The study planned to estimate biological parameters linked to rheumatoid arthritis (RA) patients, detecting the influence of MTX and biotherapy treatments on these parameters and synthesizing methotrexate bovine serum albumin nanoparticles linked to folate (FA-MTX-BSA NPs) to reduce the overwhelming expression of inflammatory cytokines. Inflammatory parameters showed significant increases in newly diagnosed and MTX-receiving groups while no changes were observed in the biotherapy-maintained group. MTX-loaded BSA nanoparticles were fabricated by the desolvation method and further linked to activated folic acid to obtain FA-MTX-BSA NPs. FA-MTX-BSA NPs were successfully characterized within the nanoscale range using different screening techniques. FA-MTX-BSA NPs showed an in vitro release in a sustained manner. The potential of MTX, MTX-BSA NPs, and FA-MTX-BSA NPs in inducing cytokine level reduction was detected. Significant decreases in interleukin- 1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α) levels were obtained in cultures treated with FA-MTX-BSA NPs compared to the untreated culture in a dose-dependent pattern. Furthermore, FA-MTX-BSA NPs comparing with MTX and MTX-BSA NPs exhibited a significant advanced effect in decreasing cytokines levels. Accordingly, the conjunction of BSA NPs and MTX linked to folate potentially reduced cytokines manifestation in RA.

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