RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Efficient and High-Speed Transduction of an Antibody into Living Cells Using a Multifunctional Nanocarrier System to Control Intracellular Trafficking

https://doi.org/10.1002/jps.24310Get rights and content

ABSTRACT:

The transduction of antibodies into living cells would represent a major contribution to both basic and applied biomedical fields, as currently available methods suffer from limitations such as low-uptake efficiency and endosomal entrapment. In this study, a liposome-based carrier was designed to overcome these issues. Liposomes were modified with octaarginine (R8), a cell penetrating peptide and GALA, a pH-sensitive fusogenic peptide. The presence of R8 enhanced the cellular uptake of antibodies, whereas GALA reduced endosomal entrapment, resulting in antibodies being released into the cytosol within 30 min. Moreover, compared with commercially available reagents for delivering antibodies, our system was superior in both cellular uptake and endosomal escape. In addition, specific antibodies delivered by R8-GALA liposomes were found to be associated with their epitope, confirming the preservation of functionality. This system for the efficient and high-speed cytosolic delivery of an antibody provides a valuable tool that can be useful in basic and applied research for analyzing the expression and function of intracellular molecules. © 2014 Wiley Periodicals, Inc. and the American Pharmacists Association.

Section snippets

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.

REFERENCES (40)

Cited by (16)

  • Antibody nanocarriers for cancer management

    2021, Current Opinion in Biomedical Engineering
    Citation Excerpt :

    Liposomes have shown promise not just for extracellular antibody delivery but also for intracellular delivery. In one notable development, antibody-loaded liposomes were modified with octaarginine (R8), a cell-penetrating peptide, and GALA, a pH-sensitive fusogenic peptide [55], to overcome cellular and endosomal membrane barriers to intracellular delivery. This system demonstrated reduced endosomal entrapment and preserved antibody functionality after in vitro delivery to HeLa cervical cancer cells.

  • Harnessing SLE Autoantibodies for Intracellular Delivery of Biologic Therapeutics

    2021, Trends in Biotechnology
    Citation Excerpt :

    To overcome endosomal entrapment of these complexes, endosomal escape enhancers could be introduced [44,64–66]. One example includes fusion with pH-sensitive GALA peptide, which becomes activated in the acidic endosomal environment and causes a breach in the endosomal membrane [67]. Since carriers have the added risk of immunogenicity and problematic pharmacokinetics implicating accumulation in the liver, no preferred method for safe delivery has emerged as yet.

  • Strategies to target bioactive molecules to subcellular compartments. Focus on natural compounds

    2019, European Journal of Medicinal Chemistry
    Citation Excerpt :

    The same group also pioneered the “decoration” of liposomes with the TPP group (incorporated as stearyl-TPP) to confer mitochondriotropic behaviour [254,255]. Mito-Porter, i.e. liposomes decorated with octa-Arg and/or other (leader) peptides [256,257] which has evolved into multifunctional envelope-type nano devices (MEND) [258–260], can also deliver DNA [261], siRNA [262] and small (e.g [263]) as well as large [264] molecules to the mitochondrial matrix in cultured cells. The MEND concept incorporates into the vehicle multiple components capable of facilitating evasion of detection by the reticuloendothelial system, interaction with the cell membrane and internalization, escape from the endosomal compartment, fusion with the mitochondria or other target subcellular compartment.

  • Endosomal acidic pH-induced conformational changes of a cytosol-penetrating antibody mediate endosomal escape

    2016, Journal of Controlled Release
    Citation Excerpt :

    Furthermore, the tight association of antibodies with the antigen receptor hampers their recycling or endosomal escape, causing lysosomal degradation, as exploited in antibody-drug conjugate technology [4]. To overcome these limitations of antibody itself, antibody delivery systems into the cytosol of living cells, such as polymeric micelles [6] and cell-penetrating and fusogenic peptide-embedded liposomes [7], have been recently developed. Some bacterial toxins and cell-penetrating peptides (CPPs) that are derived from natural proteins or have been designed have the ability to reach the cytosol of cells after endocytosis from outside of cells [2,3].

View all citing articles on Scopus

Contributed equally to this work.

This article contains supplementary material available from the authors upon request or via the Internet at http://onlinelibrary.wiley.com/.

View full text