A hydroxyl PEG version of PEGylated liposomes and its impact on anti-PEG IgM induction and on the accelerated clearance of PEGylated liposomes.

Surface decoration of liposomes with polyethylene glycol (PEG), PEGylation, is recognized as a method to bestow liposomes with a prolonged circulation time following intravenous administration. However, many reports have emphasized that a first dose of PEGylated liposomes (PL) elicits an anti-PEG IgM antibody response that can trigger a rapid systemic clearance of a second dose of PL via a phenomenon that is referred to as "accelerated blood clearance (ABC)." Such a phenomenon is usually observed with PL that has been modified with methoxy-PEG. In the current study, we introduced various functional groups, methoxy (OCH3), amino (NH2), carboxyl (COOH), and hydroxyl (OH), at the chain ends of PEG to investigate the effect on anti-PEG IgM induction. Among different PEG-modified liposomes, hydroxyl PEG-modified liposomes (PL-OH) efficiently attenuated the anti-PEG IgM response in vitro. In addition, PL-OH was less recognizable by anti-PEG IgM compared with other PLs. These findings raised the possibility that PL-OH could attenuate/abrogate elicitation of the ABC phenomenon. Nonetheless, upon repeated intravenous injection, PL-OH triggered the enhanced clearance of a subsequently injected second dose. Furthermore, in vitro studies have demonstrated that, as a complement activator, PL-OH is stronger than PL-OCH3 and induces further complement activation in the presence of anti-PEG IgM, which was the predominant contributor to the rapid clearance of a second dose of PL-OH. Our results suggest that the screening of complement activation by polymer-modified products in tandem with anti-polymer antibody production should be a prerequisite in the development of polymers that might enhance the therapeutic efficacy of nanocarriers.

Reactivity of IgM antibodies elicited by PEGylated liposomes or PEGylated lipoplexes against auto and foreign antigens.

Polyethylene glycol (PEG) is an attractive tool for the development of nanoparticle-based cancer therapy since it endows nanoparticles with extended-circulation properties. Nevertheless, recent reports have revealed that intravenous injection of either PEGylated liposomes (SLs) or PEGylated lipoplex (PLpx) could elicit an anti-PEG immunoglobulin (IgM) response in a T cell-independent (TI) manner that would substantially compromise the in vivo fate of PEGylated products upon repeated administration. In the same context, viral or bacterial infections trigger the production of polyreactive IgM that binds both self and foreign antigens. The polyreactivity of IgM elicited by SLs or PLpx, to bacteria and other polymers, however, is yet to be elucidated. In this study, the polyreactivity of IgM elicited by SLs or PLpx was challenged against different bacteria (TI antigens) and against synthetic polymer composed of repetitive structures (PVP-360 or FITC-dextran). Results demonstrated that anti-PEG IgM elicited by either SLs or PLpx showed no reactivity to various bacteria examined, while the IgM showed remarkable reactivity to both PVP-360 and FITC-dextran. In addition, interestingly, anti-PEG IgM elicited by either SLs or PLpx showed no antinuclear antibody-like immune reactivity, and, therefore, treatment with either SLs or PLpx was not expected to exacerbate autoimmune diseases such as systemic lupus erythematosus. Collectively, our findings could provide information supporting the safety of PEGylated nanoparticle-based pharmaceutics, particularly in patients with autoimmune diseases.

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.