Pharmaceutical Science
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ItemDesign, Development and Evaluation of Targeted Delivery System for the Treatment of Lung Cancer( 2017-02)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.
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ItemDevelopment and evaluation of polymeric hybrid a-Difluoromethylornithine (DFMO) & Etoposide loaded nanocarriers for the treatment of Neuroblastoma( 2016-08)Neuroblastoma (NB) is the most common extra-cranial solid cancer in childhood and infancy with patients having an average age of 17 months. Most are diagnosed with advanced stage NB when tumor progression is aggressive, making treatment of NB even more difficult. Up to 45% of patients are in the high-risk category with MYCN gene amplification being observed. The FDA-approved drug difluoromethylornithine (DFMO) exhibits anticancer activity against MYCN-amplified NB cells. DFMO is a suicide inhibitor of ornithine decarboxylase (ODC), a rate-limiting enzyme in the biosynthesis of polyamines. ODC gene expression is directly activated by MYCN suggesting that MYCN amplification is connected to high ODC expression. ODC expression produces high polyamine levels that contribute to the malignant phenotype and maintenance of NB tumorgenesis. This MYCN-ODC connection suggests that ODC may be a suitable new target for the treatment of NB with the administration DFMO. Etoposide, a topoisomerase inhibitor is often used in front-line therapy in the treatment of NB. The use of DFMO/Etoposide in vivo is currently limited due to the short half-lives (fast elimination/clearance) of both drugs which may explain why antitumor in vivo were not synergistic as observed in vitro. iRGD peptide-conjugated PEGylated polymeric hybrid nanocarriers loaded with synergistically acting DFMO and Etoposide drugs (iRGD-PEG-HNC-D-E) were characterized at 81ﾱ7nm in size, +12ﾱ2.5mV in zeta potential with a mean polydispersity index 0f 0.354ﾱ0.03. The developed nanocarriers had a 10 and 6-fold decrease in initial drug concentrations, DFMO and Etoposide respectively, with similar efficacy as compared to free drugs alone against various NB cell-lines over a 72h period. The current formulation shows stability suspended in phosphate buffer saline (PBS) 7.4 over 6 days. iRGD-PEG-HNC-D-E formulations can be modified (polymer: polymer ratio) to alter drug release profiles with the developed formula having a 90% release of both drugs after 72h.