Comprehensive analysis of PEGylated liposome-associated proteins relating to the accelerated blood clearance phenomenon by combination with shotgun analysis and conventional methods.

PEGylated liposome, sterically stabilized by polyethylene glycol (PEG), results in reduced recognition of the liposome by the mononuclear phagocyte system. Recently, we reported regarding the accelerated blood clearance (ABC) phenomenon that PEGylated liposome is cleared very rapidly from blood circulation upon repeated injection. Anti-PEG IgM production and subsequent complement activation were crucial in causing the ABC phenomenon. However, there still remains the possibility that unknown plasma factors might affect the fate of PEGylated liposome that is subjected to the ABC phenomenon. A label-free approach to shotgun analysis is a great tool for characterizing proteins in a biological system. In this study, therefore, a shotgun analysis was employed to identify plasma protein bound on PEGylated liposome after the ABC phenomenon was induced in the mouse model. The analysis revealed that immunoglobulin and complement components (C1 and C3) are the major proteins. Subsequent analysis with enzyme-linked immunosorbent assay and Western blotting showed that the immunoglobulin was IgM and that the complement system was mainly activated via an anti-PEG IgM-mediated classical pathway. These results support our earlier assumptions-anti-PEG IgM and complement activation were the major causes of the ABC phenomenon. Our proposed analytical strategy would be expected to provide useful information for the development and design of the nanocarrier drug delivery system.