Abstract
In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.
Highlights
In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood
We focused on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brush-functionalised nanoparticles as cationic model systems
Proteomic analysis of the pristine cytosolic fractions confirmed the presence of expected cytoplasmic proteins, including a large pool of proteins associated with cytoskeleton assembly and organisation, either originating from monomeric soluble proteins, or potentially oligomers that may not have been separated in our fractionation protocol
Summary
In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Amongst the range of viral and non-viral vectors that have been designed, polycationic systems are attractive for in vitro work[4,5,6,7], their translation and efficacy in vivo has been limited To some extent, this is due to the more difficult control of biodistribution and oligonucleotide delivery that can be achieved with these systems, in comparison with lipid-based and viral systems[8]. Plasmid DNA was found to release rapidly, within seconds, upon cytosolic entry, confirming the short burst effect typically achieved[15] This may be appropriate for DNA delivery requiring fast release to enable nuclear translocation and expression, such behaviour severely limits the sustained release of RNA. We demonstrate that engineering of the binding strength of oligonucleotides enables the modulation of competitive binding and the sustained release and knockdown efficiency of siRNA
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