Aptamer-mediated delivery of chemotherapy to pancreatic cancer cells

Abstract

Gemcitabine is a nucleoside analog that is currently the best available single-agent chemotherapeutic drug for pancreatic cancer. However, efficacy is limited by our inability to deliver sufficient active metabolite into cancer cells without toxic effects on normal tissues. Targeted delivery of gemcitabine into cancer cells could maximize effectiveness and concurrently minimize toxic side effects by reducing uptake into normal cells. Most pancreatic cancers overexpress epidermal growth factor receptor (EGFR), a trans-membrane receptor tyrosine kinase. We utilized a nuclease resistant RNA aptamer that binds and is internalized by EGFR on pancreatic cancer cells to deliver gemcitabine-containing polymers into EGFR-expressing cells and inhibit cell proliferation in vitro. This approach to cell type-specific therapy can be adapted to other targets and to other types of therapeutic cargo.

FIG. 1.

E07–gemcitabine (Gem) polymer binds to the epidermal growth factor receptor (EGFR)-expressing MiaPaCa-2 cells. (a) Western blots were done using the cell-free extracts from MiaPaCa-2 and HPAF-2 cells. EGFR was detected in the MiaPaCa-2 but not in the HPAF-2 cells. ß-tubulin was used as the loading control. (b) E07-Gem polymer (blue) and E07 (green) showed similar binding to the EFGR expressing MiaPaCa-2 cells. The controls, mE07–Gem polymer (orange) and the mE07 (pink) represent background (non-specific) binding (upper panel). There was a minimal shift in binding to HPAF-2 cells by E07 aptamer alone (green) but not E07–Gem polymer (blue) over background binding (pink and orange, lower panel). Cells were analyzed by FACSCalibur on the FL2-H channel. The number of events counted is plotted on the y-axis. Quantitation of the histograms is presented in tabular form as geometric mean (Geo Mean) for the corresponding peaks.

FIG. 2.

Imaging MiaPaCa-2 cells with E07 and E07–Gem polymer: (a) E07 and (b) E07–Gem polymer aptamers labeled with the fluorophore Alexa-488 bound to the MiaPaCa-2 cells were detected by fluorescence microscopy. No detectable staining above the background was observed when the mutant aptamer (mE07) was used. DAPI was used for the nuclear staining. DAPI, 4',6'-diamindino-2-phenylindole.

FIG. 3.

E07–Gem polymer is internalized by the MiaPaCa-2 cells. E07–Gem polymer (green and pink) and the mE07–Gem polymer (blue and orange) labeled with the fluorophore PE were incubated with the MiaPaCa-2 cells either on ice (upper panel) or at 37°C (lower panel) for 30 minutes. After the binding reactions, the cells were treated with Riboshredder (RS) for 10 minutes at room temperature (pink and orange). The cells were analyzed by using FACSCalibur. The arrow indicates the E07-gem polymer bound to the cells after RS treatment at 37°C (pink), presumably due to aptamer internalization. Quantitation of the histograms is presented in tabular form as Geo Mean for the corresponding peaks.

FIG. 4.

Sequence of the Gem-containing polymer. (a) The template DNA used for in vitro transcription to synthesize the Gem-containing polymer. The T7-promoter sequence (red) is underlined. The 3′-“wing” sequence used for annealing the gem-containing polymer to the E07 aptamer is in green font color. The Gem molecules are denoted as C (blue) in the RNA sequence. (b) The Gem polymer and the corresponding 2′F polymer (control) were run on a 10% denaturing gel to visualize the products. The arrow denotes the Gem polymer and the 2′F polymer of approximate 45 nt length.

FIG. 5.

E07-gem polymer inhibits MiaPaCa-2 cell growth. (a) MiaPaCa-2 cells were treated with the indicated concentrations of E07-Gem polymer to establish a dose-response curve. The percent inhibition {[(untreated – treated)/untreated]×100} is ploted in the y-axis. (b) MiaPaCa-2 and HPAF-2 cells were treated with E07-Gem polymer (400 nM), mE07–Gem polymer (400 nM), Gem-polymer (400 nM), E07–2′F polymer (400 nM), and unpolymerized Gem (Gemzar, 1000 nM) and analyzed for the percentage of cell growth inhibition.

FIG. 6.

Model for the E07 mediated delivery of the GEM-containing polymer. E07 aptamers are represented in green with the 3′ wing extension in red. The GEM-containing polymers (blue) are annealed by Watson-Crick base paring to the E07 aptamers via the wing extension, represented by short vertical lines. Gemcitabine monophosphates (dFdCMP) are represented by the yellow circles distributed along the blue strand. (1) E07–Gem containing polymer binds to the EGFR expressing cells on the plasma membrane and is (2) internalized to the endosome, where the Gem-containing polymer is (3–4) degraded to release dFdCMP into the cytoplasm.