br Corresponding author Corresponding author at Department o
∗ Corresponding author. ∗∗ Corresponding author at: Department of Chemical Engineering, Feng Chia Uni-versity, 100 Wenhwa Road, Taichung 40724, Taiwan.
sponse of patients, which provides a reliable means to effectively adjust the treatment strategy based on the individual feedback.
Quantum dots (QDs) are luminescent semiconductor nanocrys-tals with unique optical characteristics and are well recognized as an ideal probe for bioimaging applications . They have a narrow and size-tunable emission spectrum, exhibit the strong and long-lasting luminescence, and tolerate the photo and chemical degra-dation. The solubilization of hydrophobic QDs requires surface modification. QDs conjugated with solubilizer such as the diagnos-tic or therapeutic agents have a wide application for cancer diag-nosis and therapy . On the other hand, magnetic NPs (MNPs) are attractive to many biomedical applications mainly including bioimaging, magnetic hyperthermia, drug delivery, and biodetec-tion . They consist of the iron oxide core and can be properly reshaped by surface modification to reduce their toxicity and to in-crease their stability and solubility. MNPs can be used as the mag-netic resonance imaging (MRI) GW3965 agent whereas they have poor signal intensity and are inapplicable to long-term tracking . Therefore, one entity combined with the advantage of QDs and MNPs is appealing for the theranostic application.
The methods for incorporation of QDs and MNPs into one entity have been reported by the multi-step and one-pot chemical reac-tions [9–11]. However, “green nanomedicine” instructs the imple-mentation of nanotechnology in compliance with the 12 principles of green chemistry . In this study, the issue was addressed by
development of a hybrid peptide carrying QDs, MNPs, and a target-ing ligand. As a poof-of-concept approach, the nanomaterials-based peptide was investigated for its e cacy on HER2/nue-positive breast cancer cells. HER2/nue belongs to the family of human epi-dermal growth factor tyrosine kinase receptor , and its abnor-mal expression level increases cell proliferation and decreases cell apoptosis . Consequently, this multifunctional peptide enabled detecting and destroying HER2/nue-positive breast cancer cells.
2. Materials and methods
2.1. Plasmid construction
The bivalent anti-HER2/neu a body (ZH2) containing two identical domains of ZHER2:342  was synthesized by Mission Biotech Co. (Nangang, Taiwan). The synthesized DNA subject to the XhoI-XmnI digestion was subcloned into plasmid pDW363 . It resulted in plasmid pDW-ZH which harbors the fusion of the BirA-mediated biotinylation motif (BP) and ZH2 (BP-ZH2). The BP-ZH2 DNA was amplified by the polymerase-chain reaction (PCR) with designed primers (5 -GGGGGCCTGAACGATATTTCG-3 and 5 -CCAGTGCCAAGCTTTTATTT-G-3 ). Cleaved by HindIII, the PCR DNA was ligated into plasmid pJF-TrxFus (Lab. Collection) pretreated with HindIII and SmaI. It produced plasmid pJF-TZH2 with the construction of TrxA fused with BP-ZH2 (TrxA-BP-ZH2). After the NdeI-HindIII cleavage, the TrxA-BP-ZH2 DNA was recovered from plasmid pJF-TZH2 and incorporated into plas-mid pET28 to give plasmid pET28-TZH2. Finally, the dockerin (Doc) domain from Clostridium thermocellum DSM1237 was am-plified from plasmid pGPB-DocI  using PCR with designed primers (5 -ATATGCTGCAGAAAG-TACCTGGTACTCCTT-3 and 5 - TGAACAAGCTTAGTTCTTGTACGGCAAT-GT-3 ). Meanwhile, PCR was conducted to amplify the DNA backbone of plasmid pET28-TZH2 using designed primers (5 -CTAATCTGCAGCATGCCGAGCCTT-TTC-3 and 5 -AAACGTCGTCAGCGTCGTCG). These two PCR DNAs were treated with the PstI-HindIII cut and spliced together to obtain plasmid pET28-TZH2 for expression of peptide A. Al-ternatively, plasmid pET-TCoh for expression of peptide B was constructed with the tri-fusion of TrxA, BP, and the cohesin (Coh) domain. This plasmid was obtained by incorporation of Coh, recovered from plasmid pRed-Coh  by BglII-HindIII di-gestion, into the corresponding sites of plasmid pET28-TZH2. In addition, birA was amplified from E. coli by PCR with designed primers (5 -ATATCATATGAAGGATAACACCGTGC-3 and 5 -CGCG-AAGCTTATTTTTCTGCACTACGCAGGG-3 ) and subcloned into the NdeI-HindIII site of plasmid pTH18cr  to produce pTH-BirA.