Modular Hepatitis B Virus-like Particle Platform for Biosensing and Drug Delivery

The hepatitis B virus-like particle (HBV VLP) is an attractive protein nanoparticle platform due to the availability of 240 modification sites for engineering purposes. Although direct protein insertion into the surface loop has been demonstrated, this decoration strategy is restricted by the size of the inserted protein moieties. Meanwhile, larger proteins can be decorated using chemical conjugations; yet these approaches perturb the integrity of more delicate proteins and can unfavorably orient the proteins, impairing active surface display. Herein, we aim to create a robust and highly modular method to produce smart HBV-based nanodevices by using the SpyCatcher/SpyTag system, which allows a wide range of peptides and proteins to be conjugated directly and simply onto the modified HBV capsids in a controlled and biocompatible manner. Our technology allows the modular surface modification of HBV VLPs with multiple components, which provides signal amplification, increased targeting avidity, and high therapeutic payload incorporation. We have achieved a yield of over 200 mg/L for these engineered HBV VLPs and demonstrated the flexibility of this platform in both biosensing and drug delivery applications. The ability to decorate over 200 nanoluciferases per VLP improved detection signal by over 1500-fold, such that low nanomolar levels of thrombin could be detected by the naked eye. Meanwhile, a dimeric prodrug-activating enzyme was loaded without cross-linking particles by coexpressing orthogonally labeled monomers. This along with a epidermal growth factor receptor-binding peptide enabled tunable uptake of HBV VLPs into inflammatory breast cancer cells, leading to efficient suicide enzyme delivery and cell killing.