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Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.

Sundaram S, Lee LK, Roth CM - Nucleic Acids Res. (2007)

Bottom Line: Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries.While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry.Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.

ABSTRACT
The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.

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Mean hydrodynamic diameter (nm) of complexes (charge ratio 10: 1, final ON concentration 50 μg/ml in PBS) of PEI with (A) PO and (B) PS ONs. Data represent mean ± SD (n ≥ 3).
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Figure 2: Mean hydrodynamic diameter (nm) of complexes (charge ratio 10: 1, final ON concentration 50 μg/ml in PBS) of PEI with (A) PO and (B) PS ONs. Data represent mean ± SD (n ≥ 3).

Mentions: In order to characterize further the PEI/ON complexes, particle sizes were estimated in the form of mean hydrodynamic diameter using dynamic light scattering. Complexes prepared at various PEI/ON charge ratios in PBS were subjected to particle size measurements for 15 min at a regular interval of 3 min. For charge ratios below 10 : 1, complexes were found to aggregate, as indicated by the rapid increase in particle size (data not shown). Although the OliGreen binding assay indicated PEI-ON association at these charge ratios, stable, submicron sized complexes were formed only at a charge ratio of 10: 1 or above. At a charge ratio of 10 : 1, only 10 K/PO complexes displayed particle aggregation, with the particle diameter increasing from 200 to 450 nm within 15 min. Irrespective of the PEI MW and ON chemistry, all other particles were stable in the presence of salt and maintained a mean diameter of approximately 200 nm (Figure 2). All further studies were therefore performed at a charge ratio of 10 : 1.Figure 2.


Interplay of polyethyleneimine molecular weight and oligonucleotide backbone chemistry in the dynamics of antisense activity.

Sundaram S, Lee LK, Roth CM - Nucleic Acids Res. (2007)

Mean hydrodynamic diameter (nm) of complexes (charge ratio 10: 1, final ON concentration 50 μg/ml in PBS) of PEI with (A) PO and (B) PS ONs. Data represent mean ± SD (n ≥ 3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1935005&req=5

Figure 2: Mean hydrodynamic diameter (nm) of complexes (charge ratio 10: 1, final ON concentration 50 μg/ml in PBS) of PEI with (A) PO and (B) PS ONs. Data represent mean ± SD (n ≥ 3).
Mentions: In order to characterize further the PEI/ON complexes, particle sizes were estimated in the form of mean hydrodynamic diameter using dynamic light scattering. Complexes prepared at various PEI/ON charge ratios in PBS were subjected to particle size measurements for 15 min at a regular interval of 3 min. For charge ratios below 10 : 1, complexes were found to aggregate, as indicated by the rapid increase in particle size (data not shown). Although the OliGreen binding assay indicated PEI-ON association at these charge ratios, stable, submicron sized complexes were formed only at a charge ratio of 10: 1 or above. At a charge ratio of 10 : 1, only 10 K/PO complexes displayed particle aggregation, with the particle diameter increasing from 200 to 450 nm within 15 min. Irrespective of the PEI MW and ON chemistry, all other particles were stable in the presence of salt and maintained a mean diameter of approximately 200 nm (Figure 2). All further studies were therefore performed at a charge ratio of 10 : 1.Figure 2.

Bottom Line: Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries.While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry.Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biochemical Engineering, Rutgers University, 599 Taylor Road, Piscataway, NJ 08854, USA.

ABSTRACT
The widespread utilization of gene silencing techniques, such as antisense, is impeded by the poor cellular delivery of oligonucleotides (ONs). Rational design of carriers for enhanced ON delivery demands a better understanding of the role of the vector on the extent and time course of antisense effects. The aim of this study is to understand the effects of polymer molecular weight (MW) and ON backbone chemistry on antisense activity. Complexes were prepared between branched polyethyleneimine (PEI) of various MWs and ONs of phosphodiester and phosphorothioate chemistries. We measured their physico-chemical properties and evaluated their ability to deliver ONs to cells, leading to an antisense response. Our key finding is that the antisense activity is not determined solely by PEI MW or by ON chemistry, but rather by the interplay of both factors. While the extent of target mRNA down-regulation was determined primarily by the polymer MW, dynamics were determined principally by the ON chemistry. Of particular importance is the strength of interactions between the carrier and the ON, which determines the rate at which the ONs are delivered intracellularly. We also present a mathematical model of the antisense process to highlight the importance of ON delivery to antisense down-regulation.

Show MeSH
Related in: MedlinePlus