Cell-Dependent Differential Cellular Uptake of PNA, Peptides, and PNA-Peptide Conjugates

Abstract

Peptide nucleic acid (PNA) oligomers were conjugated to cell-penetrating peptides: pAnt, a 17-residue fragment of the Drosophila protein Antennapedia, and pTat, a 14-amino acid fragment of HIV protein Tat. A 14-mer PNA was attached to the peptide by disulfide linkage or by maleimide coupling. The uptake of (directly or indirectly, via biotin) fluorescein-labeled peptides, PNAs, or PNA-peptide conjugates was studied by fluorescence microscopy, confocal laser scanning microscopy, and fluorometry in five cell types. In SK-BR-3, HeLa, and IMR-90 cells, the PNA-peptide conjugates and a T1, backbone-modified PNA were readily taken up (2 microM). The PNA was almost exclusively confined to vesicular compartments in the cytosol. However, the IMR-90 cells also showed a weak diffuse staining of the cytoplasm. In the U937 cells, we observed a very weak and exclusively vesicular staining with the PNA-peptide conjugates and the T(lys)-modified PNA. No evident uptake of the unmodified PNA was seen. In H9 cells, both peptides and the PNA-peptide conjugates quickly associated with the membrane, followed by a weak intracellular staining. A cytotoxic effect resulting in artificial staining of the cells was observed with fluoresceinated peptides and PNA-peptide conjugates at concentrations above 5-10 microM, depending on cell type and incubation time. We conclude that uptake of PNAs in many cell types can be achieved either by conjugating to certain peptides or simply by charging the PNA backbone using lysine PNA units. The uptake is time, temperature, and concentration dependent and mainly endocytotic. Our results also show that proper controls for cytotoxicity should always be carried out to avoid misinterpretation of visual data.

Metal-containing peptide nucleic acid conjugates

“…Up to now, numerous studies were undertaken not only with the aim to get more insight into the transport mechanisms of CPP-PNA conjugates but also for improved understanding of the nature of CPPs that can be employed. [90][91][92][93] The conjugation to CPPs was found to efficiently improve cell penetration of PNAs. However, the cellular uptake was usually dominated by an endosomal pathway with poor endosomal escape.…”

Combination of a new generation of PNAs with a peptide-based carrier enables efficient targeting of cell cycle progression

“…[13][14][15] In order to improve delivery of PNAs into cells and to reduce the doses required for detectable effects, several chemical modifications based on covalently linked cell-penetrating peptides have been successfully used. 10,12,[16][17][18] Linkage of nuclear localization or polylysine sequences to PNAs have also been reported to improve both their cellular internalization and nuclear translocation. 14,19,20 Moreover, it has recently been shown that the cellular uptake of PNAs depends on the cell type and that a carrier system is required for efficient uptake.…”

“…14,19,20 Moreover, it has recently been shown that the cellular uptake of PNAs depends on the cell type and that a carrier system is required for efficient uptake. 18 The solubility of PNAs and their tendency to self-aggregate are important factors to be considered for the biological use of PNAs. [21][22][23] PNA solubility is often sequence-dependent, which usually leads to several restrictions in PNA probe design and can be improved by incorporation of Lys residues or ethylene glycol linkers (eg1 ¼ 8 amino-2-6 dioxaoctanoic acid).…”

Cell-penetrating peptides for nanomedicine – how to choose the right peptide

“…Koppelhaus et al, for example, investigated Tat-and penetratin-mediated cellular uptake of peptide nucleic acid oligomers in five different cell lines (HeLa, SK-BR-3, IMR-90, H9 and U937) demonstrating poor or no uptake at all [44]. The authors concluded that the efficiency of the internalization largely depends on the composition of the membrane (membrane-bound components and lipids).…”