Structure–Property Relationships of Oligonucleotide Polyelectrolyte Complex Micelles

Abstract

Polyelectrolyte complex micelles (PCMs), nanoparticles formed by electrostatic self-assembly of charged polymers with charged-neutral hydrophilic block copolymers, offer a potential solution to the challenging problem of delivering therapeutic nucleic acids into cells and organisms. Promising results have been reported in vitro and in animal models but basic structure–property relationships are largely lacking, and some reports have suggested that double-stranded nucleic acids cannot form PCMs due to their high bending rigidity. This letter reports a study of PCMs formed by DNA oligonucleotides of varied length and hybridization state and poly­(l)­lysine-poly­(ethylene glycol) block copolymers with varying block lengths. We employ a multimodal characterization strategy combining small-angle X-ray scattering (SAXS), multiangle light scattering (MALS), and cryo-electron microscopy (cryo-TEM) to simultaneously probe the morphology and internal structure of the micelles. Over a wide range of parameters, we find that nanoparticle shape is controlled primarily by the hybridization state of the oligonucleotides with single-stranded oligonucleotides forming spheroidal micelles and double-stranded oligonucleotides forming wormlike micelles. The length of the charged block controls the radius of the nanoparticle, while oligonucleotide length appears to have little impact on either size or shape. At smaller length scales, we observe parallel packing of DNA helices inside the double-stranded nanoparticles, consistent with results from condensed genomic DNA. We also describe salt- and thermal-annealing protocols for preparing PCMs with high repeatability and low polydispersity. Together, these results provide a capability to rationally design PCMs with desired sizes and shapes that should greatly assist development of this promising delivery technology.

Assembly and Characterization of Polyelectrolyte Complex Micelles

“…If a neutral hydrophilic block is conjugated to one or both of the polyelectrolytes, nano-scale phase separation occurs instead ( Figure 1A). The resulting self-assembled core-shell nanoparticles are variously referred to as polyelectrolyte complex micelles (PCMs), polyion complex micelles, block ionomer complexes, or coacervate-core micelles by analogy to surfactant micellization, even though all components of the system are hydrophilic 6,7 . PCMs' ability to encapsulate hydrophilic molecules such as proteins and nucleic acids, as well as the extensive tunability offered by the block copolymer carrier architecture makes them attractive candidates for delivering therapeutic molecules in vivo [8][9][10][11][12][13] .…”

“…Recent work by our group and others in the field has begun to address this problem by developing structure-property, and in some cases structure-property-function relationships for PCMs formed from nucleic acids and various cationic-neutral polymers 7,[25][26][27] . Two consistent themes that have emerged from these studies are the importance of developing well-controlled, repeatable protocols for PCM assembly and the benefit of using multiple techniques to characterize the resulting nanoparticles.…”

“…The polyelectrolytes are mixed in high salt conditions where electrostatic attractions are screened, then the salt concentration is slowly lowered to allow the polyelectrolytes to settle into their most energetically favorable configurations, analogous to the slow cooling process of thermal annealing. Using this protocol, we are regularly able to achieve exceptionally low polydispersity and high repeatability for oligonucleotide PCMs 7,26 . Finally, we describe how four separate measurement techniques can be used to characterize PCMs over a very wide range of length scales, from external morphology to internal structure: DLS, multi-angle light scattering (MALS), small angle X-ray scattering (SAXS), and transmission electron microscopy (TEM).…”

“…generated in a high throughput manner 28 . The choice of charged group strongly affects the stability of ion pairing and shape of micelles 26 , and PCM size has been shown to increase with the length of the charged block 5,7,26 (Figure 2), thus allowing PCM properties to be tuned for the requirements of a desired application. For linear diblocks, we have found that the charged block should have at least 10 charges and be strongly charged at the desired pH.…”

“…When using oligonucleotides as a polyanion, the hybridization state and chemical structure help determine the propensity for self-assembly and the characteristics of the resulting PCM 5,7,26 . Optimizing these, within the requirements for biological efficacy (if using PCMs for delivery) will increase the likelihood of forming the desired structures.…”