Analyzing siRNA Concentration, Complexation and Stability in Cationic Dendriplexes by Stem-Loop Reverse...

Area of Science:

• Biochemistry
• Molecular Biology
• Pharmaceutical Sciences

Background:

• RNA interference (RNAi) utilizes small interfering RNAs (siRNAs) for messenger RNA (mRNA) regulation.
• Non-viral carriers, such as dendriplexes, are used for siRNA delivery.
• Accurate quantification of siRNA within delivery systems is crucial for developing effective RNAi therapeutics.

Purpose of the Study:

• To develop a novel, sensitive, and specific method for quantifying intact siRNA in drug formulations.
• To enable indirect assessment of siRNA stability and concentration within carriers like dendriplexes.
• To support the development and quality control of siRNA-based drug delivery systems.

Main Methods:

• A reverse transcription real-time PCR (RT-qPCR) based application was developed for siRNA quantification.
• The method specifically quantifies released, uncomplexed target siRNA using a dilution series.
• Limits of detection (LOD) and quantification (LOQ) were determined for both uncomplexed siRNA and siRNA within dendriplexes.

Main Results:

• The RT-qPCR method demonstrated high sensitivity and specificity for intact target siRNA.
• Achieved LOD of 4.2 pM and LOQ of 77.8 pM for uncomplexed siRNA; LOD of 31.6 pM and LOQ of 44.4 pM for dendriplex samples.
• The method accurately quantified siRNA complexation in dendriplexes and assessed siRNA protection from degradation.

Conclusions:

• The novel RT-qPCR application provides accurate and sensitive quantification of siRNA in drug delivery systems.
• This method is valuable for assessing siRNA concentration, stability, and complexation within carriers.
• It serves as an important tool for the development and quality control of siRNA-based therapeutics.