Validation of a Galectin-8 Reporter As a Measure of Nanocarrier Endosomal Escape and Biologic Drug Intracellular Bioavailability

Introduction: Endosomal escape is a critical step in the delivery of intracellularly active biomacromolecules, such as nucleic acids, peptides, and proteins. We and others recently utilized Galectin 8 (Gal8) recruitment to detect endosomal vesicle disruption by nonviral carriers^1,2. Here, we sought to validate a Gal8 recruitment-based assay as a direct measure of endosome escape and predictor of biologic drug intracellular bioactivity (siRNA gene silencing); in addition, this methodology was utilized to rapidly optimize polymer composition and molecular weight for siRNA knockdown activity building from our recent work on siRNA nanocarriers (si-NPs)³.

Materials and Methods: A library of poly[(ethylene glycol)-b-[(2-(dimethylamino)ethyl methacrylate)-co-(butyl methacrylate)] [PEG-(DMAEMA-co-BMA)] polymers of equivalent MW were synthesized with 0, 25, 40, 50, 60, and 75 mol% BMA using RAFT polymerization³. A second polymer library was synthesized with 50 mol% BMA composition with varied molecular weight within the DMAEMA-co-BMA si-NP core-forming polymer block (S, M, L, XL block length, 19-24 kDa range). Polymers were complexed with siRNA into si-NPs , which were analyzed for pH-dependent membrane disruption (hemolysis assay), siRNA loading efficiency (ribogreen), endosome disruption in living cells (Gal8-YFP recruitment assays), lysotracker studies, cell viability, and knockdown bioactivity (luciferase assay).

Results and Discussion: The balance of cationic and hydrophobic content, as well as the core block lengths affect endosomal escape and gene knockdown characteristics of polymeric si-NPs. Multiple polymers were both nontoxic and exhibited switch-like pH-dependent hemolysis. However, only one polymer (50B) was both nontoxic and had robust knockdown. This polymer was also the only polymer found to have robust Gal8-YFP recruitment. As total block length is increased in the 50B composition, endosomal escape and model gene knockdown increases. We also find that directly measuring endosomal escape as measured by Gal8 -YFP recruitment is an accurate predictor of model gene knockdown, and correlates with standard lysotracker studies.

Conclusions: Gal8-YFP recruitment is a robust new tool to screen polymer libraries for cytosolic delivery, especially for cargo whose cytosolic delivery and bioactivity may not be easily measured. It is straight forward, rapid and does not require expensive fluorescently labelled cargo. It can also work with peptides¹. The results confirm that Gal8-YFP recruitment is a sensitive method of assessing how effective polymers are at endosomal escape, as a predictor of in vitro bioactivity.

Refs: 1. Kilchrist et al. Cell Mol Bioeng. 2016. 2. Wittrup et al. Nat. Biotech 2015. 3. Nelson et al. ACS Nano 2013