UCSF

Abstract

Introduction: Liver cancer is on the rise in the US with an increase of 2.7% new cases per year and 2.6% increase of death rates (4). The current treatments include surgical, tumor ablation, embolization, radiation, and chemotherapy. Many patients are diagnosed much too late in their progression of liver cancer and cannot undergo surgical treatments. Chemotherapy has become a promising possible treatment for liver cancer although its largest limitation is systemic toxicity. It has been found that 50-70% of the chemotherapy agent is not absorbed by the liver cancer cells and is circulated through the rest of the body (6). This amount of chemotherapy agent in the blood circulation can cause severe cardiac toxicity. Luckily, chemotherapy filter devices are currently being researched and may solve the limitation of systemic toxicity. Current devices include ionic based, magnetic based, and DNA based (6). The experiments described in this paper will be focusing on DNA based chemotherapy filters and Doxorubicin as the chemotherapy agent. The goal of this research is to create a simple and quick assay to measure the efficiency of a functionalized DNA chemotherapy filter device.

Materials and Methods: In order to simulate an in-vivo usage of a DNA chemotherapy filter device, 50 mg of genomic DNA (Salmon sperm, Sigma-Aldrich, St. Louis, MO) was mixed together with 50 mL of 0.05M Doxorubicin in a beaker. The solution was mixed using a magnetic stir bar, allowing the DNA to bind Doxorubicin. In order to optimize the assay, three different types of extraction agents were tested; 0.3M solution of sodium acetate, 10%wt/vol solution of trichloroacetic acid (TCA), and 35%wt/vol solution of silver nitrate. These extraction agents were mixed with the DNA and Doxorubicin solution to analyze how efficient they were at removing bound Doxorubicin from DNA. The resulting samples were analyzed using both a fluorospectrometer (SPECTRA max M2, Molecular Devices) and higher performance liquid chromatography (HPLC) (SPECTRA max M2, Molecular Devices). Finally, all resulting graphs were created using Microsoft Excel (Microsoft Office 2011).

Results and Discussion: It was found that the 35%wt/vol solution of silver nitrate was the most effective in releasing Doxorubicin from DNA. The 10% wt/vol solution of TCA was found to be the second most efficient followed by the 0.3M solution of sodium acetate. Silver nitrate was able to retrieve 98.12% of the Doxorubicin initially bound to DNA according to the HPLC data, and 80.66% according to the fluorospectrometer data. TCA was able to retrieve 79.21% of the initial Doxorubicin from DNA according to the HPLC data and 85.91% according to the fluorospectrometer data. Sodium acetate was able retrieve 74.35% of the initial Doxorubicin from DNA according to the HPLC data and 17.27% according to the fluorospectrometer data. The discrepancy between the HPLC data and the fluorospectrometer data is most likely due to the quenching limitation of the fluorospectrometer causing a lower Doxorubicin concentration reading than the actual concentration. HPLC appears to be the more reliable and accurate device for measuring Doxorubicin concentration in solution (13).

Conclusion: This paper describes a simple and quick method of extracting Doxorubicin from DNA and then measuring the amount of unbound Doxorubicin. This assay will assist in the optimization of a functionalized DNA-based chemotherapy filter device by allowing the users to test how efficient a device is at absorbing excess Doxorubicin in a simple and quick method. This research uses genomic DNA in place of iron oxide bound DNA. The current model of the functionalized DNA-based chemotherapy filter device uses iron oxide bound DNA that allows the DNA to be magnetically bound to the filter device. Future investigations will test this assay using iron oxide bound DNA instead of genomic DNA. This will allow for a more accurate representation of the efficiency of the extraction agents ability to release Doxorubicin from DNA. This is done in order to measure unbound Doxorubicin and thus the amount of Doxorubicin the DNA is able to bind.