The Structure and Morphology of Single-Component Oligomeric RNA Delivery Vectors Derived from Amphiphilic...

Advances in nucleic acid delivery have inspired efforts to mimic the function of natural viruses through the development of self-assemblies capable of gene delivery. RNA assemblies based on amphiphilic polymers are emerging as alternatives to lipid nanoparticles, but the factors that govern the self-assembly of RNA with polymeric amphiphiles are poorly understood. Here, we describe the structure of coacervate nanoparticle assemblies derived from RNA and synthetic cationic polymer amphiphiles based on Charge Altering Releasable Transporters (CARTs). CARTs are effective gene delivery agents derived from block copolymer amphiphiles. Cryogenic electron microscopy and tomography (CryoEM, CryoET), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS) reveal that the self-assembly of RNA with low molar mass (≤10,000 g/mol) CART amphiphiles generates nanoparticles with disordered bicontinuous internal morphologies composed of interpenetrating lipid and aqueous coacervate domains. Systematic variation of the cationic and lipophilic blocks in low molar mass CART amphiphiles demonstrates that both the internal domain spacings (6 to 8 nm) and the order of the resulting bicontinuous CART-RNA assemblies depend on the CART chemical structure and the oligonucleotide cargo (mRNA vs siRNA). Notably, the presence of RNA drives the formation of bicontinuous morphologies. In contrast, CART/RNA assemblies with higher molar mass (≥28,000 g/mol) CART amphiphiles fail to generate bicontinuous assemblies, instead yielding aggregates composed of particles approximately 10 to 20 nm in diameter. This work illuminates the internal morphologies of RNA assemblies with synthetic block copolymer amphiphiles, with implications for the rational design of polymer-based RNA delivery systems.