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

Seong‐Eun Kim,Koo Chul Kwon,Jeewon Lee,Sung Duk Jo,You‐Yeon Won

doi:10.1002/advs.201600471

Abstract

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.

Highlights

We found that the fluorescence intensity reached maximum when no peptide linker was used, which is probably because the separation between adjacent fluorescent proteins (FPs) in the capsid interior is maximized, and the self-quenching effect becomes minimized (Scheme 1a,b)
The additional genetic insertion of FP to the N-terminus of capsid subunit resulted in the synthesis of DL-FPNPs having significantly enhanced fluorescence intensity and photostability compared to the native FPs (Scheme 1c)
The cancer cell receptor-binding peptides, a tandem repeat of affibody peptide with specific and strong affinity for human epidermal growth factor receptor I (EGFR) that is overexpressed on the surface of many types of cancer cells,[14b,15] were genetically presented on the outer surface of the DL-FPNP (Scheme 1a,c), enabling the DL-FPNP to have dual modality of cancer cell targeting and imaging

Summary

Introduction

The HBVC subunits[14] that are genetically conjugated to FPs (enhanced green fluorescent protein/ eGFP or mCardinal/mC) were self-assembled to mono- or double-layered FP nanoparticles (ML- or DL-FPNPs, respectively) in the cytoplasm of Escherichia coli. Transmission electron microscope (TEM) shows that the modified capsid subunits by inserting eGFP into the site between Cys48 and Ser49 (Scheme 1a) were self-assembled to spherical protein nanoparticles (named eGFPin-HBVCs) in E. coli (Figure 1a–c).

Results

Conclusion