Developing Porous\nProtein Cage Nanoparticles as Cargo-Loadable\nand Ligand-Displayable Modular Delivery Nanoplatforms

Protein cage nanoparticles, self-assembled from protein\nsubunits,\nprovide distinct exterior and interior spaces and can carry diagnostic\nand/or therapeutic cargo agents through chemical conjugation, <i>in vitro</i> disassembly/reassembly process, or assembly-mediated\nencapsulation. Here, we developed porous SpyCatcher-mi3 (SC-mi3) as\nmodular delivery nanoplatforms, capable of loading cargos through\npores and displaying targeting ligands using SpyCatchers (SC) as anchors\nfor SpyTagged (ST) ligands. Fluorescent dyes (F5M and A647) and a\npH-sensitive prodrug (Aldox) were conjugated to the interior surface\ncysteines of SC-mi3, forming F5M@SC-mi3, A647@SC-mi3, and Aldox@SC-mi3.\nSubsequently, EGFR-binding affibody molecules (EGFRAfb) were displayed\non the exterior surface of F5M@SC-mi3 and Aldox@SC-mi3 using the SC/ST\nprotein ligation system, forming F5M@mi3/EGFRAfb and Aldox@mi3/EGFRAfb,\nrespectively. F5M@mi3/EGFRAfb selectively bound to EGFR-overexpressing\nMDA-MB-468 cells, visualizing the target cancer cells, while Aldox@mi3/EGFRAfb\nselectively delivered doxorubicin, leading to target-specific cancer\ncell death. To encapsulate large proteins within SC-mi3, biotins were\ninitially conjugated to the interior surface (BPM@SC-mi3) and mSA2-fused\nprotein cargo molecules (mSA2-HaloTag and mSA2-yCD) were successfully\nintroduced through the pores and securely encapsulated, forming TMR-H@SC-mi3\nand yCD@SC-mi3, respectively. Subsequent display of EGFRAfb on their\nsurface allowed the visualization of target cancer cells using fluorescent\nHaloTag ligand labeling and facilitated the killing of target cancer\ncells by converting the prodrug 5-FC to the cytotoxic drug 5-FU. Modular\nfunctionalization of the two distinct spaces in porous SC-mi3 may\noffer opportunities for developing target-specific functional cargo-delivery\nnanoplatforms in biomedical fields.