Vector unpacking as a potential barrier for receptor-mediated polyplex gene delivery

Abstract

Ligand‐conjugated polymer (polyplex) gene delivery vectors have strong potential as targeted, in vivo gene transfer vehicles; however, they are currently limited by low delivery efficiency. A number of barriers to polyplex‐mediated delivery have been previously identified, including receptor binding, internalization, endosomal escape, and nuclear localization. However, based on understanding of viral gene delivery systems, yet another potential barrier may exist; a limited ability to unpackage the plasmid DNA cargo following localization to the nucleus. We have developed a model system that employs a cationic polymer linked to epidermal growth factor (EGF) as a ligand to target delivery of plasmid DNA encoding the green fluorescent protein to mouse fibroblasts bearing the EGF receptor. Using fluorescence microscopy to simultaneously trace both the plasmid and polymer during gene delivery in combination with an in vitro transcription assay, we provide evidence that plasmid unpackaging can indeed be a limiting step for gene expression for sufficiently large polymer constructs. Short‐term expression is significantly enhanced by using short polycations that dissociate from DNA more rapidly both in vitro and in vivo. Finally, we describe a thermodynamic model that supports these data by showing that shorter polycations can have a higher probability of dissociating from DNA. This work demonstrates that vector unpackaging should be added to the list of barriers to receptor‐mediated polyplex gene delivery, thus providing an additional design principle for targeted synthetic delivery vehicles. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 598–606, 2000.

A Combinatorial Polymer Library Approach Yields Insight into Nonviral Gene Delivery

“…It has been shown that polymeric vector/DNA complexation significantly inhibits RNA synthesis and that above an optimal length, overall transfection decreases as polylysine length increases. 33 Similarly, 25 kDa PEI vectors are known to have higher transfection efficacy than those formed with higher molecular weight PEI. 34 However, as previously mentioned, it is also known that PEI and PBAEs with molecular weights below 8 kDa typically exhibit poor transfection compared with higher molecular weight versions.…”

Quantitative comparison of polyethylenimine formulations and adenoviral vectors in terms of intracellular gene delivery processes

“…The processes of vesicular degradation were included to contribute to plasmid and vector loss during transfection (Figures 2 and 3). Nuclear import protein association kinetics were separated for vector and plasmid to capture nuclear uptake behavior observed for various vectors previously 11,16 while maintaining the vector-independent characteristics of plasmid import within C3A cells.…”

Improved chitosan-mediated gene delivery based on easily dissociated chitosan polyplexes of highly defined chitosan oligomers

“…12 We speculated that these drawbacks could be eliminated if chitosans of lower molecular weights were used, that is, chitosans that were long enough to form stable polyplexes, but sufficiently short to be soluble at neutral pH, give a reduced viscosity, form less aggregated shapes more typical for multivalent ions, and form more easily dissociated polyplexes. 23,24 Chitosan oligomers shorter than 14 monomer units were recently found to fulfill many of these pharmaceutical requirements, but formed only weak complexes with DNA, resulting in physically unstable polyplexes that transfected cells at low efficiencies in vitro and in vivo. 20 A larger chitosan oligomer fraction (approximately a 24-mer; 4.7 kDa) formed physically stable polyplexes of comparable efficiency to very stable polyplexes based on higher molecular weights of the chitosan.…”