Developing Porous Protein Cage Nanoparticles as Cargo-Loadable and Ligand-Displayable Modular Delivery Nanoplatforms

Protein cage nanoparticles, self-assembled from protein subunits, provide distinct exterior and interior spaces and can carry diagnostic and/or therapeutic cargo agents through chemical conjugation, <i>in vitro</i> disassembly/reassembly process, or assembly-mediated encapsulation. Here, we developed porous SpyCatcher-mi3 (SC-mi3) as modular delivery nanoplatforms, capable of loading cargos through pores and displaying targeting ligands using SpyCatchers (SC) as anchors for SpyTagged (ST) ligands. Fluorescent dyes (F5M and A647) and a pH-sensitive prodrug (Aldox) were conjugated to the interior surface cysteines of SC-mi3, forming F5M@SC-mi3, A647@SC-mi3, and Aldox@SC-mi3. Subsequently, EGFR-binding affibody molecules (EGFRAfb) were displayed on the exterior surface of F5M@SC-mi3 and Aldox@SC-mi3 using the SC/ST protein ligation system, forming F5M@mi3/EGFRAfb and Aldox@mi3/EGFRAfb, respectively. F5M@mi3/EGFRAfb selectively bound to EGFR-overexpressing MDA-MB-468 cells, visualizing the target cancer cells, while Aldox@mi3/EGFRAfb selectively delivered doxorubicin, leading to target-specific cancer cell death. To encapsulate large proteins within SC-mi3, biotins were initially conjugated to the interior surface (BPM@SC-mi3) and mSA2-fused protein cargo molecules (mSA2-HaloTag and mSA2-yCD) were successfully introduced through the pores and securely encapsulated, forming TMR-H@SC-mi3 and yCD@SC-mi3, respectively. Subsequent display of EGFRAfb on their surface allowed the visualization of target cancer cells using fluorescent HaloTag ligand labeling and facilitated the killing of target cancer cells by converting the prodrug 5-FC to the cytotoxic drug 5-FU. Modular functionalization of the two distinct spaces in porous SC-mi3 may offer opportunities for developing target-specific functional cargo-delivery nanoplatforms in biomedical fields.