RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical TechnologyEfficient and High-Speed Transduction of an Antibody into Living Cells Using a Multifunctional Nanocarrier System to Control Intracellular Trafficking
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INTRODUCTION
The high specificity of antibodies explains their extensive use in both basic and applied research for analyzing the expression and function of proteins. However, their use for studying intracellular phenomena in living cells requires a delivery system that can overcome the cell membrane barrier and release functional antibodies into the cytosol. To date, several attempts to transduce antibodies into intact cells have been reported, including mechanical approaches such as electroporation1 and
Materials
Cholesteryl hemisuccinate (CHEMS), amiloride, and Fillipin III were purchased from Sigma (St. Louis, Missouri). 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and rhodamine–DOPE were purchased from Avanti Polar Lipids Inc. (Alabaster, Alaska). Stearyl octaarginine12., 33. and cholesteryl–GALA29., 31. were obtained from Kurabo Industries Ltd. (Osaka, Japan). Goat IgG was purchased from Rockland (Gilbertsville, Pennsylvania). HeLa human cervix carcinoma cells were obtained from RIKEN Cell
Construction of R8-Modified Liposomes Loaded with Antibody and the Cellular Uptake Evaluation
Liposomes (DOPE/CHEMS, 9/2 molar ratio) loaded with non-specific antibodies [Lip(IgG)] were prepared by the lipid film hydration method. Stable liposomes, approximately 200 nm in diameter, were obtained using antibody solutions up to 0.5 mg/mL (Fig. 1a, open bars). The ζ-potential of the carriers was highly dependent on the antibody concentration (Fig. 1b, open symbols). The modification of Lip(IgG) with R8 resulted in an inversion in the ζ-potential from negative to positive values, because the
DISCUSSION
In the analysis of the ζ-potential of liposomes after loading of the antibody solution (Fig. 1b), it was assumed that the antibodies were absorbed on the surface of the liposomes. Empty liposomes (hydrated with HEPES buffer) were highly negative particles (∼ − 70 mV). On the contrary, after the loading of almost neutral antibodies, the apparent surface charge of the liposomes quickly became neutral (Fig. 1b, open symbols). A similar tendency was observed in the case of R8-modified liposomes (
CONCLUSION
Our findings demonstrate that R8-GALA liposomes represent a potentially useful system for the efficient cytosolic delivery of antibodies. The synergistic combination of R8 and GALA overcomes the major barriers to the transduction of antibodies, namely, cellular uptake and endosomal entrapment. This system, characterized by its high speed, ease of preparation, and high performance, provides an alternative tool to continue exploring possibilities in the fields of research with antibodies. Future
ACKNOWLEDGMENTS
This work was supported, in part, by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, Japan (NIBIO), a Grant-in-Aid for Young Scientists (A) and a Grant-in-Aid for Scientific Research (S) from the Ministry of Education, Culture, Sports, Science and Technology of Japanese Government (MEXT). We also thank Dr. Milton Feather for his helpful advice in writing the manuscript.
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Contributed equally to this work.
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