Production and Engineering of Therapeutic Protein Nanoparticles

Abstract

Protein nanoparticles were proposed as therapeutic protein or enzyme delivery vehicles. The production of protein-enzyme nanoparticles via desolvation and self-assembly was investigated and optimized. First, β-galactosidase –a model enzyme– was incorporated into enhanced green fluorescent protein (eGFP) nanoparticles prepared via desolvation. Particle size was shown to be sensitive to type of cross-linker, cross-linking time, and the presence of imidazole. Protein-enzyme nanoparticles are shown to effectively deliver active enzyme to multiple cell lines in vitro. Then the potential of protein nanoparticles for therapeutic protein and enzyme delivery was studied in two diseases models: inflammatory bowel disease (IBD) and breast cancer. Bacterial effector proteins, AvrA and YopJ, were proposed as potential therapeutic agents because of their ability to efficiently subvert the MAPK and NF-κB pathways, which have been implicated in the pathogenesis and progression of IBD and breast cancer. AvrA was incorporated into eGFP nanoparticles and the resulting particles were shown to effectively deliver the effector to target cells in vitro and in vivo, inhibit inflammatory signaling and decrease inflammation in murine colitis models. YopJ was fused to Glutathione-S-Transferase (GST), which caused self-assembly of the fusion into stable nanoparticles. These particles were shown to induce cell death in a panel of breast cancer cell lines, but were not cytotoxic to non-breast cancer cells. Furthermore, these particles decreased cell migration in vitro and performed better or as well as a chemotherapeutic agent, doxorubicin. This data suggests that protein nanoparticles are a biocompatible, high efficiency alternative for intracellular delivery of active enzyme therapeutics, and demonstrates the potential of effector proteins as therapeutic agents.