Cellular Delivery

It was clear from the onset of PNA research that cellular delivery was not trivial, as the PNA molecule is a largely hydrophilic, "non-small" molecule (typically with a molecular weight of 2,000-3,000 Da for antisense use). Therefore it is no surprise that simple PNA oligomers do not passively cross the lipid bilayer membrane of living cells (Wittung et al. 1995). It is in some cases possible to attain biological cellular effects of unmodified PNAs, but extremely high concentrations are then required (20-100 pM; Kaushik et al. 2002). Consequently, a delivery vehicle or system is desired for effective cellular delivery of antisense PNA, and a large variety of such agents and systems is now available. Broadly, they may be divided into two categories that depend on whether or not they require cationic lipids (liposomes). Cationic liposomes constitute the effective delivery vehicle of choice for anionic oligonucleotides, such as phosphodiester antisense agents and siRNA, but they are not effective for PNA delivery because these oligomers are not anionic, and therefore do not "self-assemble" with the cationic lipids. However, by forming a quasi-stable duplex with a partly complementary DNA oligonucleotide, quite effectively deliver the PNA-DNA complex into the cell, where the PNA following dissociation from the DNA may hybridize to the RNA (or DNA) target (Hamilton et al. 1999). This type of PNA delivery has been used extensively, in particular by the Corey group (e.g. Doyle et al. 2001; Liu et al. 2004). PNA oligomers conjugated to fatty acids can also be delivered via cationic lipids, but the conjugates have poor aqueous solubility and tend to aggregate (Ljungstrom et al. 1999). However, it was recently discovered that PNA acridine conjugates are quite efficiently delivered by cationic lipids (Shiraishi and Nielsen 2004). Due to the protonation of the acridine at neutral pH, these compounds do not in general suffer from the severe solubility problems exhibited by the fatty acid conjugates.

A larger number of especially cationic peptides have been conjugated to PNA oligomers and reported to enhance "spontaneous" uptake significantly or even dramatically (Koppelhus et al. 2002; Lundberg and Langel 2003; Kilk et al. 2004; Kaihatsu et al. 2004). Originally it was reported that peptides such as pTat (RQIKIWFQNRRMKWKK, part of the Tat protein of HIV), pAntp (GRKKRRQRRRPPQ, part of the Drosophila antennapedia transcription factor and oligo arginine (e.g. Arg9)) are taken up by cells via an energy-independent and non-carrier-mediated pathway. However, accumulating recent evidence clearly and unanimously points to the conclusion that the major port of cellular entry by these peptides is the endosomal/lysosomal pathway (Koppelhus et al. 2002; Gait 2003). Therefore, such PNA-peptide conjugates are indeed taken up effectively by the cells, but the majority of the material remains inactive in the endosomes/lysosomes from which they are only slowly and poorly released into the cytoplasm. Eventually most of the material is discarded from the cells via the lysosomal excretion. Consequently, it should be possible to increase the potency of such conjugates significantly by agents that are known to disrupt endosomes and/or lysosomes, such as chloroquine or certain photosensitizers (Folini et al. 2003). Specific cellular targeting is also possible using e.g. cancer cell-specific peptides (Mier et al. 2003) or other ligands, such as carbohydrates, that bind to specific cell receptors (van Rossenberg et al. 2003; Hamzavi et al.

2003). However, although it has not been demonstrated, this type of delivery is also expected to occur via endocytosis. Most interestingly, it was also recently reported that organic, lipophilic cations such as triphenylphosphonium are also facilitating cellular uptake when conjugated to PNA (Filipovska et al.

2004). Finally, it is noteworthy that a quite simple synthetic cationic peptide [(KFF)3K] rather effectively delivers short PNAs (10-12 bases) to bacterial cells (Escherichia coli and to a certain extent also to Staphylococcus aureus) (Good et al. 2001; Nekhotiaeva et al. 2004). Using such systems, it may be possible to develop novel antibacterial drugs.

Nonetheless—even with the less-than-perfect delivery systems discovered to date and developed for PNA as described above—it is indeed possible to perform efficacy studies in cells in culture in order to optimize the gene and sequence target (gene screening and gene walk), and typically sub- to low micromolar concentrations of PNA are required to observe (molecular) biological effects.

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