Effective Integration of Targeted Tumor Imaging ... - ACS Publications

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Effective Integration of Targeted Tumor Imaging and Therapy using Functionalized InP QDs with a VEGFR2 Monoclonal Antibody and a miR-92a Inhibitor Yi-Zhou Wu, Jie Sun, Yaqin Zhang, Maomao Pu, Gen Zhang, Nongyue He, and Xin Zeng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02641 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on April 6, 2017

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ACS Applied Materials & Interfaces

Effective Integration of Targeted Tumor Imaging and Therapy using Functionalized InP QDs with a VEGFR2 Monoclonal Antibody and a miR-92a Inhibitor

Yi-Zhou Wu,1† Jie Sun,2,3† Yaqin Zhang,4 Maomao Pu,1 Gen Zhang,1* Nongyue He,5* and Xin Zeng,6*

1

Department of Cell Biology, School of Basic Medicine, Nanjing Medical University, Nanjing

211166, China 2

The Center for Hygienic Analysis and Detection, School of Public Health, Nanjing Medical

University, Nanjing 211166, China 3

Safety Assessment and Research Center for Drug, Pesticide and Veterinary Drug of Jiangsu Province,

School of Public Health, Nanjing Medical University, Nanjing 211166, China 4

Department of Biochemistry and Molecular Biology, School of Basic Medicine, Nanjing Medical

University, Nanjing 211166, China 5

The State Key Laboratory of Bioelectronics, Department of Biological Science and Medical

Engineering, Southeast University, Nanjing 210096, China 6

Maternal and Child Health Institute, Nanjing Maternity and Child Health Care Hospital, Nanjing

210029, China

* Corresponding author: 1. Gen Zhang, Ph.D. E-mail: [email protected] Department of Cell Biology, School of Basic Medicine, Nanjing Medical University, Nanjing 211166, China

† Yi-Zhou Wu and Jie Sun contributed equally to this work

Keywords: InP, near infrared fluorescent, VEGFR2, microvesicle, apoptosis. 1

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ABSTRACT: Rapid diagnosis and targeted drug treatment require agents that possess multiple functions. Nanomaterials that facilitate optical imaging and direct drug delivery have shown great promise for effective cancer treatment. In this study, we first modified near infrared fluorescent indium phosphide quantum dots (InP QDs) with a vascular endothelial growth factor receptor 2 (VEGFR2) monoclonal antibody to afford targeted drug delivery function. Then, a miR-92a inhibitor, an antisense microRNA that enhances the expression of tumor suppressor p63, was attached to the VEGFR2-InP

QDs

via

electrostatic

interactions.

The

functionalized

InP

nanocomposite (IMAN) selectively targets tumor sites and allows for infrared imaging in vivo. We further explored the mechanism of this active targeting. The IMAN was endocytosed and delivered in the form of microvesicles via VEGFR2-CD63 signaling. Moreover, the IMAN induced apoptosis of human myelogenous leukemia cells through the p63 pathway in vitro and in vivo. These results indicate that the IMAN may provide a new and promising chemotherapy strategy against cancer cells, particularly by its active targeting function and utility in non-invasive three-dimensional tumor imaging.

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1. INTRODUCTION Quantum dots (QDs) have been previously used for cellular imaging, such as for labeling and tracking specific molecular targets in live cells1. Due to tissue absorption and diffusion, visible QDs provide a poor signal-to-background ratio in thicker tissue sections and thus cannot be used to examine deeper tissues, particularly within the visible range of fluorescence2. Near infrared (NIR) light can penetrate living tissues by several centimeters due to the low absorbance of intrinsic tissue chromophores such as oxy- and deoxy-hemoglobin, melanin, water and lipids3. Based on this unique deeper tissue-penetrative property and non-ionizing and non-radioactive advantages, NIR imaging techniques have attracted extensive attention for real-time and dynamic monitoring/tracing of biological signals in living animals4. Among many NIR quantum dots (QDs), colloidal indium phosphide (InP) QDs have a narrow photoluminescence range that is tunable across near and mid-infrared regions5. Moreover, InP QDs do not contain heavy metal elements that may induce cytotoxicity6. Developing effective cancer therapies remains one of the most challenging tasks of the scientific community. The major limitation inherent to most conventional anticancer chemotherapeutic agents is their lack of tumor selectivity. Conventional chemotherapy delivers anticancer drugs indiscriminately to tumors and normal tissue. Therefore, it is necessary to exploit tumor-targeted drug delivery techniques to avoid the undesirable systemic side-effects. Immunological targeting of cancer cells with monoclonal antibodies (mAb) has been explored. Singh et al. initially reported

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vascular endothelial growth factor receptor 2 (VEGFR2) as a characteristic molecule of tumor angiogenesis7. VEGFR2 is largely considered to be the primary VEGF family receptor that drives angiogenesis and vascular permeability. Biological and preclinical evidence have suggested that blocking of VEGFR2 could be a promising strategy for inhibiting tumor-induced angiogenesis8-9. Treatment with DC101, a rat mAb against murine VEGFR2, has consistently induced robust inhibition of the spread and growth of metastases in several angiogenesis and tumor models10-11. A second-generation of fully human IgG1 mAb against VEGFR2, Ramucirumab, has recently emerged as a novel antiangiogenic treatment. Numerous phase I-II trials in various advanced human solid malignancies, such as in breast cancer, gastric cancer, renal cell carcinoma, hepatocarcinoma, colorectal cancer, and non-small cell lung cancer, have reported promising clinical antitumoral efficacy and tolerability of Ramucirumab12-13. Leukemia is a hematopoietic malignancy that leads to acute death within a few weeks to several months14. In this study, we attempted to target leukemia K562 cells that highly expressed VEGFR2 using VEGFR2 antibody-modified InP QDs. Inhibition of oncogenic miRNAs (OncomiRs) have potential applications for cancer treatment15. The expression of OncomiR miR-92a is up-regulated in many leukemia cell lines. miR-92a inhibits the expression of tumor suppressor p63 to increase the proliferation of myeloid cells16-17. In this study, a miR-92a inhibitor was used to suppress the intracellular expression of miR-92a. Based on biological mechanisms, we designed an InP nanocomposite (IMAN) containing both the

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VEGFR2 monoclonal antibody and the miR-92a inhibitor to target on K562 cells and induce apoptosis. It is of interest and important to determine how the IMAN, as a drug carrier, enters cancer cells. There are currently two ways for nanomaterials to enter cells: by causing the formation of “holes” on the surface of cell membranes and by internalization and endocytosis by cell membranes. However, the precise molecular mechanism has not been fully elucidated. Tetraspanin CD63 was recently found to be involved in intracellular vesicles and the endocytosis pathway18. Here, we revealed that VEGFR2-CD63 signaling is the key factor mediating entry of the IMAN into cells. Our findings demonstrated that the IMAN simultaneously enabled three-dimensional NIR visualization and induced cancer cell apoptosis in vitro and in vivo. Hence, the IMAN bridges the gap between drug delivery and targeted imaging modalities, representing a novel and promising chemotherapy strategy against cancer.

2. RESULTS 2.1. Synthesis and characterization of IMAN First, functionalized InP QDs comprising an InP core and a ZnS shell, were synthesized as previously described19-21. Water-soluble InP QDs were fabricated by a ligand exchange reaction with mercaptopropionic acid at pH=10-1120. The InP QDs were then characterized by TEM imaging. The images revealed a homogeneous distribution of InP QDs and an average particle size of approximately 7 nm (Fig. 1a). The crystal lattice distance was further evaluated by HRTEM imaging (0.32 nm in

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Fig. 1b), similar to a previous report22. The energy dispersive X-ray spectrum (EDS) collected from the InP QDs clearly showed enhanced In and P signals (Fig. 1c). Second, the InP QDs were modified with a high affinity VEGFR2 monoclonal antibody. To determine the modification efficiency, the VEGFR2-InP QDs and free VEGFR2 antibody were separated by centrifugation. SDS-PAGE analysis of the precipitate containing the VEGFR2-InP QDs showed that VEGFR2-InP QDs ran slower at 24 h than at 1 h, indicating that an increasing amount of antibody bound with the InP QDs (Fig. 1d). In the supernatant containing free VEGFR2 antibody, protein quantification assay showed that less free antibodies remained in the reaction system at 24 h (Fig. 1e). Both results demonstrated that VEGFR2 antibody could effectively bind with InP QDs and that the binding efficiency increased with time. Third, the IMAN for modifying the VEGFR2-InP QDs with the miR-92a inhibitor was synthesized. The miR-92a inhibitor loading efficiency was further evaluated by OD analysis, which indicated that the loading rate of the miR-92a inhibitor onto the IMAN was approximately 10%. The 600- to 700-nm light emitted by the InP QDs is particularly interesting for biomedical labeling because this range of light can rapidly penetrate human tissues20. The photoluminescence peak at approximately 640 nm corresponded to the most stable wavelength of light emitted from the IMAN (Fig. 1f). According to a previous report, the interaction between nanoparticles and host materials could cause a redshift of the absorption peak, which could be used to preliminarily determine whether the antibody interacted with the nanoparticles23-24. InP QDs solution had a

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notable absorption peak at 600 nm (Fig. 1g). Due to the surface plasma resonance absorption, the UV-visible absorption peak was redshifted after binding with the VEGFR2 antibody (Fig. 1g). The intercoagulation increased the grain diameter of the InP QDs, resulting in a redshift of the absorption peak. The IMAN was also characterized via nanoparticle tracking analysis (NTA) to measure the hydrodynamic diameter of the nanocomposites in their dispersion state. The average particle size of the IMAN measured in the cell culture medium was approximately 91 nm (87.04%) (Fig. 1h). To determine whether the miR-92a inhibitor could be effectively released from the IMAN, an electrophoretic mobility shift assay (EMSA) was performed (Fig. 1i). Compared to free miR-92a inhibitor moving to the positive electrode (lane 1), the VEGFR2-InP QDs completely prevented the mobility of the miR-92a inhibitor (lane 2). This result was probably due to the negative charges of the miR-92a inhibitor being counteracted by the positively charged InP QDs or because the newly formed IMAN complex was too large to enter the gel. As expected, a small amount of miR-92a inhibitor was initially detected after incubation with 0.2 mM GSH (lane 3) and further increased with 1 mM GSH (lane 4). After incubation with 2 mM GSH, which corresponds to the intracellular concentration of GSH in erythrocytes25, the mobility of the miR-92a inhibitor was completely recovered (lane 5). The EMSA results were consistent with our experimental design. Because the negatively charged GSH contains a thiol ligand, which has a stronger affinity to the InP core, GSH might counteract the positive charge of the InP QDs via place exchange and resulted in

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dissociation of the miR-92a inhibitor from the IMAN. The release rate of the miR-92a inhibitor from the IMAN was further revealed to be time-dependent (Fig. 1j). Thus, it was established that incubation with GSH at intracellular concentrations facilitates the release of the miR-92a inhibitor, which is then able to inhibit the expression of miR-92a.

Figure 1. Synthesis and characterization of the IMAN. (a) TEM image of InP QDs. Scale bar: 20 nm. (b) HRTEM image of InP QDs. Scale bar: 5 nm. (c) EDS analysis of InP QDs. (d) The VEGFR2 antibody bound with InP QDs for 0 h (1), 1 h (2), 6 h (3) or 24 h (4) and followed by centrifugation. The precipitate containing the VEGFR2-InP QDs were analyzed by SDS-PAGE and

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coomassie brilliant blue staining. (e) The supernatant containing free VEGFR2 antibody was quantified using a BCA assay (*, P

Effective Integration of Targeted Tumor Imaging ... - ACS Publications

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