Development and Evaluation of Self-assembled Stabilized Supramolecular Hydrogels of Polyoxometalates

Abstract

Polyoxometalates (POMs) are discrete anions and have got immense attraction in biomedical research due to their structural diversity rendering them highly active against viral, cancer, bacterial, and HIV infections. In this study 36 different hydrogel formulations of Polyanion (P2W15) were prepared with four different cationic polymers including gelatin, polyethyleneimine (PEI) and two water soluble chitosan derivatives i.e. carboxymethyl chitosan (CMCh) and chitosan hydrochloride (ChCl). For induction of pH responsiveness in prepared hydrogel formulations methacrylic acid (MAA) and acrylic acid (AA) were grafted on polymer backbone. The swelling and in–vitro dissolution experiments of prepared formulations were conducted at pH 1.2 and pH 7.4 showing maximum swelling and cumulative % in–vitro release at pH 7.4. Out of all 36 developed formulations, the maximum swelling and cumulative % in–vitro release (92%) was observed for MAA–ChCl–POM hydrogel formulation with sample ID CHCP6. Prepared hydrogels were physically cross–linked using electrostatic interactions between POM and polycations that were further characterized by scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FTIR), thermal analysis (thermogravimetric analysis, (TGA), differential scanning calorimetry (DSC) and X–ray diffraction (XRD). FTIR spectroscopy confirmed the interaction among different functional groups of polymer, monomer and POM. Thermal properties of the prepared hydrogels were higher than their individual components as suggested by TGA/DSC curves. The hydrogel possessed glassy smooth surface morphology supporting an equal distribution of POM throughout hydrogel network. Safety evaluation of the POM solution and oral acute toxicity/tolerability of the hydrogel system (MAA–ChCl–POM) were also conducted in rabbits using both male and female species. For oral tolerability/ acute toxicity of hydrogel dispersion, maximal tolerated dose method was used suggesting 4000 mg/kg body weight dose as safe maximal tolerated dose (MTD). Histopthological examinations of the rabbit‘s vital organs like heart, liver, kidney, spleen and lungs showed no gross significant signs of toxicity. The findings of serum xi chemistry and haematological investigations confirmed that blood, liver and kidney functions for both oral POM solution and hydrogel dispersion were normal except reduction in blood glucose level which is due to inhibition of glucose–6–phosphatase enzyme by POM. In-vitro cytotoxicity studies were also performed to check the cytotoxic potential of the free as well as encapsulated POM (hydrogel) showing the dose dependent cytotoxicity against two different cancer cell lines (MCF–7 and HeLa cells) with minimal effects on normal cells (Vero). The highest in-vitro release hydrogel formulation (sample ID CHCP6) was further selected for in–vivo evaluation of POM for pharmacokinetic parameters through a validated and sensitive ion pair HPLC method in rabbits. Kinetica 5.1 was used to determine the pharmacokinetic profile of the POM. After oral administration of hydrogel disc having 20 mg POM, relatively longer plasma concentration profiles were observed. Area under the curve (AUC) and mean retention time (MRT) obtained for hydrogel formulation were 2.87 times and 1.91 times greater respectively than oral POM solution. Time for maximum plasma concentration (Tmax) obtained for oral solution was 9.6 hours while for hydrogel disc it was 14.6 hours. Half–life (t1/2) recorded for oral POM solution was 2 hours while in the case of hydrogel formulation, extended t1/2 of 4.87 hours was observed. Apparent volume of distribution (Vz) obtained for oral POM solution was 0.003L while for hydrogel system that was 0.0038L. Clearance of oral POM solution occurred at the rate of 0.0012 L/h while for hydrogel it was 0.00056 L/h. Elimination half–life (Lz) observed for oral solution was 0.345 (hr) and that for the hydrogel enclosed POM was 0.147 (hr). Cmax values calculated for oral POM solution and hydrogel disc were lying in close proximity. The findings of the current study indicated that prepared hydrogel system showed controlled encapsulated POM release. The hydrogel system and concentration of POM used were safe from in–vivo point of view. The cytototoxity data of normal Vero cells suggest that toxicity issue of POMs can be addressed by closing it inside polymeric network and controlling its release pattern.