Dataset Information

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes.

ABSTRACT: There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by some form of endocytosis; however, the mechanism for delivery of small oligonucleotide molecules has not been well established. In this study, splice-shifting oligonucleotides have been formulated into cationic lipoplexes and polyplexes, and their internalization mechanisms have been examined by using pharmacological and genetic inhibitors of endocytosis. The results showed that intercellular distribution of the oligonucleotides to the nucleus governs their pharmacological response. A mechanistic study revealed that oligonucleotides delivered by lipoplexes enter the cells partially by membrane fusion and this mechanism accounts for the functional induction of the target gene. In contrast, polyplexes are internalized by unconventional endocytosis pathways that do not require dynamin or caveolin. These studies may help rationally design novel delivery systems with superior transfection efficiency but lower toxicity.

SUBMITTER: Ming X

PROVIDER: S-EPMC3133857 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes.

Ming Xin X Sato Katsuya K Juliano Rudolph L RL

Journal of controlled release : official journal of the Controlled Release Society 20110504 1

There is mounting interest in developing antisense and siRNA oligonucleotides into therapeutic entities; however, this potential has been limited by poor access of oligonucleotides to their pharmacological targets within cells. Transfection reagents, such as cationic lipids and polymers, are commonly utilized to improve functional delivery of nucleic acids including oligonucleotides. Cellular entry of large plasmid DNA molecules with the assistance of these polycationic carriers is mediated by s ...[more]

PMID: 21571016

Similar Datasets

Project description:Lipoplexes (LDs) have been prepared from DNA and positively charged vesicles composed of the helper lipid, dioleoyl l-?-phosphatidylethanolamine (DOPE) and either a dichain (DC) oxyethylated cationic lipid or their corresponding novel trichain (TC) counterpart. This is the first study using the TC lipids for the preparation of LDs and their application. Here the results of biophysical experiments characterising the LDs have been correlated with the in vitro transfection activity of the complexes. Photon correlation spectroscopy, zeta potential measurements and transmission electron microscopy studies indicated that, regardless of the presence of a third chain, there were little differences between the size and charge of the TC and DC containing LDs. Small angle neutron scattering studies established however that there was a significant conformational re-arrangement of the lipid bilayer when in the form of a LD complex as opposed to the parent vesicles. This re-arrangement was particularly noticeable in LDs containing TC lipids possessing a third chain of C12 or a longer chain. These results suggested that the presence of a third hydrophobic chain had a significant effect on lipid packing in the presence of DNA. Picogreen fluorescence and gel electrophoresis studies showed that the TC lipids containing a third acyl chain of at least C12 were most effective at complexing DNA while the TC lipids containing an octanoyl chain and the DC lipids were least effective. The transfection efficacies of the TC lipids in the form of LDs were found to be higher than for the DC analogues, particularly when the third acyl chain was an octanoyl or oleoyl moeity. Little or no increase in transfection efficiency was observed when the third chain was a methyl, acetyl or dodecanoyl group. The large enhancement in transfection performance of the TC lipids can be attributed to their ability to complex their DNA payload. These studies indicate that presence of a medium or long third acyl chain was especially beneficial for transfection.

Project description:Synthetic nucleic acids have shown great potential in the treatment of various diseases. Nevertheless, the selective delivery to a target tissue has proved challenging. The coupling of nucleic acids to targeting peptides, proteins, and antibodies has been explored as an approach for their selective tissue delivery. Nevertheless, the preparation of covalently coupled peptides and proteins that can also undergo intracellular release as well as deliver more than one copy of the nucleic acid has proved challenging. Recently, we have developed a novel method for the rapid noncovalent conjugation of nucleic acids to targeting single chain antibodies (scFv) using chemically self-assembled nanostructures (CSANs). CSANs have been prepared by the self-assembly of two dihydrofolate reductase molecules (DHFR(2)) and a targeting scFv in the presence of bis-methotrexate (bis-MTX). The valency of the nanorings can be tuned from one to eight subunits, depending on the length and composition of the linker between the dihydrofolate reductase molecules. To explore their potential for the therapeutic delivery of nucleic acids as well as the ability to expand the capabilities of CSANs by incorporating smaller cyclic targeting peptides, we prepared DHFR(2) proteins fused through a flexible peptide linker to cyclic-RGD, which targets ?v?3 integrins, and a bis-MTX chemical dimerizer linked to an antisense oligonucleotide (bis-MTX-ASO) that has been shown to silence expression of eukaryotic translation initiation factor 4E (eIF4E). Monomeric and multimeric cRGD-CSANs were then prepared with bis-MTX-ASO and shown to undergo endocytosis in the breast cancer cell line, MDA-MB-231, which overexpresses ?v?3. The bis-MTX-ASO was shown to undergo endosomal escape resulting in the knock down of eIF4E with at least the same efficiency as ASO delivered by oligofectamine. The modularity, flexibility, and common method of conjugation may prove to be a useful general approach for the targeted delivery of ASOs, as well as other nucleic acids to cells.

Dataset Information

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes.

Publications

Unconventional internalization mechanisms underlying functional delivery of antisense oligonucleotides via cationic lipoplexes and polyplexes.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets