Addition of “Charge-Shifting” Side Chains to Linear Poly(ethyleneimine) Enhances Cell Transfection Efficiency

Abstract

We reported recently that the addition of ester-functionalized, "charge-shifting" side chains to linear poly(ethyleneimine) (LPEI) can be used to design polyamines that promote both self-assembly and self-disassembly with DNA in aqueous environments. This investigation sought to characterize the influence of charge-shifting side chains on the ability of LPEI to mediate cell transfection and understand the extent to which increases (or decreases) in levels of transfection could be understood in terms of time-dependent changes in the net charges of these polymers. We report that the addition of "charge-shifting" side chains to LPEI leads to significant increases in levels of LPEI-mediated transfection. In particular, polymer 1e, functionalized with 20 mol % ester-functionalized side chains, mediates levels of transgene expression in vitro up to 8-fold higher than LPEI. Experiments using an amide-functionalized analog of polymer 1e demonstrated that the esters in polymer 1e play an important role in promoting increased levels of transfection. These results, in combination with the results of additional gel electrophoresis experiments, provide support for the view that increases in transfection result from time-dependent changes in the net charge of polymer 1e and the disruption of ionic interactions in polyplexes. Additional support for this view is provided by the results of confocal microscopy experiments and measurements of fluorescence resonance energy transfer, which suggest that polymer 1e promotes the disruption of polyplexes in intracellular environments effectively. The approach reported here provides a means of addressing one important "late-stage" obstacle to polyplex-mediated transfection (polyplex unpackaging). If integrated successfully with methods that have been developed to address other important barriers to transfection, this general approach could lead to the development of multifunctional polyplexes that mimic more effectively the range of functions of viruses as agents for the delivery of DNA.

Ultralow‐Fouling, Functionalizable, and Hydrolyzable Zwitterionic Materials and Their Derivatives for Biological Applications

“…to make the polymers less positively charged [106,107] or negatively charged (charge reversal) [108] upon hydrolysis of ester groups into negatively charged groups to facilitate gene unpacking. The advantage of pCB-ester polymers is that they will be hydrolyzed into zwitterionic polymers, which have been shown to be ultralow fouling and biocompatible.…”

Effect of peptide content on the regulation of transgene expression by protein kinase Cα-responsive linear polyethylenimine-peptide conjugates

“…Intelligent polymers that can alter their interactions with therapeutic genes by sensing changes in pH [14], redox potential [15], temperature [16], UV irradiation [17], and enzyme activity [18][19][20][21][22][23][24][25], have been promoted as gene carriers. We have extensively studied polymer-peptide conjugates that release pDNA in response to protein kinases (PKs) [18][19][20][21][22][23][24] and proteases [25], both specifically and abnormally activated in diseased cells.…”

Release of DNA from polyelectrolyte multilayers fabricated using ‘charge-shifting’ cationic polymers: Tunable temporal control and sequential, multi-agent release

“…Our group and others have demonstrated that it is possible to design ‘charge-shifting’ polymers, and exert control over the rates and extents of changes in charge state, by incorporating hydrolyzable side chains into the structures of cationic or anionic polymers [38–47]. Several different approaches to the design of cationic ‘charge-shifing’ polymers have been reported, largely in the context of developing new materials for the polyplex-mediated delivery of DNA (e.g., to provide control over the disassembly of cationic polymer/DNA complexes in solution or in cells) [41–47]. …”