Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Pharmaceutical Sciences

Research Advisor

Ram I. Mahato, Ph.D.

Committee

Ivan C. Gerling, Ph.D. Wei Li, Ph.D. Duane D. Miller, Ph.D. Yongmei Wang, Ph.D.

Keywords

RNA Interference, Islet Transplantation, Polymeric Micelles, Cancer, Multiple Drug Resistance

Abstract

Ex vivo gene transfer has been used to improve the outcome of islet transplantation for treating type I diabetes. RNA interference is an effective approach for reducing gene expressions at the mRNA level. The application of RNA interference to improve the outcome of islet transplantation was reviewed in Chapter 2, where I summarized biological obstacles to islet transplantation, various types of RNAi techniques, combinatorial RNAi in islet transplantation, and different delivery strategies.

Upregulation of inducible nitric oxide synthase (iNOS) and subsequent product of radical nitric oxide (NO) impair islet β cell function. Therefore, we hypothesized that iNOS gene silencing could prevent β cell death. In Chapter 3, we designed and used siRNA to silence iNOS gene in a rat β cell line and human islets. We found that siRNA inhibited rat iNOS gene expression and NO production in rat β cells in a dose and sequence dependent manner. iNOS gene silencing also protected these β cells from inflammatory cytokine‑induced apoptosis and increased their capacity to secret insulin. Although there was also dose and sequence dependent iNOS gene silencing and NO production in human islets, the effect of iNOS gene on apoptosis of islets was only moderate, as evidenced by 25‑30% reduction in caspase 3 activity and in the percentage of apoptotic cells. Since an islet is a cluster of 20‑1000 cells, the transfection efficiency of lipid/siRNA complexes into human islets was only 21‑28%, compared to effective transfection efficiency (> 90%) in β cells.

Gene delivery vectors which can express a growth factor gene to promote revascularization and silence of proapoptotic genes might be good for ex vivo genetic modification of islet prior to transplantation. Thus, in Chapter 4, we constructed bipartite plasmid vectors to co‑express a vascular endothelial growth factor (VEGF) cDNA and shRNA targeting iNOS gene. Firstly, shRNA sequences against human iNOS gene were screened. Then, we determined the effect of different promoters and shRNA backbones on gene silencing. The shRNA with H1, U6 and CMV promoters showed similar efficiency in iNOS gene silencing. In addition, a conventional shRNA showed better silencing of iNOS gene, compared to shRNA containing mir375 and mir30 backbones. A bipartite plasmid was also constructed with mir30‑shRNA and a VEGF cDNA controlled by a single CMV promoter. This plasmid showed a better silencing effect compared with plasmid without VEGF cDNA. In conclusion, we have successfully constructed bipartite vectors co‑expressing a VEGF cDNA and a shRNA against iNOS gene. These vectors could be an attractive candidate to improve the survival of transplanted islets.

The second part of research was focused on the study of polymeric micelle formulations for treating cancers. Two key elements were integrated in my research projects: development of polymeric micelle delivery systems; discovery of new therapeutics for better treatment of cancers.

The project described in Chapter 5 was carried out in collaboration with Dr. Miller's group at The University of Tennessee Health Science Center. In this study, we showed that SMART‑100 effectively inhibited HepG2 cell proliferation and was able to circumvent multiple drug resistance (MDR) in cancer cells. SMART‑100 inhibited P‑gp activity, which may be responsible for its ability to overcome MDR. Since SMART‑100 is poorly soluble in water, it was formulated in poly(ethylene glycol)‑b‑poly(D, L‑lactide) (PEG‑PLA) micelles. The solubility of SMART‑100 was increased by more than 1.1x105folds. SMART‑100 loaded PEG‑PLA micelles could effectively inhibit HepG2 cell growth and arrest cell cycle progression at G2/M phase, followed by cell apoptosis. Increased caspase 3 activity was also observed when HepG2 cells were treated with SMART‑100. The anticancer activity of SMART‑100 loaded PEG‑PLA micelles was also evaluated on luciferase expressing C4‑2‑Luc cell lines by IVIS imaging. Our results suggest that SMART‑100 has the potential to treat resistant cancers and PEG‑PLA micelles can be used to formulate SMART‑100.

We are not only interested in using commercial polymers but also interested in designing new polymers for micellar drug delivery. Therefore, the objective of study described in Chapter 6 was to design lipopolymers for hydrophobic drug delivery. In this study, poly(ethylene glycol)‑block‑poly(2‑methyl‑2‑carboxyl‑propylene carbonate‑graft‑dodecanol) (PEG-PCD) lipopolymers were synthesized and characterized by 1H NMR, FTIR, GPC, and DSC. The critical micelle concentration (CMC) of PEG‑PCD micelles was around 10-8 M and decreased with increasing length of hydrophobic block. PEG‑PCD micelles could efficiently load a model drug embelin into its hydrophobic core and significantly improve its solubility. The drug loading capacity was dependent on the polymer core structure, but the length of hydrophobic core had little effect. PEG‑PCD formed both spherical and cylindrical micelles, which were dependent on the copolymer structure and composition. Lipopolymers PEG‑PCD with various hydrophobic core lengths showed similar drug release profiles, which were slower than that of poly(ethylene glycol)‑block‑poly(2‑methyl‑2‑benzoxycarbonyl‑propylene carbonate) (PEG‑PBC) micelles. Embelin loaded PEG‑PCD micelles showed significant inhibition of C4‑2 prostate cancer cell proliferation, while no obvious cellular toxicity was observed for blank micelles.

In Chapter 7, we studied the use of paclitaxel and lapatinib loaded lipopolymer micelles for treating MDR prostate cancers. Although paclitaxel remains effective in treating prostate cancer, its prolonged treatment develops MDR due to the over‑expression of P‑gp. Our hypothesis is that combination of paclitaxel and lapatinib, which is a potent P‑gp inhibitor, can overcome MDR in prostate cancers. Paclitaxel and lapatinib loaded lipopolymer micelle formulations were developed and evaluated in vitro with cell‑based assay. The paclitaxel and lapatinib combination effectively inhibited in vitro MDR cancer cell proliferation, induced cell cycle perturbation and cell apoptosis. In contrast, monotherapy with paclitaxel or lapatinib alone showed minimal anticancer effect. The combination therapy was further investigated in vivo with athymic nude mice xenograft MDR tumor model. Similar to in vitro study, paclitaxel (5 mg/kg) and lapatinib (5 mg/Kg) combination therapy significantly inhibited tumor growth in vivo when compared to high dose paclitaxel (10 mg/kg) monothearpy. These studies indicate that the paclitaxel and lapatinib loaded PEG‑PCD lipopolymer micelle formulation could be used to treat MDR prostate cancers.

DOI

10.21007/etd.cghs.2011.0180

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