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Drug Delivery

Drug Delivery - Poster Session A

Poster X12 - Combinatorial discretization of ternary complex nanoparticle structure enables targeted saRNA vaccination

Thursday, October 24, 2024
10:00 AM - 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction: Structure-function relationships in polymeric nucleic acid delivery nanoparticles are poorly understood in comparison to viruses and lipid nanoparticles (LNPs), limiting application of their unique strengths. Multivalent condensation of nucleic acids by polycations into stable polyplexes enables straightforward lyophilization and potent endosomal escape, yet the positive surface charge of such formulations restricts their mechanisms of uptake and routes of administration. Several groups have demonstrated that polyanions such as poly(glutamic acid) can be used to neutralize polyplex surface charge by forming ternary complex nanoparticles (TCNs) that can be actively targeted with ligands. Building on evidence that subtle differences in cationic ionizable lipid chemistry can tune RNA biodistribution by altering the effective pKa and structure of LNPs, we hypothesized that tuning the chemical properties of anionic "cloaking polymers" could synergistically enhance TCN transport, targeting, and transfection.

Materials and

Methods: In this work, we applied high throughput PET-RAFT polymerization to synthesize diblock polyanion libraries with combinatorically varied anionic charge density, hydrophobicity, molecular weight, and PEG architectures, and formulated these into TCN libraries based on polyplex-formulated self-amplifying RNA (saRNA). Optimal formulations parameters were identified by screening TCN library size stability (evaluated by DLS) and saRNA packaging (evaluated via FRET) and lead formulations were selected on the basis of effective cloaking of in vitro uptake. Using Small Angle Neutron Scattering (SANS), Fluorescence Cross-Correlation Spectroscopy (FCCS), and Molecular Dynamics (MD) modeling, we investigate the interplay between polyanion and PEG chemistry in pH-responsive TCN structural transitions. Finally, we engineer the corona of stable TCNs with polymeric mannose to target uptake in myeloid antigen presenting cells in a tissue engineered lymph node model in vitro and through intramuscular injection in vivo

Results, Conclusions, and Discussions: We demonstrate that a precise balance of hydrophobicity, charge density, and PEG length are required to form stable TCN with a hypothesized core-shell structure containing tightly packed saRNA. We further show that polyanion chemistry controls pH-responsive structural transitions that correlate with markers of endosomolysis, and that mannose-targeted TCNs achieved selective transfection of antigen presenting cells. Thus, fine-tuning discrete balances between polyanionic charge density and hydrophobicity can structure a pH-responsive ternary polyelectrolyte core to support surface chemistry engineering. These findings establish synthetic and analytical techniques towards the programmable biodistribution of polymeric saRNA delivery systems with broad applications.

Acknowledgements (Optional): David Peeler is supported by the EU Horizon 2020 research and innovation programme under Marie Skłodowska-Curie grant agreement No 101027174. Lijun Hu is funded by a Rosetrees Trust PhD Plus Scholarship.