(586b) Bioreversible Anionic Conjugation Platform for Functional Protein Delivery with Ionizable Lipid Nanoparticles

Protein-based therapeutics comprise a rapidly growing subset of pharmaceuticals, accounting for 20% of all drugs on the market today. However, due to their large, hydrophilic structures, delivery of proteins across cell membranes has been a longstanding issue in the field. As such, protein-based drugs are limited primarily to extracellular applications. The potential of proteins as intracellular therapeutic agents has been highlighted in the use of novel protein scaffolds that inhibit “undruggable” biological targets and more recently by gene-editing machinery for treating human disease. Translating many of these promising technologies for clinically relevant applications requires the development of delivery methods capable of translocating proteins into the cytosol of a cell for functional activity. Several protein delivery approaches have been investigated over the years, yet none have been approved for clinical in vivo use.

To overcome these delivery challenges for ultimately translating protein therapeutics for intracellular applications, we were interested in adopting lipid nanoparticle formulations that have found great success in the delivery of nucleic acids in many clinical models. It has long been demonstrated that mixing nucleic acids in the presence of cationic lipids results in the formation of hydrophobic nanoparticle structures within which nucleic acid cargos are packed. The formed lipid nanoparticles (LNPs) can then mediate the entry of nucleic acid cargos into cells for their intended therapeutic effect. To utilize LNPs for protein delivery, we explored the possibility of modifying protein surface charge to mimic the anionic nature of nucleic acids, which would enable electrostatic complexation with cationic lipids to form LNPs.

To this end, we sought to utilize a bioconjugation-based approach for attaching anionic chemical moieties onto surface-exposed residues of protein cargos. We designed a panel of lysine-reactive chemical linkers containing anionic sulfonate groups and a self-immolative, cleavable disulfide bond. The designed compounds would hypothetically provide an anionic “cloak” to protein cargos to enable LNP formation with cationic lipids. Once protein cargos are delivered into cells, the reductive environment of the cytosol will cause cleavage of the disulfide bond, which can further self-immolate to release the protein cargo back in its native form.

Herein this work, we establish the proof-of-concept of our approach by demonstrating successful anionic modification and internalization of super-folder green fluorescent protein (sfGFP) with both Lipofectamine 2000, an off-the-shelf cationic lipid reagent, and LNP formulations consisting of the ionizable lipid MC3. We further expand upon our approach by investigating functional protein delivery with LNP formulations of: a) RNase A as a model cytotoxic cargo in various cancer cell models, as well as b) inhibitory β-catenin-specific antibodies for cell signaling interference in oncogenic transduction pathways.

Taken together, we demonstrate that anionic bioconjugation of proteins can enable intracellular protein delivery with LNPs. Our exciting proof-of-concept results lay the groundwork for a generalizable, bioconjugation-based platform that can be employed for the delivery of a wide range of protein cargos. Future work will involve applying this technique to deliver a wider array of various functional therapeutic proteins, with the ultimate goal of translating protein therapeutics for intracellular applications.