Engineered drug-protein nanoparticle complexes for folate receptor targeting

• We fabricated biomimetic protein-based nanoparticles with both molecular encapsulation and cell-targeting capabilities using recombinant technology and chemical synthesis.
• Functionalization of the protein nanoparticles’ external surface with folic acid increased their cellular uptake into cancer cells that overexpressed folate receptors.
• Nanoparticles demonstrated acid-responsive drug release and were cytotoxic to cancer cells.
• Our investigation demonstrates the potential of using biofabrication strategies to generate functional nanomaterials.

Nanomaterials that are used in therapeutic applications need a high degree of uniformity and functionality which can be difficult to attain. One strategy for fabrication is to utilize the biological precision afforded by recombinant synthesis. Through protein engineering, we have produced ∼27-nm dodecahedral protein nanoparticles using the thermostable E2 subunit of pyruvate dehydrogenase as a scaffold and added optical imaging, drug delivery, and tumor targeting capabilities. Cysteines in the internal cavity of the engineered caged protein scaffold (E2 variant D381C) were conjugated with maleimide-bearing Alexa Fluor 532 (AF532) and doxorubicin (DOX). The external surface was functionalized with polyethylene glycol (PEG) alone or with the tumor-targeting ligand folic acid (FA) through a PEG linker. The resulting bi-functional nanoparticles remained intact and correctly assembled. The uptake of FA-displaying nanoparticles (D381C-AF532-PEG-FA) by cells overexpressing the folate receptor was approximately six times greater than of non-targeting nanoparticles (D381C-AF532-PEG) and was confirmed to be FA-specific. Nanoparticles containing DOX were all cytotoxic in the low micromolar range. To our knowledge, this work is the first time that acid-labile drug release and folate receptor targeting have been simultaneously integrated onto recombinant protein nanoparticles, and it demonstrates the potential of using biofabrication strategies to generate functional nanomaterials.