In vitro evaluations of multifunctional nanoparticles for simultaneous EGFR gene silencing and drug delivery in pancreatic cancer

• Online Access: http://hdl.handle.net/2047/d10018736

Pancreatic cancer, a lethal malignancy is difficult to diagnose and is associated with poor prognosis due to distal dissemination of the cancer to organs such as the liver, spleen and gastrointestinal tract. Epidermal growth factor receptor (EGFR), a member of ErbB family of growth factor receptors, is over-expressed in pancreatic cancer and is an important indicator of poor prognosis and therapeutic outcomes. Cetyltriethylammonium bromide (CTAB)-modified positively-charged poly(ethylene oxide)-poly(epsilon-caprolactone) (PEO-PCL) nanoparticles loaded with both EGFR-silencing siRNA and paclitaxel (PTX) were prepared and characterized by determining particle size and surface charge found to be approximately 260 nm and 10 mV respectively. When 1 nM siRNA was loaded on 2 mg of CTAB-modified PEO-PCL nanoparticles, the loading capacity and efficiency were 6.5 μg/2 mg and 97%, respectively. Human pancreatic adenocarcinoma (Panc-1) cells were incubated for 3 hours with PEO-modified PCL nanoparticles loaded with siGLO siRNA and OregonGreen®-labeled PTX and viewed under a fluorescence microscope to confirm nanoparticle uptake and distribution in the cells. The baseline EGFR expression in Panc-1 cells was confirmed by Western blot and immunocytometric analysis. The EGFR silencing efficacy studies using aqueous solution and nanoparticle formulations was examined with RT-PCR and Western blot analysis. These studies were carried out by extracting RNA and protein from Panc-1 cells treated with EGFR silencing siRNA- and scrambled siRNA-loaded CTAB-modified-PEO-PCL nanoparticles. For the Western blot and PCR EGFR band intensity was the least for protein extracted from cells treated with EGFR siRNA loaded nanoparticles compared to band intensity from protein extracted from both untreated cells and cells treated with scrambled siRNA loaded nanoparticles. The results of an MTS assay showed that PTX loaded CTAB-modified-PEO-PCL nanoparticles had a 3-fold lower IC50 value of 100nM compared to PTX in solutions, which had an IC50 of 300nM. Similarly siRNA loaded CTAB-modified PEO-PCL nanoparticles showed a greater reduction in percent cell viability compared to siRNA in solution. And finally combination of EGFR siRNA + PTX loaded CTAB-modified PEO-PCL nanoparticles showed greater reduction in percent cell viability compared to using either alone or PTX loaded nanoparticles, scrambled siRNA loaded nanoparticles and combination of scrambled siRNA + PTX loaded nanoparticles. Lastly, both quantitative and qualitative apoptosis studies were carried out using treated and untreated Panc-1 cells. Caspase 3/7 assay was performed to quantitatively determine that maximum apoptosis occurs in cells incubated with a combination of EGFR siRNA and PTX loaded nanoparticles. Qualitative TUNEL assay was performed to evaluate the enhancement in apoptotic cell death due to combination therapy. In comparison to the other treatments combination of EGFR siRNA + PTX loaded nanoparticles showed the most apoptotic cells based on the brown stained nuclei.