Examination of a DNA Aptamer (TLS11a) as a Cancer-Specific Targeting Agent within Cultured BNL 1ME A. 7R.1 Liver Cancer Cells
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Hepatocellular carcinoma (HCC), the most common form of liver cancer, kills over 650,000 people worldwide every year, primarily in regions where viral hepatitis infections are common. Unfortunately, most HCC cases remain un-diagnosed until late stages of the disease when patient outcome is poor, such that death typically occurs within months to one year of initial diagnosis. In order to better care for these patients, new target-specific approaches are needed to improve early detection and therapeutic intervention. In this thesis, polymer nanoparticles functionalized with a HCC-specific aptamer were examined as potential targeted drug delivery vehicles. In particular, the in vitro interactions (i.e. binding and uptake) of an HCC-specific aptamer, designated TLS11a, were characterized in a hepatoma cell line via live-cell fluorescence imaging. Building of the aptamer-AlexaFluor®546 to the cell surface was found to occur within 20 minutes of aptamer introduction. Shortly thereafter, the aptamer was taken up, presumably by endocytosis, and localized to late endosomes of lysosomes using a pH-sensitive LysoSensor™ Green dye. Next, the cell surface epitope(s) recognized by the TLS11a aptamer were characterized using SDS-PAGE and western blot procedures. A prominent band just over 21 kilodaltons (kDa) was revealed in the cell surface protein fraction, the hydrophilic fraction and in whole cell lysate but not in hydrophobic membrane fraction. Implications of this result are still being examined, but suggest that the TLS11a may recognize an extracellular peripheral membrane protein. Using a modified nanoprecipitation technique, aptamer-functionalized polymer nanoparticles containing poly(lactic-co-glycolic-acid) (PLGA) and poly(lactide)-b-poly(ethylene glycol) (PLA-PEG) were prepared in an organic acetone solvent then loaded with the chemotherapeutic agent, doxorubicin. Following treatment of cultured HCC cells with drug-loaded nanoparticles, there was no apparent difference in cytotoxicity between nanoparticles with or without the aptamer targeting agent; however, both nanoparticle-based treatments showed greater cytotoxicity over free doxorubicin in solution or blank nanoparticles supporting the nanoparticle itself as a viable drug delivery vehicle. Additional studies are needed to fully optimize the nanoparticle design to ensure sufficient amounts of aptamer are present on each nanoparticle and that they are readily available for molecular recognition of the target. Taken together, these preliminary studies provide support for further examination of the TLS11a as a HCC-specific targeting agent for use in polymer nanoparticle drug delivery.