Simultaneous quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems.

Area of Science:

• Analytical Chemistry
• Nanotechnology
• Biochemistry

Background:

• Robust analytical methods are crucial for RNA drug delivery systems.
• Simultaneous quantification of multiple RNA cargos in nanoparticles presents a significant challenge.
• Accurate quantification is essential for developing effective nanoparticle-based therapeutics.

Purpose of the Study:

• To develop and validate a method for simultaneous quantification of single- and double-stranded RNA cargos.
• To establish a reliable analytical technique for RNA quantification in nanoparticle drug delivery systems.
• To overcome the challenge of analyzing multiple RNA types co-loaded within nanoparticles.

Main Methods:

• Ion-pair reversed-phase high-performance liquid chromatography with UV detection (IP-RP-HPLC-UV).
• Phenol:chloroform:isoamyl alcohol for complete RNA extraction from nanoparticle carriers.
• Separations using C18 or PLRP-S columns with triethylammonium acetate (TEAA) and acetonitrile gradients.

Main Results:

• The IP-RP-HPLC-UV method achieved simultaneous quantification of various RNA types (mRNA, polyinosinic:polycytidylic acid, oligodeoxynucleotides, CRISPR guide RNAs).
• The method demonstrated high sensitivity (limit of quantification < 60 ng), linearity (R² > 0.997), and accuracy (≈ 100% recovery).
• Successful application in quantifying co-loaded RNA cargos in lipid-polymer hybrid and lipid nanoparticles.

Conclusions:

• The developed IP-RP-HPLC-UV method provides a robust, sensitive, and versatile solution for quantifying multiple RNA cargos in nanoparticles.
• This analytical approach facilitates the advancement of nanoparticle-based drug delivery systems.
• The validated method can simplify the analysis of complex RNA mixtures within nanocarriers.