Quantitative analysis of oligonucleotide delivery into isolated mitochondria: a proof-of-concept method

Area of Science:

• Mitochondrial biology
• Drug delivery systems
• Molecular therapeutics

Background:

• Mitochondria are key targets for nucleic acid therapies due to their DNA.
• Delivering therapeutic oligonucleotides to mitochondria is difficult because of dual membranes and matrix localization.
• Accurate quantification of delivered oligonucleotides is needed to evaluate delivery vectors.

Purpose of the Study:

• To establish a reliable method for quantifying fluorescent oligonucleotide delivery into isolated mitochondria.
• To assess the efficiency and localization of oligonucleotide delivery using dendrimer vectors.
• To provide insights for improving mitochondrial drug delivery vectors.

Main Methods:

• Utilized a microfiltration apparatus for fluorescent analysis of oligonucleotide delivery.
• Employed a range of dilutions to ensure accurate quantification within concentration limits.
• Validated the method by comparing two amphiphilic dendrimer vectors for oligonucleotide delivery.

Main Results:

• The developed method reliably quantifies delivered fluorescent oligonucleotides in isolated mitochondria.
• The protocol allows visualization of oligonucleotide localization within mitochondria, assessing membrane penetration.
• Biological data demonstrated differences in delivery efficiency between two tested dendrimer vectors.

Conclusions:

• The microfiltration-based method offers a reliable and convenient way to quantify mitochondrial oligonucleotide delivery.
• This technique is crucial for evaluating and optimizing vectors for mitochondrial nucleic acid therapies.
• The findings provide essential data for advancing precision therapies targeting mitochondrial DNA.