Design, Development and Evaluation of Targeted Delivery System for the Treatment of Lung Cancer

Gandhi, Nishant
Koomoa-Lange, Dana L.
Chougule, Mahavir B.
Pharmaceutical Sciences
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Lung cancer is the leading cause of cancer-related deaths in the world. Lung cancer alone causes more deaths than pancreas, colon, prostate, and breast cancer deaths combined. Only 15% of lung cancer patients survive for 5 or more years after diagnosis. Lung cancer is further divided into small cell lung cancer and non-small cell lung cancer (NSCLC). The NSCLC accounts for 85% of all lung cancer cases. The current mainstay of treatment of lung cancer is multi-drug therapy. Chemotherapy is the preferred option for the treatment of lung cancer. However, severe side effects caused by chemotherapy demands developing novel methods for the treatment. The Luteinizing hormone-releasing hormone (LHRH) is overexpressed in non-small cell lung cancer (NSCLC). This thesis is focused on investigating the ability of poly (amino ether) (PAE) polymer based formulation of small-interfering RNA (siRNA) to silence the mammalian target of Rapamycin (mTOR) in NSCLC cell lines in vitro. In the first part of this thesis, we modified and developed a bio-reducible polymer by introducing a sulfhydryl group (-SH) to PAE polymer. The modified PAE polymer (mPAE) showed decreased cytotoxicity and improved buffering capacity compared to the widely used transfection polymer poly-ethyleneimine (PEI). In the second part, cationic bio-reducible polymer modified Poly (amino-ether) was used to formulate bio-reducible nanoparticles. The mPAE was used to deliver mTOR siRNA to the non-small cell lung cancer cell lines (A549 and H460) and access their potential as a siRNA deliver carrier for lung cancer therapy. The mPAE and mTOR siRNA formed stable, bio-reducible nanoparticles (NPs) at a polymer to siRNA weight ratio of 45:1, with average diameter 114 nm and surface charge of around +27mV. The mTOR siRNA showed increase release in the presence of 10mM GSH. By optimizing the concentration of the mPAE polymer, we were able to fabricate polymeric NPs capable of efficient gene knockdown (60% and 64%) in A549 and H460 cells, respectively without significant cytotoxicity at 30µg/ml concentrations. The MS-MP-NPs showed improved cell growth inhibition (31% and 32%) in A549 and H460 cells in vitro, respectively. The MS-MP-NPs also showed time-dependent cellular uptake as determined by FACS for up to 24 hours. The results demonstrate that the mPAE polymer based NPs show strong potential for future modification with Poly (ethylene glycol) and targeting ligand to improve the gene delivery and achieve higher lung cancer growth inhibition in vitro and in vivo. In the third part, PEGylated-targeted NPs (MS-MP-PG-LR) were prepared. To attach PEG on the surface of the nanoparticles, first, the PEG was conjugated with AMAS and then allowed to attach on the MS-MP NP surface. Further, cysteine-terminated LHRH was subsequently conjugated on the surface of the PEG group through a maleimide reaction with the cysteine group. We found that PEGylation of the mPAE based nanoparticles allows increased delivery of the siRNA. 22% PEGylated prevented the cytotoxic effect of 60 µg/ml concentrations of the nanoparticle system. Western blot results confirmed the overexpression of the LHRH receptor on the non-tumor (Breast adipocytes) and the tumor cells (SKOV-3). As shown before, PEGylation also provided serum stability to the NP system. The optimal particle size of the MS-MP-PG and MS-MP-PG-LR20 nanoparticles was found to be 124 ± 5.9and 132 ± 6.8 respectively. The zeta potential of the respective nanoparticles was positive (19.5 ± 4.6 and 20.2 ± 5.4). The FACS analysis showed that the targeted MS-MP-PG-LR20 nanoparticle system selectively internalizes in the LHRH-R overexpressed A549 and H460 cells compared to the SKOV-3 cells (significantly lower LHRH-R expression). The mTOR siRNA encapsulated, targeted MS-MP-PG-LR20 NPs showed significantly increased cell growth inhibition and mTOR gene silencing compared to the SS-MP-PG-LR20 nanoparticle system. The caspase activity assay confirmed the apoptotic pathway mediated cell growth inhibition of the MS-MP-PG-LR20 nanoparticles.
Pharmaceutical sciences, LHRH, Lung Cancer, mTOR, nanoparticles, polymer, siRNA
185 pages
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