Abstract
Cellular uptake and antisense activity of d-octaarginine conjugated peptide nucleic acids (PNAs) is shown to exhibit pronounced cooperativity in serum-containing medium, in particular by being enhanced by analogous mis-match PNA–cell-penetrating peptide (PNA–CPP) conjugates without inherent antisense activity. This cooperativity does not show cell or PNA sequence dependency, suggesting that it is a common effect in cationic CPP conjugated PNA delivery. Interestingly, our results also indicate that Deca-r8-PNA and r8-PNA could assist each other and even other non-CPP PNAs as an uptake enhancer agent. However, the peptide itself (without being attached to the PNA) failed to enhance uptake and antisense activity. These results are compatible with an endosomal uptake mechanism in which the endocytosis event is induced by multiple CPP–PNA binding to the cell surface requiring a certain CPP density, possibly in terms of nanoparticle number and/or size, to be triggered. In particular the finding that the number of endosomal events is dependent on the total CPP–PNA concentration supports such a model. It is not possible from the present results to conclude whether endosomal escape is also cooperatively induced by CPP–PNA.
Highlights
The DNA mimic peptide nucleic acid (PNA) has shown promising properties for development of efficient mRNA splice modulation antisense agents in a range of cellular systems and in in vivo studies [1,2,3,4,5,6,7]
A previous study in serum-free medium during delivery has shown that enhanced cellular antisense activity can be obtained by attaching a lipid domain to cell-penetrating peptides (CPPs)–PNAs forming
The increased aggregation and cellular uptake was directly correlated with increased cellular antisense activity (Figure 7C). These results strongly suggest that the observed cooperative cellular antisense activity may at least in part be ascribed to increased cellular uptake in terms of endocytosis events driven by PNA-conjugate aggregation/nanoparticle formation
Summary
The DNA mimic peptide nucleic acid (PNA) has shown promising properties for development of efficient mRNA splice modulation antisense agents in a range of cellular systems and in in vivo studies [1,2,3,4,5,6,7]. Like antisense agents in general PNAs are not readily taken up by eukaryotic cells. Cationic lipid complexes are employed as effective vectors to deliver negatively charged antisense oligonucleotides (ASOs), while delivery of PNA using cationic lipid formulations requires conjugation to lipophilic or anionic domains, or prehybridization to a carrier oligonucleotide (e.g., DNA) [9,10]. Cellular uptake can be dramatically enhanced by chemical conjugation to cell-penetrating peptides (CPPs) (e.g., Tat, Penetratin, or oligoarginines) [12]
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