Water-soluble, meso-substituted cationic porphyrins--a family of compounds for cellular delivery of oligonucleotides.

The delivery of oligonucleotides to appropriate intracellular compartments is crucial to their development as tools in gene function studies and as therapeutics. Here, we report the characterization of meso-substituted cationic porphyrins as a large class of water-soluble reagents for oligonucleotide delivery. These porphyrins form non-covalent complexes with single-stranded oligonucleotides and deliver these molecules into the nuclei of cell lines in culture. The porphyrins protect oligonucleotides from nuclease degradation, and delivery is unaffected by the presence of serum. Delivery capacity is dependent on the charge ratio and concentration of the oligonucleotide and porphyrin used to form the complex, on the chemical substituents of the oligonucleotide and on the identity of the cationic porphyrin. This class of molecules provides a versatile set of water-soluble delivery reagents that could contribute to the development of oligonucleotide drugs.

Activation of protein kinase C triggers its ubiquitination and degradation.

Treatment of cells with tumor-promoting phorbol esters results in the activation but then depletion of phorbol ester-responsive protein kinase C (PKC) isoforms. The ubiquitin-proteasome pathway has been implicated in regulating the levels of many cellular proteins, including those involved in cell cycle control. We report here that in 3Y1 rat fibroblasts, proteasome inhibitors prevent the depletion of PKC isoforms alpha, delta, and epsilon in response to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Proteasome inhibitors also blocked the tumor-promoting effects of TPA on 3Y1 cells overexpressing c-Src, which results from the depletion of PKC delta. Consistent with the involvement of the ubiquitin-proteasome pathway in the degradation of PKC isoforms, ubiquitinated PKC alpha, delta, and epsilon were detected within 30 min of TPA treatment. Diacylglycerol, the physiological activator of PKC, also stimulated ubiquitination and degradation of PKC, suggesting that ubiquitination is a physiological response to PKC activation. Compounds that inhibit activation of PKC prevented both TPA- and diacylglycerol-induced PKC depletion and ubiquitination. Moreover, a kinase-dead ATP-binding mutant of PKC alpha could not be depleted by TPA treatment. These data are consistent with a suicide model whereby activation of PKC triggers its own degradation via the ubiquitin-proteasome pathway.

Delivery of oligoribonucleotides to human hepatoma cells using cationic lipid particles conjugated to ferric protoporphyrin IX (heme).

The receptor-ligand interaction between hepatocyte heme receptors and heme was evaluated as a basis for developing a targeted cationic lipid delivery reagent for nucleic acids. Heme (ferric protoporphyrin IX) was conjugated to the aminolipid dioleoyl phosphatidylethanolamine (DOPE) and used to form cationic lipid particles with dioleoyl trimethylammonium propane (DOTAP). These lipids particles (DDH) protect oligoribonucleotides from degradation in human serum and increase oligoribonucleotide uptake into 2.2.15 human hepatoma cells (to a level of 50-60 ng oligo/10(4) cells) when compared with the same lipid particles (DD) prepared identically without heme. The DDH heme level that was optimal for oligoribonucleotide delivery was also optimal for maximum expression of plasmid-encoded luciferase. The enhancing effect of heme was evident only at net particle negative charge. Fluorescence microscopy showed that DDH delivered oligoribonucleotides into both the 2.2.15 cell cytoplasm and nucleus. DDH may thus be a potentially useful delivery vehicle for oligonucleotide-based therapeutics and transgenes, appropriate for use in such liver diseases as viral hepatitis, hepatoma, and hypercholesterolemia.