Sub-cellular temporal and spatial distribution of electrotransferred LNA/DNA oligomer

Area of Science:

• Biotechnology
• Molecular Biology
• Drug Delivery

Background:

• RNA interference (RNAi) therapy faces challenges with low biological activity and inefficient in vivo delivery.
• Optimizing oligonucleotide chemistry and delivery methods are crucial for advancing RNAi-based therapies.

Purpose of the Study:

• To evaluate the electropulsation technique (EP) for efficient cellular delivery of chemically-modified locked nucleic acid (LNA)/DNA oligomers.
• To investigate the intracellular trafficking and localization of LNA/DNA oligomers delivered via EP.

Main Methods:

• Utilized electropulsation (EP) for cellular delivery of cyanine 5 (Cy5)-labeled LNA/DNA oligomers into HeLa cells.
• Employed single-cell confocal fluorescence microscopy to track oligomer distribution.
• Used HeLa GFP-Rab7 cell lines and specific organelle markers to identify intracellular compartments.

Main Results:

• EP facilitated rapid cellular uptake of LNA/DNA oligomers, enabling direct access to the cytoplasm and nucleus.
• Delivered oligomers rapidly translocated from cytoplasm to nucleus within minutes.
• EP bypassed the endocytic pathway, unlike non-EP delivery which entered cells via endocytosis.
• Oligomers delivered without EP showed accumulation in cytoplasmic organelles over time.

Conclusions:

• Electropulsation is an effective method for delivering LNA-based oligonucleotides.
• EP offers significant advantages by avoiding endolysosomal entrapment.
• This technique provides rapid, unimpeded access of oligonucleotides to the cytoplasm and nucleus for therapeutic targeting.