Enhancing Endosomal Escape and Gene Regulation Activity for Spherical Nucleic Acids

Area of Science:

• Biotechnology
• Molecular Biology
• Nanotechnology

Background:

• Small interfering RNAs (siRNAs) show therapeutic promise but are limited by poor stability and cellular uptake.
• Spherical nucleic acids (SNAs) enhance siRNA stability and cellular entry but suffer from endosomal entrapment, hindering gene silencing.
• Endosomal entrapment is a common challenge for siRNA-based nanoparticle delivery systems.

Purpose of the Study:

• To investigate the effect of calcium chloride (CaCl2) formulation on SNA delivery and gene silencing activity.
• To determine if CaCl2 enhances cytosolic delivery of SNAs by reducing endosomal entrapment.
• To evaluate the impact of CaCl2-mediated enhancement on siRNA-based gene silencing efficacy.

Main Methods:

• Formulation of SNAs using CaCl2 instead of NaCl.
• Confocal microscopy to assess the intracellular localization and endosomal escape of SNAs.
• Gene silencing assays in U87-MG and SK-OV-3 cell lines to quantify silencing activity.
• Development and testing of CaCl2-salted SNA-based forced intercalation probes for mRNA detection.

Main Results:

• CaCl2-salted SNAs showed a 22% decrease in late endosomal accumulation compared to NaCl-salted SNAs.
• CaCl2-salted SNAs demonstrated significantly enhanced gene silencing activity (up to 20-fold) across different sequences and cell lines.
• Improved cytosolic mRNA detection was observed with CaCl2-salted SNA-based probes.

Conclusions:

• Formulating SNAs with CaCl2 enhances cytosolic delivery by mitigating endosomal entrapment.
• CaCl2-mediated SNA formulation substantially increases gene silencing efficacy compared to NaCl-based formulations.
• This approach offers a promising strategy to improve the therapeutic potential of siRNA-based nanomedicines.