Enhanced delivery of cell-penetrating peptide–peptide nucleic acid conjugates by endosomal disruption

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.

Photochemically stimulated drug delivery increases the cytotoxicity and specificity of EGF–saporin

“…PCI in vitro has also been reported as an intracellular delivery system for peptides (Berg et al 1996, Berg et al 1999, PNAs (Shiraishi & Nielsen 2006, siRNA (Oliveira et al 2007) and some chemotherapeutics, such as bleomycin , doxorubicin , Lai et al 2007) and mitoxantrone (Adigbli et al 2007). PCI has been demonstrated in vivo with the protein toxin gelonin (Selbo et al 2001b, a nonviral p53 gene (Ndoye et al 2006), and has also been shown to increase the therapeutic effect of bleomycin .…”

Specific uptake and interactions of peptide nucleic acid derivatives with biomimetic membranes

“…6,8,12 Although the mechanism of translocation of CPPs is not fully understood, their ability to successfully transport cargo (e.g., PNAs) into the cell makes them effective delivery agents. 12,[16][17][18] However, this does not necessarily 35 correlate with an improvement in PNA antisense activity. The issue is that internalization is predominantly via an endocytotic pathway, leading to the PNAs being delivered into an endosomal compartment and thereafter being trapped inside the cell.…”

“…6,11,15,17,[21][22] have shown promising improvements in cellular bioavailability, there is still a need to improve our understanding of cellular uptake for different 50 delivery vectors and to provide better correlations of their in vivo structure-activity relationships.…”

“…Overcoming the poor distribution of PNAs in either the cytoplasm or nucleus, if achieved, could enhance their bioactivity. 4,6,8,13,17,[19][20] Although recent approaches applying endosomolytic agents, such as 45 chloroquine and Ca…”

Glycosylated cell-penetrating peptides and their conjugates to a proapoptotic peptide: preparation by click chemistry and cell viability studies

“…Glycosylation of bioactive compounds has been used mainly for increasing either the hydrophilicity, the enzymatic stability of the compound, and/or its delivery into the brain [22][23][24][25]. In the context of CPPs, we have hypothesized that glycosylation may regulate and or affect the internalization of the corresponding glycosylated analogs of (R6/W3) [26][27][28][29][30]. According to the strategy depicted in Scheme 1, we have synthesized glycosylated analogs of (R6/W3), in which galactose unit(s) linked via click chemistry replace(s) one, two, or the three tryptophans.…”