Plasmid DNA Delivery Using a Stable Nanovesicle Platform: A Design-of-Experiments-Guided Investigation

Delivering plasmid DNA (pDNA) into cells is essential for numerous biotechnological and biomedical applications. Among available nanocarriers, nonviral lipid-based vesicles are particularly promising for transfecting mammalian cells. Nevertheless, further development is required to create delivery systems that are both broadly effective across cell types and scalable for clinical use. Here, we explore stable nanovesicles composed of the sterol derivative cholesteryl <i>N</i>-(2-dimethylaminoethyl)carbamate (DC-CHOL) and myristalkonium chloride (MKC) as a platform for pDNA delivery. These nanovesicles, previously shown to efficiently deliver small RNAs to neuroblastoma cells, exhibit favorable physicochemical properties, such as high morphological uniformity and long-term colloidal stability, positioning them as strong candidates for DNA transfection. Using suspension-adapted human embryonic kidney 293 (HEK293) cells, which are widely employed for producing viral vectors and complex biotherapeutics, we evaluated the delivery performance of DC-CHOL/MKC nanovesicles with a reporter plasmid encoding enhanced green fluorescent protein. A Design of Experiments (DoE) approach was applied to identify and optimize critical transfection parameters, namely, the DNA concentration, DNA-to-vesicle ratio, and NaCl concentration in the complexing medium. This study demonstrates the capability of these nonviral vectors to deliver double-stranded plasmid DNA and emphasizes the critical role of the physicochemical characteristics of the pDNA/lipid complex in achieving efficient transfection.