Charge-Shifting Cationic Polymers That Promote Self-Assembly and Self-Disassembly with DNA

Abstract

Synthetic materials that assemble reversibly with polyanions under physiological conditions are of interest for a broad range of biotechnical applications. Cationic polymers are used widely as agents for the condensation of DNA, but polycations also introduce practical limitations in applications for which subsequent dissociation or disassembly of polycation/DNA complexes is desired. The design of cationic polymers that promote the release of associated DNA presents a challenge because it requires the introduction of functionality that is inherently opposed to that required for efficient DNA condensation. Here, we report the synthesis and biophysical characterization of linear poly(ethylenimine) (LPEI) functionalized with methyl ester side chains. The gradual hydrolysis of the ester functionality in these materials under physiologically relevant conditions results in a controlled reduction in cationic charge density and a change in the nature of the electrostatic interactions between the polymers and plasmid DNA, as determined by agarose gel electrophoresis. Using this approach, it is possible to mediate the dissociation of DNA from polymer over a period of hours to days by varying the mole percentage of methyl esters incorporated into the polymer. Polymers having a high degree of substitution (e.g., 80 or 100 mol %) release DNA more rapidly than less-substituted polymers (e.g., 40 and 60 mol % functionalized). Polymers having 20 mol % substitution did not release DNA in these experiments. These charge-shifting materials could provide a basis for the design and fabrication of polyelectrolyte complexes and assemblies that sustain the release of DNA in solution and at interfaces.

Ultrathin Multilayered Films that Promote the Release of Two DNA Constructs with Separate and Distinct Release Profiles

“…[21][22][23] The approach reported here is based not upon the use of degradable cationic polymers, but on the fabrication of multilayers using a new class of ester-functionalized, 'charge-shifting' polyamines. We [24,25] and others [26][27][28][29][30] have demonstrated that it is possible to disrupt ionic interactions in polyelectrolyte assemblies in physiologically relevant media using cationic polymers designed to undergo gradual reductions in net charge upon exposure to aqueous media. In general, approaches to the design of these 'charge-shifting' polymers have taken one of two basic routes: (i) the attachment of amine-functional side chains to polymer backbones through cleavable linkages, [25][26][27][28][29][30] or (ii) the conjugate addition of ester-functionalized 'charge-shifting' side chains to the backbones of cationic polymers.…”

“…In general, approaches to the design of these 'charge-shifting' polymers have taken one of two basic routes: (i) the attachment of amine-functional side chains to polymer backbones through cleavable linkages, [25][26][27][28][29][30] or (ii) the conjugate addition of ester-functionalized 'charge-shifting' side chains to the backbones of cationic polymers. [24] Polymer 1 is exemplary of this second design approach; gradual reductions in the net charge of this polymer can be made to occur upon hydrolysis of ester-functionalized side chains and the introduction of anionic charge (Eq 1; full protonation of amine functionality shown for illustrative purposes). [24] We demonstrated recently that this polymer can promote both self-assembly and timedependent disassembly with DNA in solution in ways that could be understood in terms of side chain hydrolysis and subsequent changes in the net charges of the polymer.…”

“…[24] Polymer 1 is exemplary of this second design approach; gradual reductions in the net charge of this polymer can be made to occur upon hydrolysis of ester-functionalized side chains and the introduction of anionic charge (Eq 1; full protonation of amine functionality shown for illustrative purposes). [24] We demonstrated recently that this polymer can promote both self-assembly and timedependent disassembly with DNA in solution in ways that could be understood in terms of side chain hydrolysis and subsequent changes in the net charges of the polymer. [24] This approach also permits tunable control over the nature of electrostatic interactions with DNA by control over the number of charge-shifting side chains added to the polymer.…”

“…[24] We demonstrated recently that this polymer can promote both self-assembly and timedependent disassembly with DNA in solution in ways that could be understood in terms of side chain hydrolysis and subsequent changes in the net charges of the polymer. [24] This approach also permits tunable control over the nature of electrostatic interactions with DNA by control over the number of charge-shifting side chains added to the polymer. (1) This investigation sought to determine whether this approach to the disruption of ionic interactions in polyelectrolyte assemblies could be exploited to exert control over the timedependent stability of polyelectrolyte multilayers in aqueous environments.…”

“…[25] In the context of designing films that provide tunable control over film disassembly, we reasoned that the approach used to design polymer 1 could provide potential practical advantages relative to the approaches noted above (which require the synthesis of specialized monomers) because this approach is (i) modular and (ii) it can be used to introduce 'chargeshifting' side chains to a broad range of commercially available polyamines. [24] Below, we demonstrate that the addition of ester-functionalized 'charge-shifting' side chains to poly (allylamine hydrochloride) (PAH) (polymer 2) can be used to provide control over the erosion of DNA-containing films and design multilayered films that orchestrate the release of multiple different DNA constructs with separate and distinct release profiles.…”