title: Incorporation of thrombin cleavage peptide into a protein cage for constructing a protease-responsive multifunctional delivery nanoplatform.
abstract: Protein cages are spherical hollow supramolecules that are attractive nanoscale platforms for constructing cargo delivery vehicles. Using ferritin isolated from the hyperthermophilic archaeon  Pyrococcus furiosus  (Pf_Fn), we developed a multifunctional protein cage-based delivery nanoplatform that can hold cargo molecules securely, deliver them to the targeted sites, and release them to the targeted cells. The release is triggered by cleavage induced by the protease, thrombin. The thrombin cleavage peptide (GGLVPR/GSGAS) was inserted into the flexible loop region of Pf_Fn, which is located at a 4-fold axis of symmetry exposed on the surface of protein cages (Thr-Pf_Fn). Subsequently, the C-terminal glycine, which is situated in the interior cavity, was substituted with cysteine (G173C) to permit site-specific conjugation of cargo molecules. The introduced cysteine (G173C) was labeled with a fluorescent probe (F5M-Thr-Pf_Fn) for cell imaging and cargo release monitoring. The surface of F5M-Thr-Pf_Fn was further modified with biotins (F5M-Thr-Pf_Fn-NPB) as targeting ligands. The specific binding of dual functionalized F5M-Thr-Pf_Fn-NPB to the MDA MB 231 cell line, which overexpresses biotin-specific receptors on its surface, was confirmed by fluorescence microscopic analyses. The inserted thrombin cleavage peptides were effectively cleaved by thrombin, resulting in the release of the C-terminal helix in buffer and on the targeted cells without disruption of the cage architecture. Protein cage scaffolds that combine genetic and chemical modifications may serve as stimulus-responsive delivery nanoplatforms and provide opportunities for developing new types of theranostic nanoplatforms.
authors.affiliation: School of Nano-Bioscience and Chemical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, 689-798, Korea.
authors.forename: Young Ji
authors.initials: YJ
authors.lastname: Kang
authors.forename: Dae Cheul
authors.initials: DC
authors.lastname: Park
authors.forename: Hyun-Hee
authors.initials: HH
authors.lastname: Shin
authors.forename: Jongnam
authors.initials: J
authors.lastname: Park
authors.forename: Sebyung
authors.initials: S
authors.lastname: Kang
mesh_terms: D045744:Cell Line, Tumor; D016503:Drug Delivery Systems; D005293:Ferritins / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D008024:Ligands; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D036103:Nanotechnology / Q000379:methods*; D010455:Peptides / Q000737:chemistry; D020836:Protein Structure, Quaternary; D019711:Pyrococcus / Q000737:chemistry; D017978:Receptors, Growth Factor / Q000235:genetics / Q000378:metabolism; D013917:Thrombin / Q000737:chemistry*
chemical_list: D008024:Ligands; D010455:Peptides; D017978:Receptors, Growth Factor; C052136:biotin receptor; D005293:Ferritins; D013917:Thrombin