Pathway Complexity Defines the Shape of Nucleic Acid-Loaded Polyion Complex Micelles: Spheroids vs. Hexagonal Disks.

(Macro)molecular self-assembling systems are typically subjected to pathway complexity. The final structural outcome is not only determined by the system's composition but also depends on the details and kinetics of the pathway chosen to navigate the energetic landscape during the assembly process. In this study, a distinct manifestation of pathway complexity is presented in the formation of short double-stranded DNA (dsDNA)-loaded polyion complex (PIC) micelles. Besides the conventional assembly pathway of rapidly mixing dsDNA with preformed polycationic block copolymers, compositionally equivalent PIC micelles are prepared via polymerization-induced electrostatic self-assembly (PIESA). A combination of synchrotron time-resolved small-angle x-ray scattering (TR-SAXS), cryogenic transmission electron microscopy (cryo-TEM), and fluorescence correlation spectroscopy (FCS) reveals the formation of spheroidal particles during direct mixing, whereas PIESA generates hexagonal disk-shaped colloids with internal crystalline dsDNA ordering. A molecular assembly mechanism for PIESA is postulated, which relies on the continuous formation of charge-neutralized complexes that act as primary building blocks for the colloidal hexagons. Mechanistic insights into pathway complexity during PIC micellization establish assembly pathway control as a strategy to tune the particle's physicochemical properties beyond conventional design parameters, such as polymer size and chemistry. This approache expands the opportunities for therapeutic oligonucleotide delivery through control over morphology and cargo loading.