Mixing-sequence-dependent nucleic acid complexation and gene transfer efficiency by polyethylenimine.

Area of Science:

• Biomaterials Science
• Gene Therapy
• Nanotechnology

Background:

• Nonviral gene delivery vectors, known as polyplexes, are formed by complexing nucleic acids with cationic polymers.
• Current research focuses on novel cationic polymers and delivery pathways, often overlooking polyplex preparation methods.
• Polyplex formation significantly influences gene delivery efficacy and safety.

Purpose of the Study:

• To investigate the impact of mixing sequence on polyplex characteristics and gene delivery.
• To determine how different addition orders of nucleic acids and cationic polymers affect complexation, size, and charge.
• To correlate polyplex properties with transfection efficiency and cytotoxicity.

Main Methods:

• Comparing polyplexes formed by adding nucleic acids to polymers versus polymers to nucleic acids.
• Utilizing plasmid DNA and small interfering RNA (siRNA) as model nucleic acids.
• Employing polyethylenimine (PEI) as a standard cationic polymer for comparison.

Main Results:

• The mixing sequence critically affects nucleic acid complexation, polyplex size, and surface charge.
• Adding PEI to plasmid DNA and siRNA resulted in larger polyplexes, enhanced gene expression and silencing.
• This specific order also led to increased cytotoxicity compared to the reverse addition method.

Conclusions:

• A model was developed suggesting gradual addition of cationic polymers to nucleic acids leads to larger polyplexes with higher gene delivery efficiency and cytotoxicity.
• This preparation strategy incorporates more nucleic acids, resulting in enhanced cellular expression and silencing.
• The findings offer crucial insights for optimizing polyplex formulation for efficient and safe nonviral gene delivery using various cationic polymers and nucleic acids.