Nanoparticles of compacted DNA transfect postmitotic cells

Area of Science:

• Biotechnology
• Molecular Biology
• Cell Biology

Background:

• Gene transfer into non-dividing cells remains a challenge.
• Nuclear uptake is limited by the 25-nm nuclear membrane pore size.
• DNA nanoparticles offer potential for enhanced gene delivery.

Purpose of the Study:

• To investigate the efficacy of charge-neutral DNA nanoparticles for gene transfer into growth-arrested cells.
• To elucidate the mechanism of enhanced transfection mediated by DNA nanoparticles.
• To determine the size limitations for nuclear entry and subsequent gene expression.

Main Methods:

• Development of charge-neutral DNA nanoparticles by compacting single DNA molecules.
• Transfection of growth-arrested neuroblastoma and hepatoma cells with DNA/liposome mixtures encoding luciferase.
• Microinjection of naked or compacted plasmids encoding enhanced green fluorescent protein into HuH-7 cells.
• Assessment of transgene expression and the effect of nuclear pore inhibitors (wheat germ agglutinin).

Main Results:

• Compacted DNA demonstrated robust transfection in growth-arrested cells (6,900-360-fold increase compared to naked DNA).
• Cytoplasmic microinjection of DNA nanoparticles yielded a 10-fold improvement in transgene expression versus naked DNA.
• This enhancement was inhibited by wheat germ agglutinin, indicating nuclear pore-dependent transport.
• Transgene expression decreased significantly as nanoparticle size approached the 25-nm nuclear pore diameter.

Conclusions:

• Suitably sized DNA nanoparticles can effectively transfect growth-arrested cells.
• Nuclear uptake across the nuclear membrane pore is a key mechanism for enhanced gene transfer.
• DNA nanoparticles represent a promising strategy for gene delivery into postmitotic cells.