title: Selection of a Novel Aptamer Against Vitronectin Using Capillary Electrophoresis and Next Generation Sequencing.
abstract: Breast cancer (BC) results in ~40,000 deaths each year in the United States and even among survivors treatment of the disease may have devastating consequences, including increased risk for heart disease and cognitive impairment resulting from the toxic effects of chemotherapy. Aptamer-mediated drug delivery can contribute to improved treatment outcomes through the selective delivery of chemotherapy to BC cells, provided suitable cancer-specific antigens can be identified. We report here the use of capillary electrophoresis in conjunction with next generation sequencing to develop the first vitronectin (VN) binding aptamer (VBA-01; Kd 405 nmol/l, the first aptamer to vitronectin (VN; Kd= 405 nmol/l) , a protein that plays an important role in wound healing and that is present at elevated levels in BC tissue and in the blood of BC patients relative to the corresponding nonmalignant tissues. We used VBA-01 to develop DVBA-01, a dimeric aptamer complex, and conjugated doxorubicin (Dox) to DVBA-01 (7:1 ratio) using pH-sensitive, covalent linkages. Dox conjugation enhanced the thermal stability of the complex (60.2 versus 46.5°C) and did not decrease affinity for the VN target. The resulting DVBA-01-Dox complex displayed increased cytotoxicity to MDA-MB-231 BC cells that were cultured on plasticware coated with VN (1.8 × 10-6mol/l) relative to uncoated plates (2.4 × 10-6mol/l), or plates coated with the related protein fibronectin (2.1 × 10-6mol/l). The VBA-01 aptamer was evaluated for binding to human BC tissue using immunohistochemistry and displayed tissue specific binding and apparent association with BC cells. In contrast, a monoclonal antibody that preferentially binds to multimeric VN primarily stained extracellular matrix and vessel walls of BC tissue. Our results indicate a strong potential for using VN-targeting aptamers to improve drug delivery to treat BC.
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.|Department of Molecular Medicine and Translation Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.|Current address: RayBiotech, Norcross, Georgia, USA.
authors.forename: Christopher H
authors.initials: CH
authors.lastname: Stuart
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina, USA.|Current address: Department of Chemistry and Biochemistry, Swarthmore College,Swarthmore, Pennsylvania, USA.
authors.forename: Kathryn R
authors.initials: KR
authors.lastname: Riley
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.|Current address: Department of Food Engineering, Faculty of Engineering, Adnan Menderes University, Aydin, Turkey.
authors.forename: Olcay
authors.initials: O
authors.lastname: Boyacioglu
authors.affiliation: Brain Tumor Center of Excellence, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Denise M
authors.initials: DM
authors.lastname: Herpai
authors.affiliation: Brain Tumor Center of Excellence, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Waldemar
authors.initials: W
authors.lastname: Debinski
authors.affiliation: Department of Pathology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Shadi
authors.initials: S
authors.lastname: Qasem
authors.affiliation: Wake Forest Institute of Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: Frank C
authors.initials: FC
authors.lastname: Marini
authors.affiliation: Department of Chemistry, Wake Forest University, Winston-Salem, North Carolina, USA.
authors.forename: Christa L
authors.initials: CL
authors.lastname: Colyer
authors.affiliation: Department of Cancer Biology, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.|Department of Molecular Medicine and Translation Science, Wake Forest School of Medicine, Winston-Salem, North Carolina, USA.
authors.forename: William H
authors.initials: WH
authors.lastname: Gmeiner