Tracking the DNA complexation state of pBAE polyplexes in cells with super resolution microscopy

Area of Science:

• Biotechnology
• Cell Biology
• Materials Science

Background:

• Gene therapy requires effective and safe delivery vectors.
• Cationic polymers like Poly(β-amino ester)s (pBAE) form nanoparticles (polyplexes) for oligonucleotide delivery.
• Understanding polyplex complexation and DNA release within cells is vital for gene expression.

Purpose of the Study:

• To develop a method for visualizing and quantifying polyplex components (pBAE and DNA) inside cells.
• To track the dynamic complexation state of polyplexes during cellular uptake and nuclear entry.
• To reveal the spatiotemporal fate of pBAE polymers and DNA within mammalian cells.

Main Methods:

• Utilized two-colour direct stochastic reconstruction super-resolution microscopy (dSTORM).
• Applied dSTORM to achieve nanometre-scale resolution for visualizing intracellular polyplexes.
• Tracked pBAE polyplexes from cell binding through DNA release and nuclear entry.

Main Results:

• Successfully visualized and quantified pBAE and DNA components within polyplexes inside cells.
• Monitored the dynamic changes in polyplex complexation state over time.
• Determined the fate of pBAE polymers and DNA following cellular internalization and nuclear transport.

Conclusions:

• The developed dSTORM method enables detailed analysis of polyplex behavior within cells.
• This technique provides insights into the critical complexation and release dynamics for gene therapy vectors.
• The findings contribute to the advancement of efficient and safe gene delivery systems.