Although polycationic vectors display excellent performance in vitro with many cellular systems, their clinical use remains very restricted. To some level, this is due to the poor compatibility of such systems with biological fluids and tissues. In addition, in contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, such as poly(ethylene imine) and poly(dimethylaminoethly methacrylate), the detailed molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, gene delivery vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. To explore cytosolic release mechanisms, we characterised the cytoplasmic interactome associated with a polycationic vector based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) brushes grafted from nanoparticles. Such cationic brushes were found to be particularly effective at trapping small RNAs, resulting in high knock down efficiencies<sup>1-3</sup>. However, how such stable association is disrupted in the cytosol was not clear. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we used fluorescence microscopy and developed a kinetic model based on competitive binding. We propose that the molecular structure and architecture (in particular the high surface density) of cationic brush-decorated nanoparticles, together with the cytosolic molecular crowding, modulate competitive binding and, in turn, the long term release of RNA. Based on these observations, we chemically designed polymer brushes with improved RNA retention in the cytosol, avoiding burst release, and enabling to achieve long term (at least 10 days) knock down (&gt;70%) with one single transfection. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

Polymer Brush Design for Gene Delivery Applications

ACS Spring 2022

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