Advisor: Matt Tirrell
Pritzker School of Molecular Engineering, The University of Chicago
Polyelectrolyte complexation describes the preference for oppositely-charged polymers to associate with each other in aqueous solution rather than with small counterions, due to their lower translational entropy per unit charge. If the attraction is strong enough, phase separation occurs, resulting in a hydrated polymer-rich phase that can either be liquid-like (complex coacervate) or a solid precipitate. Nucleic acids are strongly-charged polyanions, and phase-separated complexes have been observed when DNA molecules are mixed with cationic polymers. Complexation neutralizes the nucleic acids’ charge, and the resulting complexes can be internalized by cells via endocytosis. If the polycation is conjugated to a neutral hydrophilic polymer such as poly(ethylene glycol) (PEG), nanoscale phase separation occurs forming nanoparticles (termed polyelectrolyte complex micelles, PCMs) with the neutral block forming a corona around a neutralized polyion core. Conceptually, PCMs assembled with nucleic acids as the polyanions are attractive delivery vehicles: in addition to the charge neutralization and steric protection from nucleases afforded by the polyion core, the neutral corona provides colloidal stability and size control to allow optimization of circulation properties, as well as a platform for attaching targeting ligands to further improve biodistribution.
Several promising results in vitro and in small animal models confirm the potential of this strategy, but much work remains to optimize PCMs as safe, efficacious nucleic acid delivery vehicles, as well as to improve our understanding of the physics of PCM self-assembly. Few systematic studies have been conducted on how parameters such as polymer charge density, hydrophobicity, and molecular structure influence PCM properties, despite evidence that these strongly influence the complexation behavior of polyelectrolyte homopolymers. Using small angle X-ray scattering (SAXS) and electron microscopy we investigate the relationship between the physical properties of oligonucleotide PCMs and chemical structure of the nucleic acids and block copolymers that form them. These observations narrow the design space for optimizing therapeutic PCMs and provide new insights into the rich polymer physics of polyelectrolyte self-assembly.
In short, we compare poly-l-lysine (pLys) and poly-VinylBenzylTriMethylAmmonium (pVBTMA) as two polycations previously used for nucleic acid complexation that differ in charge density, hydrophobicity, and charged group. We show many similarities and some marked differences when comparing PCMs formed by the two polycations. Oligonucleotide PCMs formed with pVBTMA-PEG show similar trends to those formed with pLys-PEG; micelle radius is controlled by the length of the charged block but largely insensitive to oligonucleotide length, and hybridization state of the nucleic acid determines the shape of micelles. We observe major discrepancies for PCMs with double-stranded oligonucleotides, where pLys-PEG forms micron-scale worm-like micelles, but pVBTMA-PEG forms much shorter cylindrical micelles that are highly polydisperse, implying some mechanical instability in this system. pVBTMA-PEG PCMs and pVBTMA-DNA macro scale complexes also proved to be less stable in high NaCl conditions, suggesting the interactions between nucleic acids and pVBTMA are weaker than those with pLys, despite higher charge density, increased hydrophobicity, and non-susceptibility to neutralization. The large structural differences between pLys and pVBTMA suggest that the similarities in PCM properties we observe may be universal, while the differences provide useful leads for further investigation into the physics of polyelectrolyte self-assembly as well as possible solutions to the pressing problem of nucleic acid delivery.
Nucleic acid modifications:
Chemical modifications to therapeutic nucleic acids are frequently used to resist nuclease degradation. We find a substantial effect on PCM formation and stability from these atomic differences. This manuscript is in preparation, more information available soon.
Complexation between nucleic acids (polyanion) and hydrophilic cation-neutral block copolymers forms phase separated structures. This study explores the effect of chemical parameters determining the physical properties of polyelectrolyte complexes.
Fares, H.M., Marras, A.E.*, Ting, J.M.*, Tirrell, M.V., Keating, C.D. “Impact of wet-dry cycling on the phase behavior and compartmentalization properties of complex coacervates” Nature Communications. 11:5423 (2020) — link — pdf
Ting, J. M.*, Marras, A. E.*, Mitchell, J. D., Campagna, T., Tirrell, M.V. “Comparing zwitterionic and PEG exteriors of polyelectrolyte complex micelles” Molecules. 25:2553 (2020) *equal contribution — link — pdf — preprint — data
Marras, A.E., Vieregg, J.R., Ting, J.M., Rubien, J.D., Tirrell, M.V. “Polyelectrolyte complexation of oligonucleotides by charged hydrophobic – neutral hydrophilic block copolymers” Polymers. 11:83 (2019) — link — pdf — data