4.2 software was utilized for performing the semi-rigid docking studies [37]. The highest binding energy is well thought-out as the most excellent docked conformer of the ligand. The BSA structure was taken from RCSB protein data bank (PDB ID: 3V03). The ligand molecule (Complex) was optimized in Gaussian 09 software and the optimized structure was further using in docking [38]. The grid sizes along three axes were kept as 126 while the grid spacing was 0.375 Å. The conformation of most excellent docked conformer was examined using PyMOL software [39] out of 50 different conformations.

Culture of cell and maintenance
Dulbecco's Modified Eagle Medium (DMEM) along with 10% heatinactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 μg/ ml streptomycin were involved to culture SiHa cells (cancerous tissues of the cervix uteri). 310 K temperature was maintained during culture of the cells in a CO 2 incubator with a 5% CO 2 supply.

Cell proliferation assay
With the help of MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), the cell proliferation was investigated. Seeding of cell was performed in 0.1 mL of DMEM consisting of 10% FBS, 50 U/ mL penicillin and 50 μg/mL streptomycin at the confluences of 70-80% cells per well. These were incubated at 310 K in 5% CO 2 . For accuracy in the obtained results all the treatments were performed in triplicate. To remove the dead cells, media of each well were substituted by 100 μL fresh media. Then in order to produce water-insoluble formazan, 0.5 mg/ mL of MTT solution in DMEM was supplemented to every well and incubated for extra 3 h at 310 K. This formazan of each well is then mixed with DMSO and absorbance was recorded at 570 nm by a micro plate reader. By using the following formula (Eq. (5)), percentage of cell viability was calculated.
where, OD represents specimen optical density.

Fluorescence imaging
A fluorescence microscope was used to visualize cell proliferation efficiency on the specimen. Cancer cells were seeded onto the sample in 12-well plates for 24 h at 310 K. Fresh solution of PBS (phosphate-buffer saline) was used twice to wash test samples for removing the dead or floating cells. The 4% paraformaldehyde solution fixed the adhered cells for 20 min. Then they were washed again with PBS and marked by AO (acridine orange) and EtBr (ethidium bromide) (100 μg/mL) fluorescent dye for 10 min. Again, these were rinsed with PBS for twice and consequently incubated for 5 min in dark. After that with a fluorescence microscope (Leica, Germany) images were collected.

Structure analyses and spectroscopic characterization
Several techniques of spectroscopy were employed to characterize the synthesized ligand and the complex. The complex structure was firmed and analyzed by X-ray diffraction and HS analyses studies.
The FTIR spectrum is shown in S1 for the complex from which it is observed that the strong peak for azomethine (C--N) group obtained at 1682 cm À1 indicating the formation of Schiff base. The other prominent peaks such as C-C at 1209 cm À1 , C-H aromatic at 3012 cm À1 , C--C at 1313 cm À1 , C-H aliphatic at 2972 cm À1 and bending peak for C-H at 1343 cm À1 which were obtained at their normal areas also support the formation of the complex.
UV-vis results reveal band for π-π* transitions at around 370 nm for C--C and C--N groups in the complex. The band characteristic for ligand appears at 334 nm (n-π*) which is absent in spectrum of complex.
ESI-MS spectra (S2 and S3) for ligand and the complex clearly demonstrate the prominent peaks for expected m/z values (slightly different from calculated values) for both ligand and the complex. This slight difference is due to Hþ adducts from solvent as the structures of both ligand and the complex contain some probable centre to accept those Hþ adducts.
The refinement details related to crystallographic studies are listed in Table 1. The parameters related to geometry of complex show close proximity to the literature values. The crystalline pattern of complex is centrosymmetric with P-1 triclinic space group. Crystalline structure contains a central Cu(II) with four co-ordinations resulting in a square planar geometry with distortion (Figure 1 (4) indicating that the {CuN 2 O 2 } moiety is distorted (** S1). Both oxygen atoms O1 and O2 participate in the H-bonding with the H atoms of an adjacent molecule (Figure 2 and S2). Additionally, C9-H9A⋅⋅⋅Cl2 interaction is stabilizing the crystal structure. Strong and weak interactions contributed in lattice are highlighted by HS. Figure 3 represents the contribution of different interactions attained from HS fingerprint plot. The C-H⋯O and C-H⋯Cl interactions constitute 33% of total interactions present in the crystal structure. Further, S4 represents Hirshfeld surface obtained by recording d norm (S4a) shape-index (S4b) and curvedness properties (S4c).

DFT analyses of the complex
The DFT calculations of the complex were performed to understand the electronic construction of the complex. Besides, to get an insight of kinetic stability, chemical reactivity and electrical transport properties of the complex, the understanding of frontier molecular orbitals (HOMOs and LUMOs) is very essential. The small energy gap of HOMO and LUMO orbital of a complex makes it more polarizable. Such molecules are soft molecules that are more capable of chemical and biological reactivity because they have high electron donation (high E HOMO ) and acceptor (low E LUMO ) ability. MO (molecular orbital) pictures are depicted in S5. As observed from the figure, in HOMO, density of electron is delocalized on the C-centre and the O centers of the salicylaldehyde ring. The same electronic structure was observed in case of single occupied molecular orbital (SOMO). Further, in LUMO the density of electron is localized on C-centers of the salicylaldehyde ring and N centers of the ligand. However, in HOMO -1, the density of electron is transferred to metal centre (i.e., Cu centre) of the complex along with the coordinating atoms of the ligand (N and O). This observation suggests that aromatic rings of the ligand and the metal ions are dynamic parts for biological activities like protein interactions, anti-cancerous properties etc. The HOMO (À6.041 eV) and LUMO (À1.941 eV) molecular orbitals have an energy gap of 4.1 eV indicating capability of the studied complex towards chemical and biological reactivity. The SOMO orbital of the complex is in the energy level of -5.757 eV.

BSA interaction analyses
BSA shows strong absorption at~280 nm which increases with increasing concentrations of complex and the complex itself shows absorption around 370 nm (S6a). Upon excitation at 295 nm (which deals exclusively with Tryptophan (W) residue of the protein), the fluorescence Table 2. Binding parameters of the interaction between BSA and complex. Inset picture shows decrease in average lifetime of BSA with increase in complex concentration.  emission centered at 350 nm decreases (S6b) with addition of complex as a result of Tryptophan quenching [35]. Quenching can be attributed by the formation of complex between the quencher and fluorophore in ground state or due to excited state collision, which can be predicted by the Stern-Volmer Eq. (6) [35]: F and F 0 represent intensities of the fluorophore (Tryptophan here) in presence and absence of quencher Q (here the complex) respectively. The plot (Figure 4a) of F 0 /F vs. quencher concentration will give a linear nature of the plot from where one can calculate the Stern-Volmer quenching constant (Table 2) [35]. The linearity of the plot gives only one type of quenching phenomenon. The binding parameters were obtained from Eq. (7) below ( Figure 4b) and the parameters were tabulated in Table 2: K b denotes binding constant and binding sites number is denoted by n.
To elucidate the exact nature of binding and quenching phenomenon, we further performed time-resolved studies. The lifetime for Tryptophan of BSA in excited state decreases with adding the complex. Reduction in the lifetime values may be attributed by the occurrence of interaction between BSA and complex at excited state by collision and thereby shows reduction in the lifetime of BSA ( Figure 5, Table 3). Moreover, we have plotted F 0 /F or τ 0 /τ as function of complex concentration (Figure 4c), which shows similar trend enabling that the quenching occurs through excited state collision or diffusion. Thus, it can be inferred that the quenching governing the said interaction is dynamic in nature [35].
Furthermore, we have investigated the conformation of the protein upon the said interaction by circular dichroism (CD) spectroscopy. BSA  shows helical nature in the CD spectra ( Figure 6) with two negative ellipticity bands at 222 nm and 208 nm corresponding to n → π* and π → π* transitions, respectively [40]. It is important to note that CD spectra of the protein in presence of complex below 220 nm could not be monitored, as the complex shows strong absorption below this wavelength range and also due to observing high voltage values for the instrumental limitation within the mentioned wavelength range [40].
Besides, to predict the probable binding site of the complex, within the BSA scaffold, we have performed blind docking study. The complex locates itself within BSA where it finds Tryptophan and some hydrophobic amino acid residues of the protein (Figures 7a, b) with binding energy as -28.34 kJ mol À1 .

Cytotoxicity studies
To evaluate cytotoxic effect for studied complex against SiHa, prohibition of cell propagation was checked by nurturing the cells with fixed concentration of the complex for 1, 2, 3 days using MTT assay. As control, the refined cells without sample are taken. About 50 μg/mL of the complex was utilized for this study. S7 shows the results with mean AE 1-1.5 standard deviation values with respect to individual experiments. Gradual decreasing of cell viabilities with time is observed than the control demonstrating cytotoxicity of the complex. To support these data additionally, fluorescent images were collected using AO and EtBr dye. AO and EtBr recognize the normal cells from apoptotic cells on the basis of cell membrane permeability. It is generally observed that AO shows green fluorescence and EtBr demonstrates red fluorescence on binding with DNA. Generally, dead cells shine red and permeable to both the AO and EtBr dye. Whereas, the living cells shine green and permeate only AO. After treatment, the comparative number density of cells can be visualized from fluorescent images of specimen staining with AO/EtBr (Figure 8). It is significant to point out here that the number densities as well as cell health are gradually decreasing owing to the incorporation of the complex into the cells with respect to control and another interesting observation is that cell apoptosis is noticed from day 1 to day 3 gradually indicating green to yellow colour of the cell. So, the complex is significantly cytotoxic in nature showing potential anticancer activity and the cell viability obtained for the complex after days 3 against the cancer cells is very much analogous to the reported values in literature [41,42].

Conclusions
In the current study, synthesis, spectroscopic characterization, structural analyses, Hirshfeld surface, DFT analysis, BSA interaction and cytotoxicity studies of a Cu(II) complex have been investigated. The crystallographic analysis shows that a central Cu(II) with four coordinations results in square planar geometry with distortion. Fingerprint plots of HS analysis exhibit the percentage of various interactions contributed in crystal structure. The C-H⋯O and C-H⋯Cl interactions constitute 33% of entire interactions present in the complex. Besides, from DFT study it is revealed that electron clouds of HOMO and LUMO orbitals are dispersed on Cu(II) ions and aromatic rings indicating potential capability of the complex for biological activities like protein interactions, anti-cancerous properties. This inspection leads to examine BSA interaction studies of the complex through a range of spectroscopic studies and the obtained results strongly support the above prediction. Besides, the complex has been found to be significantly toxic for SiHa cancerous cell lines and effectively causes more cell death. Therefore, the complex under the present investigation is expected to show effectual anticancer properties and unlocks the possibility of its uses in chemotherapeutics. The present studies can be expanded to virus infected cell lines which may unfasten a new path in metallopharmaceutics for healing cancer by disturbing life cycle of virus.

Author contribution statement
Bidhan Chandra Samanta: Conceived and designed the experiments; Contributed reagents, materials, analysis tools or data; Wrote the paper.
Paula Brandao: Contributed reagents, materials, analysis tools or data.
Saptarshi Mukherjee: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Data availability statement
Data will be made available on request.

Declaration of interest's statement
The authors declare no conflict of interest.

Additional information
Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e11345.