Gene delivery with low molecular weight linear polyethylenimines

Area of Science:

• Biotechnology
• Molecular Biology
• Polymer Chemistry

Background:

• Linear polyethylenimine (LPEI) demonstrates superior gene transfer capabilities compared to branched forms.
• High molecular weight LPEI is limited by cytotoxicity, hindering transfection efficiency.
• Investigating lower MW LPEIs (1.0–9.5 kDa) aims to overcome cytotoxicity limitations.

Purpose of the Study:

• To evaluate the gene transfer efficiency and cytotoxicity of various LPEI molecular weights.
• To understand the role of endolysosomes in LPEI-mediated gene delivery.
• To optimize LPEI for non-viral gene delivery applications.

Main Methods:

• Polyplexes of plasmid DNA and LPEIs were analyzed for transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells using flow cytometry.
• Endolysosomal involvement was assessed using bafilomycin A1 and sucrose.
• Confocal laser scanning microscopy was employed to study polyplex size, shape, endolysosomal localization, and nuclear translocation.

Main Results:

• Transfection efficiency varied with LPEI MW and concentration, with 5.6 kDa LPEI achieving the highest efficiency (44%) at low cytotoxicity.
• Polyplexes colocalized with endolysosomes, and larger polyplexes showed faster escape from acidic organelles.
• DNA uptake after 6 hours was comparable for 5.0 kDa and 9.0 kDa LPEI, as determined by flow cytometry.

Conclusions:

• Low MW LPEIs are effective non-viral gene delivery vectors.
• Reduced molecular weight significantly enhances gene transfer efficiency.
• Lower MW LPEIs exhibit substantially lower toxicity compared to higher MW counterparts.