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

Abstract

The success of gene therapy hinges on the effective encapsulation, protection, and compression of genes. These processes deliver therapeutic genes into designated cells for genetic repair, cellular behavior modification, or therapeutic effect induction. However, quantifying the encapsulation efficiency of small molecules of interest like DNA or RNA into delivery carriers remains challenging. This work shows how super-resolution microscopy, specifically direct stochastic optical reconstruction microscopy (dSTORM), can be employed to visualize and measure the quantity of DNA entering a single carrier. Utilizing pNIPAM/bPEI microgels as model nano-carriers to form polyplexes, DNA entry into the carrier is revealed across different charge ratios at temperatures below and above the volume phase transition of the microgel core. The encapsulation efficiency also depends on DNA length and shape. This work demonstrates the uptake of the carrier entity by primary derived macro-phages and showcases the cell viability of the polyplexes. The study shows that dSTORM is a potent tool for fine-tuning and creating polyplex microgel carrier systems with precise size, shape, and loading capacity at the individual particle level. This advancement shall contribute significantly to optimizing gene delivery systems.