Stabilization of a Synthetic Protein Nanoparticle by a Polycation for Gene Delivery

Nanoparticles (NPs) have attracted significant attention in the field of gene therapy in recent years. Despite the many advances in NP-mediated non-viral gene delivery, the stability of the NPs, successful internalization of nucleic acids, and efficacy in delivery and transfection in cells remains an obstacle to their clinical translation. This work explores the surface-stabilization of a synthetic protein nanoparticle (PNP) with a polycation for improved NP stability and pDNA transfection <i>in vitro</i> and <i>in vivo</i>. Utilizing electrohydrodynamic (EHD) jetting, PNPs were used to encapsulate a large plasmid followed by stabilization by a polycation. The stabilization of the PNP in solution is enabled by the electrostatic interactions between the polycation and the protein. Electron microscopy, dynamic light scattering (DLS), and nanoparticle tracking analysis (NTA) confirm that the surface-stabilized PNPs (ssPNPs) have sub 200 nm diameters in their dry and hydrodynamic states. Additionally, it was determined that the ssPNP size and positive charge are stable in aqueous conditions for extended periods of time, without the addition of stabilizing agents. Utilizing NTA and UV-vis absorbance data, a method was established to determine the DNA amount per ssPNP, indicating an overall yield of 75-80%. In human HepG2 cells, more than 90% of cells take up the ssPNPs, partly due to the positive surface charge. Moreover, transfection of ca. 50% can be achieved while varying the amount of DNA and NP dosing, with negligible cytotoxicity. In a mouse glioblastoma model, ssPNPs demonstrated transfection effectiveness higher than JetPEI®, a commercially used product. This platform shows that using a polycation as a surface stabilizer allows for successful pDNA internalization, NP stability, and transfection efficacy. These findings demonstrate the capability of this new gene delivery system to overcome challenges that hinder the clinical translation of NPs, with the potential for the treatment of various cancers and diseases.