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

## TL;DR

Researchers developed a nanovesicle platform for delivering plasmid DNA into cells, optimizing parameters for efficient transfection using a Design of Experiments approach.

## Contribution

A novel nonviral nanovesicle platform for pDNA delivery is optimized using a Design of Experiments approach for transfection efficiency.

## Key findings

- DC–CHOL/MKC nanovesicles efficiently deliver plasmid DNA into HEK293 cells.
- Key transfection parameters like DNA concentration and DNA-to-vesicle ratio were optimized using a DoE approach.
- The pDNA/lipid complex's physicochemical properties are critical for transfection success.

## Abstract

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 N-(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.