Development of methotrexate-entrapped ultradeformable liposomes with improved skin permeation for the treatment of rheumatoid arthritis

Abstract

The purpose of this study was to develop methotrexate-entrapped ultradeformable liposomes (MTX-UDLs) with enhanced skin permeation for rheumatoid arthritis therapy. MTX-UDLs were prepared by extrusion method using phosphatidylcholine as a bilayer matrix and sodium cholate or Tween 80 as an edge activator. MTX-UDLs were prepared with different phosphatidylcholine to edge activator ratios. MTX-UDLs were characterized for physicochemical properties including particle size, polydispersity index, zeta potential, and entrapment efficiency. The deformability of MTX-UDLs was determined using steel pressure filter device, and were compared to those of methotrexate-entrapped conventional liposomes (MTX-CLs). The in vitro skin permeation of MTX-UDLs was investigated using Franz diffusion cell. The depth of skin penetration of rhodamine 6G-entrapped UDLs (R6G-UDLs) was determined by confocal laser scanning microscopy. The effects of MTX-UDLs on skin structure was evaluated by differential scanning calorimetry (DSC) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR). The optimized MTX-UDLs (phosphatidylcholine: Tween 80 = 7:3, w/w) were incorporated into 1% Carbopol gel for convenient in vivo skin application. The anti-rheumatic activity of MTX-UDLs-incorporated gel (MTX-UDLs-gel) was investigated in Complete Freund's Adjuvant (CFA) induced arthritis rat model. MTX-UDLs showed particle size of ~100 nm, with narrow size distribution. The deformability index of MTX-UDLs was two to five times higher than those of MTX-CLs. MTX-UDLs showed a significantly improved in vitro skin permeation compared with MTX-CLs and MTX solution. R6G-UDLs with optimized composition demonstrated higher fluorescence intensity at every increment of skin depth than R6G-CLs. DSC thermograms and ATR-FTIR spectra showed that MTX-UDLs did not alter lipid organization of the stratum corneum. MTX-UDLs-gel showed enhanced anti-rheumatic activities with a significant reduction in edema volume and histological scores assigned for paw edema level, leukocyte infiltration and accumulation of neutrophils in CFA induced arthritis rat model. The tissue architecture was also improved and the expression of inflammatory cytokines (TNF-α, and IL-1β) was effectively suppressed in paw tissues after MTX-UDLs-gel treatment. In conclusion, the optimized MTX-UDs could be potential transdermal nanocarriers for effective delivery of MTX to the inflamed joints to alleviate rheumatoid arthritis.