Nucleic Acid Aptamer - Mediated DNA Killing Of Doxorubicin On MUC1 - Positive Tumor Cells

Objective:Chemotherapy is the primary treatment for advanced metastatic breast cancer. However, conventional chemotherapy faces themajor obstacle of systematic toxicity. These adverse reactionsseriously limit the efficacy of chemotherapy to eliminate metastatic cancer cells because the drug dosage and treatment frequency are often curbed by patients’ intolerance to treatment-associated side effects.One strategy for overcoming the systemic toxicity of chemotherapy is targeted tumor therapy. Because a targeted drug delivery system can selectively guide therapeutics into tumor cells, the effective accumulation of anticancer agents occurs in thetumorbut not in normal tissue. MUC1 has been recognized as an important molecular target for cancer treatment. It is a cell surface glycoprotein thatis widely overexpressed in many types of adenocarcinomas, including cancers of the lung, colon, pancreas,stomach,ovary, and breast. Prior studies have demonstrated that MUC1 in cancer cells is under glycosylated, exposing the protein backbone and increasing the proteinogeneic accessibility by ligands such as antibodies or aptamers. This feature, together with the fact that MUC1 is overexpressed in most carcinoma cells, makes MUC1 an attractive therapeutic target. Aptamers (Apt) are short, single-stranded oligonucleotides (DNA and RNA) that can form complicated three dimensional structures and bind with a target molecule with high specificity and affinity. DNA tetrahedron (Td) holds some advantages as a potential drug carrier of the anticancer agent doxorubicin. Thus far, however, there have been no reports in the literature on using aptamer-guided DNA tetrahedron for targeted drug delivery to cancer cells. In this study,we attempted to construct the first aptamer-tetrahedron complex (Apt-Td) for the targeted delivery of doxorubicin to MUC1-positive cancer cells. The basic properties of the Apt-Td complex and its efficacy as a targeted drug delivery system were evaluatedin vitro, using the MUC1-expressing MCF-7 breast cancer cell line as the model system. We here report that Apt-Td delivers doxorubicin to MUC1-positive breast cancer cells in a targeted manner.Methods:Td or Apt-Td was created using the principle of DNA complementary base pairing in a self-assembled manner. Flow cytometric analysis was used to test the specific binding of Apt-Td to MUC1 positive cancer cells and MUC1 negative control cells. The drug-loading capacity of Apt-Dox, Td-Dox, or Apt-Td-Dox was evaluated according to fluorescence spectrum of the Doxorubicin and analyzed by Synergy4 analyzer. Confocal microscopy, flow cytometric, and cell co-culture experiment was utilized to monitor the cellular uptake of Apt-Td-Dox to the both type of cells. The cytotoxicity of Apt-Td-Dox to MCU1 positive cancer cells was tested by the MTS assay.Results:The complex thus formed (Apt-Td) had an average size of 12.38 nm and was negativly charged (-10.mV). Similar to the MUC1 aptamer, the Apt-Td could preferentially bind with MUC1-positive cancer cells. A drug loading experiment revealed that each Apt-Td complex could carryapproximately 25 Doxmolecules. Moreover, Apt-Td selectively delivered Dox into the MUC1-positive cancer cells but reduced Dox uptake by the MUC1-negative control cells. Dox-loaded Apt-Td also induced a significantly higher cytotoxicity to the MUC1-positive cancer cells versus the MUC1-negative control cells in vitro (p<0.01).Conclusion:Apt-Td could be constructed in the manner described in this study, and and may potentially serve as a drug carrier in the targeted treatment of MUC1-expressing tumors.