Transfection, defined as functional delivery of cell-internalized nucleic acids, is dependent on many factors linked to formulation, vector, cell type, and microenvironmental culture conditions. We previously developed a technology termed glycosaminoglycan (GAG)-binding enhanced transduction (GET) to efficiently deliver a variety of cargoes intracellularly, using GAG-binding peptides and cell penetrating peptides (CPPs) in the form of nanoparticles, using conventional cell culture. Herein, we demonstrate that the most simple GET transfection formulation (employing the FLR peptide) is relatively poor at transfecting cells at increasingly lower dosages. However, with an endosomally escaping version (FLR:FLH peptide formulations) we demonstrate more effective transfection of cells with lower quantities of plasmid (p)DNA in vitro. We assessed the ability of single and serial delivery of our formulations to readily transfect cells and determined that temperature, pH, and atmospheric pressure can significantly affect transfected cell number and expression levels. Cytocompatible temperatures that maintain high cell metabolism (20-37 °C) were the optimal for transfection. Interestingly, serial delivery can maintain and enhance expression without viability being compromised, and alkaline pH conditions can aid overall efficiencies. Positive atmospheric pressures can also improve the transgene expression levels generated by GET transfection on a single-cell level. Novel nanotechnologies and gene therapeutics such as GET could be transformative for future regenerative medicine strategies. It will be important to understand how such approaches can be optimized at the formulation and application levels in order to achieve efficacy that will be competitive with viral strategies.
© 2024 The Authors. Published by American Chemical Society.