Quality-by-Design (QbD) Assisted Synthesis of Nanoparticle for Efficient Loading, Stabilization, and Intracellular Delivery of Bioactive for the Treatment of Arthritis

Rheumatoid arthritis (RA) is an autoimmune disorder that induces joint inflammation, cartilage injury, and bone damage. Thus far, methotrexate (MTX) is a primary DMARD drug to treat RA. Despite high efficiency, its clinical application is compromised due to delivery-associated systemic side effects. This investigation reports a Quality-by-design (QbD; Box Behnken Strategy) assisted production of a novel, innovative, and multipurpose polycation-templated approach for producing stable albumin MTX nanoparticles (pT-AMTX-NP). This approach formed a highly biocompatible MTX formulation with reduced toxicity (1.81 ± 0.54% hemolysis) compared to plain MTX (13.19 ± 2.77%; SEM:1.965). pT-AMTX-NP was found to be nanometric (Particle size: 135.86 ± 5.17 nm; PDI: 0.27) with a net surface charge of ζ –10.15 ± 2.19 mV. With 4.01-fold cationization (TNBS assay), pT-AMTX-NP showed high drug loading (64.98 ± 1.25%) and sustained MTX release under physiological conditions (up to 48 h; p < 0.001). The nanoformulation followed the Higuchi release kinetics model (R², 0.9957). Significantly reduced burst release by 70.61% (p = 0.0002) and 12.89% (p = 0.0115) compared to plain MTX and conventional MTX-formulation (AMTX-NP), respectively. Interestingly, pT-AMTX-NP showed pH-responsive drug release bio-environment-responsive architectural change. Cell line studies in lipopolysaccharide (LPS) stimulated RAW264.7 macrophage showed a significant reduction in intracellular nitrite level following pT-AMTX-NP treatment (p < 0.01). Cellular uptake and cell viability confirmed selective cellular uptake potential in inflamed cells. Furthermore, compared to the control, the high intracellular ROS-generation was noted with pT-AMTX-NP (2.1485-fold; p < 0.01). Furthermore, hemolysis assay and stability assessments were also conducted to determine the hemocompatibility and suitable conditions for the storage of nanoformulation. The outcome of this study suggests that the developed multipurpose nanoformulation is a superior therapeutics approach for improved RA treatment. Suggestively, the developed strategy can also be adopted to benefit other clinical situations that demand to counter inflammation, cytostatic as well as psoriatic conditions.