In vitro immunobiological assays of methotrexate-stearic acid conjugate in human PBMCs.

In the present research, a lipophilic methotrexate (MTX) prodrug was developed by covalent conjugating of MTX to stearic acid (SA) as a lipid moiety via amid bond. The structure of synthesized conjugate, MTX-SA, was confirmed by IR and NMR spectra. To evaluate inflammatory response of MTX-SA conjugate and MTX, human PBMCs were isolated and exposed to 50, 500 and 5000 nM of MTX-SA conjugate and free MTX. The expression of four key genes involved in inflammation and apoptosis including IL-8, IL-1β, IL-10 and Bcl2 depicted that the MTX-SA had controversial behavior in different doses on the inflammatory transcription. Also, MTX-SA statically decreased the number of immune live cells in comparison to MTX. However, MTX-SA did not capture PBMCs cell cycle in G0/G1 phase. Totally, these results showed MTX-SA with long lipid chain has different effect on immune responses and it is irrefutable that detailed studies of its immunotoxicity and immunogenicity ought to be taken into account.