Intracellular processing of DNA-lipid nanoparticles: A quantitative assessment by image segmentation.

Area of Science:

• Biotechnology
• Cell Biology
• Gene Therapy

Background:

• Lipid nanoparticles (LNPs) are crucial for nucleic acid delivery in gene therapy.
• Current LNP formulations exhibit low endosomal escape rates, hindering therapeutic efficiency.
• Quantifying cellular uptake and processing of nanocarriers remains challenging.

Purpose of the Study:

• To develop an automated live-cell imaging method for analyzing nanocarrier intracellular processing.
• To compare the endosomal escape and intracellular fate of different LNP formulations.
• To identify strategies for enhancing LNP endosomal escape.

Main Methods:

• Engineered HuH7 hepatic cell lines with fluorescent reporters (Galectin, Rab) and lysosomal co-staining were utilized.
• DNA-loaded LNPs with SM-102 and ALC-0315 ionizable lipids were tracked using live-cell imaging.
• Automated analysis quantified endocytic events, endosomal escape, and intracellular trafficking.

Main Results:

• Significant differences in endosomal escape rates and intracellular processing were observed between SM-102 and ALC-0315 LNP formulations.
• Only subpopulations of target cells showed efficient endosomal escape or recycling.
• The use of recycling inhibitors enhanced endosomal escape in target cells.

Conclusions:

• The developed imaging method provides quantitative insights into LNP intracellular dynamics.
• Understanding LNP processing is key to optimizing formulations for gene therapy.
• Targeted modulation of cellular pathways, like recycling, can improve LNP therapeutic potential.