Folate Decorated Leflunomide Nanoparticles Based Hydrogel: A Neoteric Approach against Rheumatoid Arthritis

Abstract

Rheumatoid Arthritis (RA) is a complex inflammatory disorder characterized by severe inflammation, with erosion of cartilage, leading to destruction of joints. Inflammatory cells infiltration, particularly macrophages, plays a major contribution to the pathogenesis and escalation of RA. These recruited macrophages overexpressing folate receptors beta (FR-β) have gained significant attraction for ligand-targeted delivery. Leflunomide (LEF), being an immunomodulatory agent is considered the cornerstone of the therapy, however, its oral efficacy is impeded by low solubility and escalating adverse effects profile. Therefore, targeted nanoparticles were developed to improve their efficacy and minimize side effects. In the present work, folate conjugated chitosan-chondroitin sulfate nanoparticles entrapping LEF were developed, for selective targeting at inflammatory sites in RA. For this purpose, first, folate conjugated chitosan (FA-CHI) polymer was synthesized. The developed polymer was further characterized in terms of Fourier Transform Infrared Spectroscopy (FTIR), X-Ray Diffraction Analysis (XRD), Differential Scanning Calorimetry (DSC), and Nuclear Magnetic Resonance (NMR); and utilized for the preparation of Folate conjugated Leflunomide Nanoparticles (FA-LEF-NPs). The nanoparticles were prepared through ionotropic gelation technique, employing FA-CHI and chondroitin sulfate (CHS). The polymers CHI and CHS were also presented with innate anti-inflammatory and antirheumatic attributes. For successful quantification of FA and LEF from the developed NPs, there is a need to develop a fast, robust, and validated quantification method. Therefore, a highly sensitive, accurate, and reproducible analytical method for the simultaneous detection of LEF and FA from polymeric nanocarriers was also developed. Analysis was performed on HPLC (Shimadzu 10 A), having a reversedphase C-18 column (Beckmen, 250 X 4.6 mm, 5μm) equipped with a photodiode detector set at a wavelength of 260 nm (LEF) and 285 nm (FA). The isocratic mobile phase was composed of acetonitrile, water, and trimethylamine in a ratio of 65:35:0.5 at pH 4. Rapid analysis of both agents was performed, with a total run time of 10 m (FA = 2.1 ± 0.1 min, LEF = 5.9 ± 1 min) at a 1 ml/min flow rate. The assay demonstrated good linearity of 0.9989 of 0.9997 for LEF and FA respectively with recovery in the range of 95-100%. The method also depicted good specificity, and intra and inter-day precision based on relative standard deviation (RSD) values. The method is helpful in the detection and quantitation of lower values of both agents with values of the limit of xi detection and quantification of (LEF= 0.078 and 0.039 μg/ml) and (FA = 2.14 and 0.13 μg/ml) respectively. The method’s suitability was determined by simultaneous detection of LEF and FA from polymeric nanocarriers. FA-LEF-NPs were afterwards optimized employing Box-Behnken design through Design Expert software. The concentration of drug, polymers, and pH were used as independent variables, while mean particle size of nanoparticles, entrapment efficiency, and zeta potential were taken as the dependent variables. The optimized nanoparticles depicted a particle size, entrapment efficiency, and zeta potential of 246 nm, 89%, and 33.8 mV respectively. The developed nanoparticles also demonstrated considerable biocompatibility and hemocompatibility, ensuring their safety potential upon systemic delivery. These nanoparticles were further fabricated into a hydrogel, employing almond oil (A.O) as a permeation enhancer, for their application to skin. The permeation properties of FA conjugated LEF-NPs loaded hydrogel through the skin were significantly improved with A.O (p < 0.001). Fabricated hydrogel demonstrated a sustained release drug profile, with highest release at pH 5. The in vivo studies justified the preferential accumulation of folate conjugated nanoparticles at inflamed joints more than any other organ in comparison to the free LEF and LEF-NPs formulation. The efficacy of the fabricated hydrogel was also ascertained on the Complete Freund’s Adjuvant (CFA) induced mice model. FA-LEF-NPs + A.O loaded hydrogel significantly improved the pharmacological parameters of mice, with a significant reduction of inflammatory cytokines expression. Histopathological studies also illustrated the therapeutic potential of FA-LEF-NPs + A.O loaded hydrogel, without any toxicity profile of liver or lungs. The fabricated hydrogel also displayed an excellent stability profile for a longer period. These all studies confirmed that the FA-LEF-NPs + A.O loaded hydrogel can be a promising and safe therapy to target RA.