Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

Abstract

Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA-octaarginine conjugate upon varying the cell culture transfection volume (and cell density) at fixed PNA concentration. The results show that for all delivery modalities the cellular antisense activity increases (less than proportionally) with increasing volume (in some cases accompanied with increased toxicity), and that this effect is more pronounced at higher cell densities. These results emphasize that transfection efficacy using cationic carriers is critically dependent on parameters such as transfection volume and cell density, and that these must be taken into account when comparing different delivery regimes.

Effective Cellular Delivery of Antisense Peptide Nucleic Acid by Conjugation to Guanidinylated Diaminobutanoic Acid-Based Peptide Dendrons

“…The cellular delivery capacity of the Dab dendrons was assessed by the antisense splice correction activity of dendron–PNA conjugates in the pLuc705 HeLa cell system. , This HeLa cell line is stably transfected with a luciferase gene carrying the IVS2-705 mutated intron 2 from a thalassemic human β-globin gene producing an aberrantly spliced mRNA. Untreated cells express only very low levels of functional luciferase, while blocking the aberrant splice site by an antisense PNA leads to splice correction and restoration of luciferase expression and activity (Figure S1).…”

“…A SAR study of the cellular delivery properties of the dendrons was performed based on the antisense splice correction efficiency of dendron–peptide nucleic acid (PNA) conjugates in the HeLa pluc705 human cell culture system. − …”

Inhibiting mutant KRAS G12D gene expression using novel peptide nucleic acid‑based antisense: A potential new drug candidate for pancreatic cancer

“…As demonstrated, lipofectamine effectively and successfully transfected FITC-fluorescently labeled PNA into the cytoplasm of cells. Lipofectamine is a cationic, lipid-based chemical transfectant which forms liposomes that complex with negatively charged nucleic acids, and, through the endocytosis pathway, deliver DNA or RNA into the cell cytosol (47). Despite their positive charge, the PNAs were efficiently delivered into cells with the use of cationic lipofectamine, as demonstrated by florescence measurements and microscopy.…”

Nanoscale Phytosomes as an Emerging Modality for Cancer Therapy

“…Similarly, in our recent study, we utilized lipofectamine, a lipid-based, liposome-forming transfectant, to complex with peptide nucleic acid (PNA)-based antisense. Through the endocytosis pathway, the liposomes enhanced the intracellular delivery of the PNAs and, thereby, inhibited the proliferation and induced apoptosis of pancreatic cancer cells [77]. Since lipofectamine is only used for research purposes for transfection of nucleic acids into eukaryotic cells, future studies will focus on the delivery of PNA-based treatments by means of oral or intravenous phytosome formulations.…”