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Updated: Aug 11, 2026
Generation of Cationic Nanoliposomes for the Efficient Delivery of In Vitro Transcribed Messenger RNA
Published on: February 1, 2019
Delivery of phosphodiester oligonucleotides: can DOTAP/DOPE liposomes do the trick?
K Remaut1, B Lucas, K Braeckmans
1Laboratory of General Biochemistry and Physical Pharmacy, Ghent University, Harelbekestraat 72, 9000 Ghent, Belgium.
Abstract:
Delivering phosphodiester ONs (PO-ONs) remains an attractive but challenging goal in antisense therapy. Both in the literature and in our experiments, most cationic liposomes fail in generating an antisense effect with PO-ONs, while they succeed with chemically modified ONs such as phosphothioate ONs (PS-ONs). This work aims to explain the biological activity of PO- and PS-ONs delivered by DOTAP/DOPE liposomes based on a detailed understanding of their cell biological behavior by means of fluorescence correlation spectroscopy and confocal laser scanning microscopy. We conclude that DOTAP/DOPE liposomes are not suited to deliver PO-ONs due to the release of naked PO-ONs in the cytosol at the time of the endosomal escape of the liposomes and the subsequent rapid degradation of the naked PO-ONs. Carriers that would not release the PO-ONs upon endosomal escape but would continue to carry the PO-ONs until they arrive at the target mRNA could therefore be better suited to delivering PO-ONs. In the case of PS-ONs, the ONs are not degraded upon release at the time of the endosomal escape of the liposomes, creating a pool of intact, biologically active PS-ONs and thus making DOTAP/DOPE liposomes mainly suitable for delivering nuclease resistant ONs. However, the cells seemed to display an export pathway for removing intact PS-ONs from the cells, limiting the presence of naked PS-ONs in the nucleus to approximately 8 h following the delivery.
Insights
DOTAP/DOPE liposomes fail to deliver phosphodiester oligonucleotides (PO-ONs) due to rapid degradation after endosomal escape. They are more suitable for delivering nuclease-resistant phosphothioate oligonucleotides (PS-ONs), though cellular export limits their nuclear presence.
Area of Science:
- Biochemistry
- Molecular Biology
- Drug Delivery Systems
Background:
- Antisense therapy aims to deliver oligonucleotides (ONs) to modulate gene expression.
- Phosphodiester ONs (PO-ONs) are attractive but challenging to deliver effectively.
- Cationic liposomes like DOTAP/DOPE often fail with PO-ONs but succeed with modified ONs (PS-ONs).
Purpose of the Study:
- To elucidate the cell biological behavior of PO- and phosphothioate ONs (PS-ONs) delivered by DOTAP/DOPE liposomes.
- To understand why DOTAP/DOPE liposomes are ineffective for PO-ON delivery in antisense therapy.
- To compare the cellular fate and activity of PO-ONs versus PS-ONs using DOTAP/DOPE liposomes.
Main Methods:
- Utilized fluorescence correlation spectroscopy (FCS) to study ON behavior within cells.
- Employed confocal laser scanning microscopy (CLSM) for visualizing ON and liposome interactions.
- Investigated the cellular uptake, endosomal escape, and degradation pathways of delivered ONs.
Main Results:
- DOTAP/DOPE liposomes release naked PO-ONs during endosomal escape, leading to their rapid degradation.
- PS-ONs, upon release, are resistant to degradation, forming an active pool within cells.
- Cellular export mechanisms limit the nuclear accumulation of intact PS-ONs to approximately 8 hours.
Conclusions:
- DOTAP/DOPE liposomes are unsuitable for delivering PO-ONs due to premature release and degradation.
- These liposomes are primarily effective for delivering nuclease-resistant ONs like PS-ONs.
- Future carriers should prevent PO-ON release until target mRNA interaction to enhance antisense therapy efficacy.
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