Formulation of promising antibacterial, anticancer, biocompatible and bioactive biomaterial as therapeutic drug delivery system for biologically active compound loaded on clay polymer

The synthesized heterocyclic compound: 5-[4-bromobenzylidene amino)-1, 3, 4-thiadiazole-2-thiol abbreviated as BATT possessed good antibacterial activity for various Gram-negative and Gram-positive bacteria. In addition to potent antitumor activity to breast cancer, for the first time, the novelty of this study is facile low-cost formulation of safe antitumor drug delivery system (DDS) for breast cancer from such simple heterocyclic compound. Heterocyclic compound is efficiently and spontaneously incorporated into MMT clay polymer matrix forming novel therapeutic nanocomposite DDS for breast cancer. BATT successfully intercalates MMT clay polymer matrix. Electron donation ability of nanocomposites is confirmed by cyclic voltammetry in terms of small peak-to-peak redox potential ΔEpeak\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\Delta E_{{{\text{peak}}}}$$\end{document}. Adsorption of BATT on clay is carried out by batch adsorption method. Better adsorption strength at low pH 2 decreased with increasing pH. Adsorption data are analyzed by multiple mathematical models. MMT clay is an efficient carrier polymer heterogonous adsorbent for BATT at optimum conditions: contact time 2.0 h, pH 2 and initial concentration up to 1200 ppm. Adsorption rate constant, k2 equals 14.6 ×\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\times$$\end{document} 10–3 and qe 19 mg g−1 (R2 0.999). pH of zero charge of MMT at pH 3 reflected adsorption mechanism of MMT. At pH 2, BATT is loaded on MMT by physi- and chemisorption. At pH 4, BATT is loaded on MMT by chemisorption.


Introduction
Nanocomposites drug delivery system (DDS) targeting therapeutic drug for treating cancer is a promising medicinal nanotechnology [1]. In current era of molecular medicine, much efforts have developed therapeutic DDS for breast cancer as nanocomposites of biomaterials [2]. Breast cancer, BC, represents quarter percent of cancer types. It is the second common cancer type that has a high incidence rate. Two million new cases of BC appear annually. It is among the main causes of women death. Most BC begins in breast tissue of glands lobules for milk production, or in ducts connecting lobules to nipple. Remainder breast is made up of fatty, connective and lymphatic tissues [3].
BC is caused by unhealthy lifestyles, long time fertility, using preventive pregnancy hormones and hormone replacement therapy, no children and breastfeeding, obesity after menopause, physical inactivity, alcohol consumption, family history, genetic predisposition and inherited mutations [3]. BC is treated by radiation therapy, targeted chemotherapy and endocrine hormone therapy. Chemotherapy (CT) harms human health by therapeutic antitumor drugs due to poor solubility in aqueous biological fluids, rapid deactivation, low apparent permeability and non-biodegradability [4]. Limitation of CT for BC is insolubility of therapeutic drugs in aqueous media [1]. DDS reduces drug side effects and maximizing drug action. DDS design is the most important nanotechnology. Therapeutic drug should possess high electron density for efficient loading on biocompatible polymer carrier [1]. MMT clay is a hydrated aluminosilicate inorganic polymers, mixed fine-grained clay minerals and metal oxides. MMT is safe drug carrier due to large specific internal surface area, physicochemical stability and several other structural and surface properties. MMT has high adsorption capacity, enhanced rheology, intercalate and reserve large neutral water-insoluble drug molecules into interlayer space 2D aluminosilicate layers. MMT releases incorporated loaded drugs in biological fluids via ion exchange mechanism or adsorption on porous active sites. MMT has high cation exchange capacity CEC for Na + or K + ions 1.0 mg equiv g −1 . 0.96 nm and 0.94 nm platelet thickness [1]. Mechanism of DDS formulation is mainly adsorption of therapeutic drug onto pores of MMT [5]. MMT is bioactive, biodegradable, hemocompatible and non-toxic [1]. Main objectives of study are preparation and characterization of new nanocomposites of biologically active 5-[4-bromobenzylidene amino)-1, 3, 4-thiadiazole-2-thiol (BATT) compound on bioactive MMT polymer. Compound BATT is selected for adsorption on clay inorganic polymer as it is biologically active compound for simulation new antibacterial and anticancer DDS.
In vitro antibacterial activity tests at 30 °C using diffusion agar method, well 6.0 mm diameter: 100 µL BATT and ciprofloxacin standard antibiotic. Tested strains are obtained from culture collection Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt). Tested Gram-positive, G + (Staphylococcus aureus, E. coli) and Gram-negative bacteria, G − (Pseudomonas aeruginosa, Bacillus cereus) are cultured on nutrient agar. Antibacterial activity was determined after one day by cut-plug method [8].
Some parameters of optimized structure are calculated by B3LYP/6 method, basis set 31-G d, p level using GAUSSIAN 9 package software, frontier molecular orbitals, HOMO and LUMO are visualized with Gaussian view [10]. Some chemical descriptors are calculated [7]: Redox characteristic of BATT is clarified via cyclic voltammetry from 0.5 V to − 0.5 V, scan rate 20 mV s −1 using silver-silver chloride reference electrode. Sample is loaded on glassy carbon electrode 0.032 cm 2 surface area [9].
BATT is uploaded on clay polymer by adsorption to formulate antibacterial anticancer DDS. In batch adsorption method, adsorption parameters contact time, pH and initial concentration are optimized. For determination maximum adsorption capacity, 100 mg MMT is dispersed in 20 mL de-ionized water contains 15 mg BATT, agitated at room temperature for different contact time (t) up to 3.5 h. Mixture is centrifuged and filtered using poly tetra-fluoro-ethylene (PTFE) filter, 0.25 µm pore size. Residual concentration in supernatant is determined by UV-Vis spectroscopy at λ max. 320 nm [10]. Each adsorption run is carried out in triplicate (n = 3). Average value is calculated along with standard deviation.
where qt is adsorbed quantity mg g −1 , Co is initial concentration mg L −1 , Ce is equilibrium concentration, mg L −1 , m is mass adsorbent, V is solution volume, L.
To optimize pH, 100 mg MMT is treated with 20 mL 15 mg BATT solution at pH range 2-12. pH is adjusted using aqueous solutions 0.1 M NaOH or 0.1 M HCl. To optimize initial concentration, 100 mg MMT is mixed with 10 mL BATT solution containing different ppm concentration BATT.
All adsorption data are carried out in triplicate (n = 3) for calculation qt and % adsorption. All data are represented as mean x and S.D

In regression analysis for any two variables X, Y
where n is the number of data points, Y i , …, Ŷ i are observed and predicted values, respectively. MSE is calculated from a set X and Y by finding regression line, insert X values into linear regression equation to find Ŷ i .
To find the best thermodynamic or kinetic model for adsorption data, an error analysis is performed. Sum of squares errors (SSE) and chi-square ( 2 ) are achieved by equations: where q e exp and q e cal are experimental and predicted adsorption capacities at equilibrium, respectively.
Nanocomposite NC 1 is prepared [1]: An aqueous MMT dispersion (1% w/v) is prepared by dispersing 2.0 g MMT in 200 mL double distilled water. Turbid dispersion is left at room temperature for eighteen hrs. for complete swelling. At pH 4.0 of dispersion, 0.2778 g BATT equivalent to excess three CEC of MMT is dissolved in 50 mL DMF and added to MMT dispersion. Mixture is stirred for 4.0 h at 40 °C, left for settling for 18 h at room temperature then filtered. Precipitate washed several times with DMF to remove unbound BATT molecules, dried at room temperature, grinded with mortar and pestle to be fine powder [13]. NC 2 is prepared by the same method of NC 1 at pH 2.

Results and discussion
All spectral analysis in Fig [12].
BAAT showed potent antibacterial to G − bacteria than ciprofloxacin and moderate activity to G + bacteria, Table 1.
Antibacterial effect of BATT is more pronounced against G − bacteria (Fig. 2) than G + bacteria that possess thick cell wall contains massive peptidoglycan. G − bacteria have two layers cell membranes, and outer membrane comprises lipopolysaccharide protective against antibiotics and antibacterial drugs and detergents that could damage inner membrane or cell wall peptidoglycan [8]. IZ for BATT exceeds that of Ciprofloxacin for G − bacteria.  [1]. Cell lines are sub cultivated, for 120 passages plated in 96-multiwell plate 10 4 cells/well along one day for fixation on plate walls before adding 0, 1, 2.5, 5 and 10 μg/mL BATT. Each dose is added to cell monolayer triplicates wells, incubated for two days at 37 °C under 5% CO 2 atmosphere. Cells are fixed, washed and stained with sulfo-rhodamine-B stain. Excess stain is washed with acetic acid and attached stain is recovered by Tris EDTA buffer. Color optical density (O.D.) is measured in Mindray MR-96A ELISA reader at 570 nm. Plot surviving fraction-concentration gave survival curve of each treated tumor cell. IC 50 percent control of infected and uninfected response values is calculated for BATT and Doxorubicin positive standard. Method validation is tested by taking fourfold concentration of blank and control. Table 2 shows mean x and standard error SE or S.D. triplicates O.D, for each test solution in mean following Poisson distribution [13]. % viability of tumor and normal cells are determined in triplicates for each concentration, Table 2. Table 3 collected mean values x and SE of %cell viability at different concentrations.
Small error SE in mean value x of tested concentrations confirmed validity and precision of SRB assay method. Figure 3 shows BATT cytotoxicity to McF-7 cells and McF-5. Error bars in histogram of cell viability versus concentration (Fig. 3) are due to small S.D. in triplicate data and confirmed high accuracy and precision of SRB assay.
BAAT inhibited availability of McF-7 by 14% at 31.25 mg/L and 15.6 mg/L with low cytotoxicity on normal cells McF-5 [13,14]. It acts by heterocyclic rings and thiol group. It is recommended to formulation of DDS for BC using MMT safe  [16]. Efficient BATT loading on MMT matrix is confirmed from FTIR vibrational bands at characteristic frequency, cm −1 (Fig. 4) [1]. Figure 5 shows optimized 3D geometrical structure of BATT. Computations are converged upon true minimum energy − 1120 eV ensuring stable ground state. High dipole moment 17.38 Debye reflected polarity charge separation and dielectric character of BAAT electron donner Lewis base. Figure 6 shows wave functions of electron density in molecular orbitals of BATT molecule in duplicate due to trans and cis isomers. HOMO lies on thiadiazole ring and LUMO on phenyl ring containing vacant p-orbitals [7].
Geometrical parameters atomic Mulliken charge reflecting electron density on atoms, bond lengths, bond angles and dihedral angles confirming molecular structure of BATT and dihedral angle are collected in, supplementary information SI, Table SI.1, 2. [1]. BATT is electrically neutral, stoichiometric formula C 9 H 6 N 3 S 2 Br. N13 has highest charge and it is electron donating site. C-H bond length is 1.1 Å. C-C bonds in phenyl group have same bond length 1.4 Å. C-N bond length, 1.3 Å is shorter than C-C bond due to electronegative N atom. Bond angles 120° confirmed trigonal planar sp 2 hybridization. Dihedral angles ± 179° are for distorted linear sp 2 hybridization. Bond angles less than 90° reflect distorted tetrahedral sp 3 hybridization. Dihedral angles ± 180° are due to SP hybridization and molecular planarity. Dihedral angles 180°-0° suggested anti-and syn positions, respectively. Dipole moment on Cartesian coordinates is µ x 3.7161, µ y − 2.7634, µ z − 0.4974. Electron transfer occurs from HOMO BATT into LUMO MMT [1,7] (Fig. 7).
States per energy interval at each energy level available for electrons occupation density of states, DOS, and Fermi levels are viewed using Gauss Sum 2.2.5 program. Intense green colored DOS reflects many states available for electrons occupation and intense electron density [15] (Fig. 8). Some molecular chemical activity indices are calculated based on E HOMO and E LUMO . Chemical potential, Pi; electronegativity, χ; global hardness, η; softness, σ and global electrophilicity index, ω, are collected in Table 4. Electron donation ability of BATT to an appropriate acceptor sites is confirmed as η nearly equals ΔE LUMO-HOMO (band gap E g ) indicating charge transfer from BATT molecule. Softness confirmed polarizability and inversely proportional to η. Parameters E HOMO , E LUMO and ∆E LUMO-HOMO reflected electron donation ability of BAAT [7].

Fig. 3 Histogram cell bio availability-concentration
Small E g 0.1408 eV showed BATT is polarizable soft Lewis base. Adsorption center SH group chelates MMT clay via coordinate bond formation reinforced by back donation to vacant p-orbital in phenyl group. High E HOMO indicated low ionization potential and high electron donation tendency to Al atoms in MMT with low  [13]. Negative chemical potential, Pi, reflects stable molecular structure.
Redox characteristics of BATT and NCs are illustrated in Fig. 9. NC 1 showed the smallest band gap, E g peak-to-peak redox potential ΔE peak . Electron confinement enabled electron transfer from HOMO BATT to lower partially empty p-orbitals of Al atoms in MMT. NC 1 showed the best redox characteristics [16].
In Fig. 10 TEM micrographs showed NPs in NC 1 are well dispersed. Particles size ranged from 22 to 39 nm. MMT clay inorganic polymer has amorphous morphology continuous nanoscale polymeric sheets of slightly defined NPs. NC 2 had tree-like morphology and large particle size 36-91 nm more aggregated than NC 1 . Small particle size of NC 1 confirmed large surface area/volume ratio and high Lewis basicity. Larger particle of NC 2 indicated irreversible phase transition to more stable state and unsuitable DDS. It is excluded from further investigation.

Fig. 8 DOS of BATT
Colloidal stability NC 1 is established from negative zeta-potential − 26.9 mV [8] (Fig. 11). Negative charges on NC 1 protected nanoparticles against coagulation. PXRD patterns in Fig. 12 confirmed existence of NC 1 as small suspended NPs showed fine wider diffraction peaks than NC 2 [17] as confirmed by crystal data. Validity of pdF cards used to simulate pXRD patterns is confirmed through calculation interplant distance, d, diffraction angle, 2θ and h k using check cell software program. Average crystalline size is calculated using Debye Scherer equation using the most intense peaks: Average particle size of NC 1 and NC 2 is 32 nm and 65 nm, respectively, in good agreement to those obtained from TEM micrographs. Weak intense XRD peaks of NC 1 confirmed nanocrystalline size with large surface area/volume ratio. NC 2 revealed moderate higher crystallinity than NC 1 . Structure type, geometry, crystal parameters and lattice parameters are shown in Table 5 using crystallography open database, COD, which agrees well with experimental pXRD patterns.
TGA plots related weight loss of BATT and NC 1 samples on heating (Fig. 13, Table 6).
DTA plots: temperature difference between tested and reference sample versus heating temperature, t °C (Fig. 14).
Activation energy, E a , of decomposition and thermodynamic of thermal decomposition are calculated [5], SI.2. Arrhenius type plots are represented in Fig. SI.1 for decomposition steps. Table 7 demonstrates these parameters.
Nonlinear fitting in Fig. 15 showed optimizing adsorption parameters. Nonlinear plots of % adsorbed are obtained on optimizing time, pH and initial concentration. Figure 16a: Adsorption is very fast at start and adsorption equilibrium is established rapidly. Adsorption% is limited up at 2 h. equilibrium time for completion adsorbed layer.
Small error bars (± error) confirmed accuracy of batch adsorption method on BATT on MMT. Adsorption was very fast and equilibrium is established within 2.0 h when adsorbed amount is limited with optimum pH 2 and initial concentration up to 1200 ppm for maximum adsorption MMT.
To clarify behavior of BATT, data are analyzed using several adsorption isotherms Langmuir, Freundlich, Tempkin, Dubinin-Radushkevich, D-R, Halsey, Elovich and Harkins-Jura. Empirical equations are described in SI.2. Linear regression analysis is followed in linear fitting of adsorption data [18]. High value of error functions and low value of correlation coefficients (R 2 ) for adsorption or kinetic model indicate that this model is not fitted to adsorption isotherm [19].
Adsorption parameters are collected in Table 8. Adsorption data did not obey Langmuir and Harkins-Jura adsorption. Linear fitting of adsorption data to Freundlich adsorption isotherm confirmed multilayer MSE is inversely proportional to correlation coefficient R 2 . Low value MSE and high R 2 for D.R. model indicate best fitting to adsorption data.
Kinetic of adsorption informed about adsorption rate and mechanism. Adsorption data are linearly fitted to kinetic parameters pseudo-first-order-, pseudo-secondorder and Elovich kinetics models. Empirical equations are shown in SI.3. Figure 16 Variation of adsorbed amount at any time t (qt) versus t. qt showed rapid increase with time and then qt becomes limited at 2.0 h. contact time. MMT clay intercalated large neutral (BATT) molecules into interlayer space of 2D alumina-silicate layers.
Adsorption data are best fitted to pseudo-second-order kinetic model (R 2 0.999) suggesting physisorption of BATT [10]. Figure 17 shows plot t/q versus t for pseudo-second-order kinetic. Kinetic parameters K 2 , q e are obtained from slope and intercept of the obtained straight line (Table 9). Small SSE confirmed validity of pseudo-second-order kinetic model for fitting adsorption data due to good agreement between q e.exp. and q e.cal. The value of 2 0.008 less than 0.05 confirmed neglecting null hypothesis (Ho) and accepting hypothesis (H) that q e is significantly affected by contact time.
Stepwise hydration of interlayer cation in MMT enabled intercalation with positively charged molecules. BAAT is a heterocyclic compounds contains high electron density. At pH 4, BAAT is intercalated to MMT by electron donation chemisorption with no electrostatic interaction. At pH 2, BAAT linked MMT by both chemisorption and physisorption.
MMT clays are low-cost adsorbents clays keeping environment by loading various. Good adsorbent as negative charge on framework is neutralized by adsorption positively charged cations. Clay minerals such as MMT, vermiculite, illite, caulinite and bentonite adsorbents for heavy metals are low cost than activated carbon, natural and synthetic zeolites and ion-exchange resins. MMT has excellent physicochemical properties. Clays are used: as barriers to avoid contamination of underground water and soil, preparations nanofillers and nanocomposites organic polymers. MMT clay is a bioactive glass hard solid material contains SiO 2 . Bioactivity depends on various proportions and available. Silicate-rich layer is produced in human fluids such as blood. MMT is safely applied in medicine for craniofacial. MMT clay is a nature-inspired layered clay mineral. Medical MMT clay is smectite phyllosilicates clays contains Al-O and Al(OH) 3 octahedral (Oh) sheet sandwiched by 2 Si-O