Ugi reaction-enabled one-step multifunctionalization of biocatalytic VLPs for multimodal therapeutics

Abstract

Multimodal nanoplatforms hold great potential in cancer therapy, but their clinical translation is limited by complex, multi-step synthesis. While protein nanocages provide a homogeneous and multifunctional scaffold, their complex architecture restricts compatibility with many chemical reactions. Here, we report the first successful use of the Ugi four-component reaction (Ugi-4CR) for one-step multifunctionalization of biocatalytic P22 encapsulating cytochrome P450 (CYP). Functionalization included glucose oxidase (GOx) for cascade enzymatic prodrug activation and glucosamine (GA) as a targeting ligand. A parallel labeling of protein with dye, indocyanine green (ICG), for near infrared (NIR) imaging and NIR-mediated phototherapy was also performed. The reaction showed high reproducibility with similar degrees of conjugation of GOx and ICG in each reaction. Enzymatic cascade kinetic analysis showed ∼3-fold improved catalytic efficiency of Ugi-produced nanoreactors compared to conventional carbodiimide coupling, while maximum velocity was reduced by ∼2-fold. The glucose-triggered activation of the prodrug using engineered nanoreactors in the absence and presence of NIR revealed a potential for synergistic chemo-photothermal therapy. Additionally, in vitro studies exhibited improved cellular uptake of targeted nanoreactors. Overall, the results confirm the intact functional integration of all the integrated components under Ugi conditions and hint at site-selective modification on the VLP surface. This work shows that the Ugi-4CR has potential for one-step multifunctionalization of protein nanocages to develop comprehensive therapeutic modalities, offering faster and efficient alternatives to tedious multi-step functionalization strategies.