br Peptide A br Peptide
Expression TrxA removal
Peptide B + Biotin Peptide B
+ Ca2+ Qdot-MNP peptide-MNP
Fig. 1. Schematic illustration of the strategy applied in this study. Refer to text for details.
3.1. The proposed strategy of targeting cancer cells
As illustrated in Fig. 1, this study proposed a strategy to de-velop a theranostic entity which was assembled from two peptides (i.e. peptide A and peptide B) for simultaneous imagining and ther-apy of cancer cells. Plasmid pET28-TZH2 was constructed with a biotin-binding motif (BP), a HER2/neu-binding domain (ZH2), and Doc. Peptide A was expressed in E. coli harboring plasmid pET28-TZH2 and used for the diagnostic imaging of cancer cells. For cancer therapy, peptide B was produced in E. coli with plasmid pET-TCoh containing BP and Coh. BP contains 13 6-NBDG and is biotinylated by biotin synthetase (encoded by birA) of E. coli . The biotinylated motif tethers it to streptavidin- or avidin-conjugated NPs. A body consisting of 58 amino acids is derived from the IgG-binding domain of staphylococcal protein A. ZH2 is
a bivalent a body and displays a high a nity (kD ∼22 pM) to HER2/nue . Cellulosome is a complex protein assembly and composed of the scaffoldin backbone linked to multiple cellulases via the specific interaction of Coh with Doc (kD ∼ 1 × 10−5 μM) . Doc has a flexible conformation and undergoes conforma-tional adjustment upon biding to Coh. The stable conformation of Doc is calcium-dependent. Derived from C. thermocellum DSM1237, the type I Coh and Doc were applied for the non-covalent fusion of peptide A and peptide B. Moreover, TrxA and the 6xHis tag were fused to the N- and C-terminus of peptide A and peptide B, respec-tively. This approach was aimed to improve the peptide solubility and facilitate the peptide purification [21,22].
3.2. Targeting cancer cells by a functionalized peptide
As described earlier, peptide A was composed of TrxA, BP, ZH2, Doc, and 6xHis. To investigate its functionality, peptide A was
Fig. 2. Selective staining of cancer cells by peptide A. Cells were incubated with peptide A for 5 min. After fixed by paraformaldyhyde, cells were stained with DAPI and Phalloidin-TRITC for observation of nucleus (blue) and cytoskeleton (red) by fluorescence microscopy, respectively. Meanwhile, the anti-TrxA antibody was applied for detec-tion of peptide A (green). Keys: HER2/neu-positive cell (upper panel); control cell (bottom panel). The bar scale is 20 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
produced in E. coli strain and the soluble portion was purified using immobilized metal a nity chromatography (IMAC). Breast cancer cells involving MCF-7/her18 (HER2/neu-positive) and MCF-7 (control) were chosen for investigation and administrated with the purified peptide. Derived from MCF-7 cell, MCF-7/Her18 cell carries Her-2 DNA for overexpression of HER2/neu. In contrast to the control cell, HER2/neu-positive cell displayed the green flu-orescence emitted by the HRP secondary antibody upon specifi-cally binding to the anti-TrxA antibody (Fig. 2). It indicates that ZH2 on peptide A is functional to selectively target the HER2/neu biomarker of the cancer cell. In addition, the observation of the cell cytoskeleton (stained with the red fluorescence) surrounded by the green fluorescence suggests that peptide A was located on the cell surface.
Peptide A was further investigated for its feasibility in con-jugation with Qdot. Therefore, peptide A was overproduced and biotinylated in the BirA-expressing E. coli in the presence of bi-otin. By purification with IMAC, recovered peptides were digested with enterokinase to remove TrxA. Peptide A free of TrxA was isolated by IMAC and then conjugated with Qdot605-streptavidin (denoted as TZH2-Qdot) at its biotinylated site. The commercially available Qdot605-streptavidin is a core-shell material coated with a polymer and has the emission maximum near 605 nm. The poly-mer shell enables direct conjugation of streptavidin without re-ducing the optical properties of Qdot. The electrophoresis mobil-ity of the peptide conjugated with Qdot605-streptavidin shifted forwards (Fig. 3(A)) and TZH2-Qdot was subsequently isolated. Cancer cells were administrated with TZH2-Qdot. By the analy-sis of fluorescence microscopy, HER2/neu-positive cell displayed the red fluorescence whereas the red signal was undetected for the control cell (Fig. 3(B)). The Qdot-mediated red fluorescence co-existed with the green fluorescence which was emitted by the antibody against HER2/neu of the fixed cell. It suggests that TZH2-Qdot was located on the surface of cells. In contrast, the scattered red fluorescence overlaid the blue-stained nucleus of the living cells. The result indicates the internalization of TZH2-Qdot into the living cell, and this was confirmed by confocal microscopy (Fig. 3(C)). Taken together, it indicates that the functional ZH2 on peptide A enables selective binding to HER2/neu-positive tumor cells.