Polymer Nano-Carrier-Mediated Gene Delivery: Visualizing and Quantifying DNA Encapsulation Using dSTORM

Area of Science:

• Biotechnology
• Materials Science
• Cell Biology

Background:

• Effective gene therapy relies on efficient gene encapsulation and delivery.
• Quantifying gene loading into delivery carriers is a significant challenge.
• Current methods lack precision in measuring encapsulation efficiency at the single-particle level.

Purpose of the Study:

• To demonstrate the application of super-resolution microscopy for quantifying DNA encapsulation in nano-carriers.
• To investigate the influence of various factors on encapsulation efficiency.
• To assess the biocompatibility and cellular uptake of the developed gene delivery system.

Main Methods:

• Utilized direct stochastic optical reconstruction microscopy (dSTORM) for high-resolution imaging.
• Employed pNIPAM/bPEI microgels as model nano-carriers forming polyplexes with DNA.
• Investigated encapsulation efficiency based on charge ratios, temperature-induced microgel transitions, DNA length, and shape.

Main Results:

• Successfully visualized and quantified DNA entry into individual microgel carriers using dSTORM.
• Demonstrated that encapsulation efficiency is dependent on charge ratio, DNA characteristics, and microgel properties.
• Confirmed uptake of polyplexes by macrophages with good cell viability.

Conclusions:

• dSTORM is a powerful tool for precise characterization of gene delivery carriers at the single-particle level.
• This technique enables fine-tuning of polyplex microgel systems for optimized size, shape, and loading capacity.
• The findings significantly advance the development of efficient and targeted gene delivery systems.