Abstract
Improvement of cellular uptake and cellular localization is still one of the main obstacles to the development of antisense-antigene therapeutics, including peptide nucleic acid (PNA). Cell-penetrating peptides (CPPs) such as Tat peptide and polyarginine have been widely used to improve the cellular uptake of PNA and other antisense agents. Cellular uptake of most CPP conjugates occurs mainly through endocytotic pathways, and most CPP conjugate is retained in the endosomal compartments of the cell. Several methods to induce endosome disruption have been shown to improve the bioavailability of CPP conjugates to the cytosol and/or nucleus by facilitating escape from the endosomal compartments. Here we describe protocols for the delivery of CPP-PNA conjugates to adherent cultured cells using photodynamic treatment (photochemical internalization), Ca2+ treatment or chloroquine treatment to potentiate the antisense effects of CPP-PNA conjugates through increased release of CPP conjugates into the cytoplasm. This protocol, consisting of CPP-mediated delivery assisted by an endosome-disruption agent, allows the delivery of the CPP-PNA conjugates to the nucleus and/or cytosol of cultured cells. The endosome-disruption treatment improves the nuclear antisense effects of CPP-PNA conjugates by up to two orders of magnitude using 24-hour delivery.
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References
Gait, M.J. Peptide-mediated cellular delivery of antisense oligonucleotides and their analogues. Cell. Mol. Life Sci. 60, 844–853 (2003).
Koppelhus, U. & Nielsen, P.E. Cellular delivery of peptide nucleic acid (PNA). Adv. Drug Deliv. Rev. 55, 267–280 (2003).
Dietz, G.P. & Bahr, M. Delivery of bioactive molecules into the cell: the Trojan horse approach. Mol. Cell. Neurosci. 27, 85–131 (2004).
Zorko, M. & Langel, U. Cell-penetrating peptides: mechanism and kinetics of cargo delivery. Adv. Drug Deliv. Rev. 57, 529–545 (2005).
Kaihatsu, K., Braasch, D.A., Cansizoglu, A. & Corey, D.R. Enhanced strand invasion by peptide nucleic acid-peptide conjugates. Biochemistry 41, 11118–11125 (2002).
Turner, J.J. et al. Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Res. 33, 6837–6849 (2005).
Shiraishi, T., Pankratova, S. & Nielsen, P.E. Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic Tat and oligoarginine peptides. Chem. Biol. 12, 923–929 (2005).
Abes, S. et al. Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. J. Control. Release 110, 595–604 (2006).
Shiraishi, T. & Nielsen, P.E. Photochemically enhanced cellular delivery of cell penetrating peptide-PNA conjugates. FEBS Lett. 580, 1451–1456 (2006).
Kang, S.H., Cho, M.J. & Kole, R. Up-regulation of luciferase gene expression with antisense oligonucleotides: implications and applications in functional assay development. Biochemistry 37, 6235–6239 (1998).
Heasman, J. Morpholino oligos: making sense of antisense? Dev. Biol. 243, 209–214 (2002).
Christensen, L. et al. Solid-phase synthesis of peptide nucleic acids. J. Pept. Sci. 1, 175–183 (1995).
Koppelhus, U. et al. Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense Nucleic Acid Drug Dev. 12, 51–63 (2002).
Awasthi, S.K. & Nielsen, P.E. Synthesis of PNA-peptide conjugates. Methods Mol. Biol. 208, 43–52 (2002).
Acknowledgements
This work was supported by the Danish Cancer Research Foundation and the European Union commission through the 6th framework EMIL (European Molecular Imaging Laboratories) network of excellence.
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T.S. did the experimental work; T.S. and P.E.N. designed the experiments and interpreted the experimental data.
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Shiraishi, T., Nielsen, P. Enhanced delivery of cell-penetrating peptide–peptide nucleic acid conjugates by endosomal disruption. Nat Protoc 1, 633–636 (2006). https://doi.org/10.1038/nprot.2006.92
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DOI: https://doi.org/10.1038/nprot.2006.92
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