Characteristics and Stability Assessment of Therapeutic Methionine γ-lyase-Loaded Polyionic Vesicles

Pyridoxal 5'-phosphate-dependent methionine γ-lyase from Citrobacter freundii (MGL, EC 4.4.1.11) is studied as an antitumor enzyme and in combination with substrates as an antibacterial agent in enzyme pro-drug therapy. For the possibility of in vivo trials, two mutant forms, C115H MGL and V358Y MGL, were encapsulated into polyionic vesicles (PICsomes). Five pairs of polymers with the number of polymer chain units 20, 50, 70, 120, and 160 were synthesized. The effect of polymer length-PEGylated poly-l-aspartic acid and poly-l-lysine-on the degree of MGL incorporation into PICsomes and their size was investigated. Encapsulation of proteins in PICsomes is a rather new technique. Our data demonstrated that the length of the polymers and, therefore, the ratio of the hydrophobic and hydrophilic fragments most likely should be selected individually for each protein to be encapsulated. The efficiency of encapsulation of MGL mutant forms into PICsomes was up to 11%. The hydrodynamic diameter and surface potential of hollow and MGL-loaded PICsomes were evaluated by the dynamic light scattering method. The size and morphology of the PICsomes were determined by atomic force microscopy. The most acceptable for further in vivo studies were PICsomes₂₀ with a size of 57-64 nm, PICsomes₇₀ of 50-90 nm, and PICsomes₁₂₀ of 100-105 nm. The analysis of the steady-state parameters has demonstrated that both mutant forms retained their catalytic properties inside the nanoparticles. The release study of the enzymes from PICsomes revealed that about 50% of the enzymes remained encapsulated in PICsomes₇₀ and PICsomes₁₂₀ after 24 h. Based on the data obtained, the most promising for in vivo studies are PICsomes₇₀ and PICsomes_120.