Treatment with doxorubicin (Dox) results in serious systemic toxicities that limit effectiveness for cancer treatment and cause long-term health issues for cancer patients. We identified a new DNA aptamer to prostate-specific membrane antigen (PSMA) using fixed sequences to promote Dox binding and developed dimeric aptamer complexes (DACs) for specific delivery of Dox to PSMA+ cancer cells. DACs are stable under physiological conditions and are internalized specifically into PSMA+ C4-2 cells with minimal uptake into PSMA-null PC3 cells. Cellular internalization of DAC was demonstrated by confocal microscopy and flow cytometry. Covalent modification of DAC with Dox (DAC-D) resulted in a complex with stoichiometry ~4:1. Dox was covalently bound in DAC-D using a reversible linker that promotes covalent attachment of Dox to genomic DNA following cell internalization. Dox was released from the DAC-D under physiological conditions with a half-life of 8 hours, sufficient for in vivo targeting. DAC-D was used to selectively deliver Dox to C4-2 cells with endosomal release and nuclear localization of Dox. DAC-D was selectively cytotoxic to C4-2 cells with similar cytotoxicity as the molar equivalent of free-Dox. In contrast, DAC-D displayed minimal cytotoxicity to PC3 cells, demonstrating the complex displays a high degree of selectivity for PSMA+ cells. DAC-D displays specificity and stability features that may be useful for improved delivery of Dox selectively to malignant tissue in vivo.

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 × 10−6 mol/l), or plates coated with the related protein fibronectin (2.1 × 10−6 mol/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.

Doxorubicin (Dox) is widely used for breast cancer treatment but causes serious side effects including cardiotoxicity that may adversely impact patient lifespan even if treatment is successful. Herein, we describe selective conjugation of Dox to a single site in a DNA hairpin resulting in a highly stable complex that enables Dox to be used more effectively. Selective conjugation of Dox to G15 in the hairpin loop was verified using site-specific labeling with [2-15N]-2′-deoxyguanosine in conjunction with [1H–15N] 2D NMR, while 1:1 stoichiometry for the conjugate was validated by ESI-QTOF mass spectrometry and UV spectroscopy. Molecular modeling indicated covalently bound Dox also intercalated into the stem of the hairpin and stability studies demonstrated the resulting Dox-conjugated hairpin (DCH) complex had a half-life >30 h, considerably longer than alternative covalent and noncovalent complexes. Secondary conjugation of DCH with folic acid (FA) resulted in increased internalization into breast cancer cells. The dual conjugate, DCH-FA, can be used for safer and more effective chemotherapy with Dox and this conjugation strategy can be expanded to include additional anticancer drugs.

Aim:To evaluate the potential use of zinc chelation for prostate cancer therapy using a new liposomal formulation of the zinc chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN). Materials & methods: TPEN was encapsulated in nontargeted liposomes or liposomes displaying an aptamer to target prostate cancer cells overexpression prostate-specific membrane antigen. The prostate cancer selectivity and therapeutic efficacy of liposomal (targeted and nontargeted) and free TPEN were evaluated in vitro and in tumor-bearing mice. Results & conclusion: TPEN chelates zinc and results in reactive oxygen species imbalance leading to cell death. Delivery of TPEN using aptamer-targeted liposomes results in specific delivery to targeted cells. In vivo experiments show that TPEN-loaded, aptamer-targeted liposomes reduce tumor growth in a human prostate cancer xenograft model.