Genetic Assembly of Double‐Layered Fluorescent Protein Nanoparticles for Cancer Targeting and Imaging

Hepatitis B virus capsid (HBVC), a self‐assembled protein nanoparticle comprised of 180 or 240 subunit proteins, is used as a cage for genetic encapsulation of fluorescent proteins (FPs). The self‐quenching of FPs is controlled by varying the spacing between FPs within the capsid structure. Double‐layered FP nanoparticle possessing cancer cell‐targeting capabilities is also produced by additionally attaching FPs and cancer cell receptor‐binding peptides (affibodies) to the outer surface of the capsid. The generically modified HBVC with double layers of mCardinal FPs and affibodies (mC‐DL‐HBVC) exhibit a high fluorescence intensity and a strong photostability, and is efficiently internalized by cancer cells and significantly stable against intracellular degradation. The mC‐DL‐HBVC effectively detects tumor in live mice with enhanced tumor targeting and imaging efficiency with far less accumulation in the liver, compared to a conventional fluorescent dye, Cy5.5. This suggests the great potential of mC‐DL‐HBVC as a promising contrast agent for in vivo tumor fluorescence imaging. Double‐Layered Fluorescent Protein Nanoparticle (DL‐FPNP) show excellent performance in in vivo cancer targeting and imaging. Hepatitis B virus capsid is genetically engineered to synthesize DL‐FPNP that also presents the multi‐copies of a cancer cell receptor‐binding peptide on its outer surface. NIR irradiation to DL‐FPNP shows far higher fluorescence emission and photostability than a conventional fluorescent dye (Cy5.5).