br E cient targeting of
3.3. E cient targeting of cancer Methoctramine by peptide-Qdot
It was intriguing to evaluate the targeting e ciency of TZH2-Qdot. To do so, the commercially available anti-ErbB2 A body® molecule was used for comparison. Like peptide A, anti-ErbB2 A -body® molecule contains the ZH2 domain. According to the man-ufacture, this molecule is biotinylated at its unique C-terminal cysteine using a maleimide-biotin reagent. In addition, anti-ErbB2 A body® molecule was conjugated with Qdot605-steptavidin (de-noted as A body-Qdot) for cell imagining. Cancer cells were then administrated with either TZH2-Qdot or A body-Qdot. As shown in Fig. 4, both conjugates emitted red fluorescence within the HER2/neu-positive cell after the administration was conducted for 5 min. The fluorescence signal increased with the increasing time of administration. Moreover, more fluorescence spots were de-tected for the TZH2-Qdot-treated cell than those for the A body-treated cell. The result suggests the high e ciency of TZH2-Qdot (i.e. peptide A conjugated with Qdot605-steptavidin) for targeting HER2/neu-positive cancer cells.
3.4. Theranostic treatment of cancer cells with the hybrid peptide
As described earlier, peptide B was composed of TrxA, BP, Coh, and 6xHis. To achieve our ultimate goal, peptide B was overpro-duced and biotinylated in the BirA-expressing E. coli. The resulting peptide free of TrxA was generated with the enterokinase diges-tion and purified by IMAC. Peptide B was conjugated with SiMAG-streptavidin (denoted as peptide-MNP) at the biotinylated site and peptide-MNP was then recovered by the Magnetic separator (Chemicell). The commercially available SiMAG-streptavidin is the aqueous dispersion of magnetic silica particles. Conjugated with streptavidin, the particle is composed of the maghemite core and the non-porous silica matrix. Moreover, peptide-MNP was mixed with TZH2-Qdot in the presence of calcium. The specific interaction of Doc with Coh on two peptides resulted in the self-assembled material (i.e. the hybrid peptide) which was recovered by the Mag-netic separator. The analysis of fluorescence microscopy showed that the hybrid peptide (denoted as Qdot-MNP) emitted the Qdot-mediated fluorescence but the signal was absent for peptide-MNP
Fig. 3. Selective staining of cancer cells by TZH2-Qdot conjugate. (A) The electrophoresis analysis of mobility. The mobility of Qdot (left well) and TZH2-Qdot conjugate (right well) was analyzed in 1% agarose gel. (B) The analysis of cell imaging by fluorescence microscopy. The administration of cancer cells with TZH2-Qdot conjugate was carried out by co-incubation for 10 min. The anti-HER2/neu (9G6) antibody was applied to the fixed and living cancer cells for detailed observation. The fluorescence signals were detected for stained nucleus (blue), Qdot (red), and HER2/neu (green), respectively. All images were merged and shown on the right. Keys: upper panel, HER2/neu-positive cell; bottom panel, control cell. The bar scale is 20 μm. (C) The analysis by confocal microscopy. The inset in part (B) was magnified by the analysis of confocal microscopy. The arrow indicates the internalized TZH2-Qdot conjugate. The bar scale is 10 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(Fig. 5(A)). It indicates that Qdot-MNP bioconjugate comprises the non-covalent fusion of peptide-MNP with TZH2-Qdot.
It is recognized that SKBR3 cell is usually used as a preclin-ical model for investigation of the HER2/neu-targeted therapies. Therefore, breast cancer cell lines involving SKBR3 cell (HER2/neu-positive) and MDA-MB-231 cell (control) were administrated with either Qdot-MNP or peptide-MNP. The result showed that the Qdot-emitted fluorescence signal appeared in HER2/neu-positive cells receiving Qdot-MNP instead of peptide-MNP (Fig. 5(B)). Ob-viously, Qdot-MNP displays a function of cell imagining similar to TZH2-Qdot (Fig. 3(B)). The e cacy of Qdot-MNP for the cell treatment was further investigated by hyperthermia. This was car-ried out by applying the AMF generator to Qdot-MNP-treated can-cer cells. As a result, the cell viability of HER2/neu-positive cells drastically dropped to around 10%. The result indicates that the