Synthesis of Tunable Protein-Polymersome Conjugates for Enhanced Targeting of Cancerous Tissues

Protein-polymersome conjugates have the potential to impact the field of immunoengineering leading to improved therapeutics via precise targeting and systemic monitoring. Here, we began by synthesizing a library of diblock copolymers consisting of a first, polyethylene glycol (PEG) block, and a pH-responsive second block comprised of Dimethylaminoethyl methacrylate (DMAEMA) bound to an alkyl methacrylate chain. We subsequently utilized a recently established rapid micro-mixing technique – flash nanoprecipitation – to generate an array of polymersomes. Polymer properties including second-block molecular weight and alkyl chain length were varied to optimize a polymeric nanocarrier capable of delivering therapeutics intracellularly. The polymer array was characterized based on size, cytotoxicity, encapsulation efficiency, potential for endosomal escape, hemolysis, and pH responsiveness to help reveal the polymer composition yielding the optimal polymersome. The tuned nanocarrier was then functionalized with an azide group on the PEG first block to enable future conjugation of immunotherapeutics.

We next synthesized a plasmid, encoding for a fusion protein containing mCherry and an antibody fragment for affinity targeting of an upregulated cancer antigen (GD2). This plasmid was transformed into E. coli, followed by the the expression, purification, and verification of the protein of interest. We selectively ligated a single bicyclononyne (BCN) functional group onto the C-terminal of the fusion protein using an engineering Sortase A ligase to covalently link our fusion protein onto the azide-linked polymersome by strain-promoted alkyne-azide cycloaddition (SPAAC).

Our protein-polymer conjugate was found to successfully demonstrate binding affinity to GD2 receptors as well as an imageable red fluorescence in vitro, as measured by flow cytometry and fluorescence microscopy respectively. The presence of mCherry will enable a future pilot study using In Vivo Imaging Systems (IVIS) to visualize the specific pathways taken by the protein-polymersome conjugates after systemic administration. Traditionally, polymersomes lack the ability to be systemically traced or target specific areas for intracellular delivery which limits their effectiveness as nanocarriers. Further, this study provides the basis for a future exploration of polymersomes linked to selectively ligated proteins. Working as a single unit, these protein-polymersome conjugations have the potential to exploit the vast array of protein functionality to enhance the biomedical applications of polymer-based nanocarriers.