Date of Award

12-2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Engineering and Imaging

Research Advisor

Denis J. DiAngelo, Ph.D.

Committee

Joel D. Bumgardner, Ph.D. Mostafa W. Gaber, Ph.D. Warren O. Haggard, Ph.D. Satoru K. Nishimoto, Ph.D. Yunzhi Yang, Ph.D. Xin A. Zhang, Ph.D.

Keywords

Apoptosis, Biocompatibility, Cancer, Chitosan, Ellagic acid, Local drug delivery

Abstract

Current advances in the drug delivery have improved the therapeutic efficacy of the drug and minimized risks of side effects associated with toxicity of the drug. Implantable polymeric delivery system has gained increasing attentions for controlled drug release and localized treatments. In comparison to conventional chemotherapy, polymeric delivery systems are implantable at a local targeted site and biodegradable after a sufficient therapeutic span. The objectives of this project were to fabricate and characterize an implantable polymeric vehicle for a local chemotherapy and investigate its biological properties against cancer cells including human WM115 melanoma, human U87 glioblastoma, and rat C6 glioma cells in vitro andin vivo.

In this study, a natural chitosan polymer was employed as a drug vehicle and ellagic acid (EA), a naturally occurring phenolic compound, was incorporated as a therapeutic agent. The chitosan/ellagic acid composite films were developed by combining 1% (w/v) chitosan solution with different concentrations (0.05, 0.1, 0.5, 1, or 20% (w/v)) of ellagic acid for a local chemotherapy. Characterization of composite films was performed on chemical structure, crystallinity, surface morphology, degradation behavior, and release profile. Cancer cell activity on the composite films was evaluated through direct and indirect cell culture using MTS assay. Anti-cancer mechanism of the composite films against cancer cells was investigated using apoptosis assay, caspase-3 activation, western blot for p53, and anti-angiogenesis assays. In the in vivo study, an animal subcutaneous model was used to assess the anti-tumor effect of the composite film on rat C6 glioma. Treatments were initiated by implanting the composite films onto the tumor. The tumor growth was monitored by measuring tumor volume using a caliper, an ultrasound machine, and an optical imaging system.

The chitosan/ellagic acid composite films exhibited increase in amide and ester linkages, diffraction peaks of the crystallized ellagic acid, enhanced surface roughness, and hydrophilicity with increasing concentration of ellagic acid. The composite films degraded enzymatically, indicated by at least a 5 times higher concentration of free amino groups in the incubation medium at 3 weeks compared with 1 day. They also displayed a sustained slow release of ellagic acid in vitro for 3 weeks incubation. Anti-cancer activity of the composite films was ellagic acid concentration dependent by inducing apoptosis of cancer cells and suppressing angiogenesis. Significant inhibitory effect (p<0.05) was found in the composite films containing 0.5% (w/v) of ellagic acid or higher compared with other groups. Study of a rat C6 glioma model demonstrated that the composite film (Ch/EA20) significantly inhibited tumor growth compared with control groups in vivo. Tumor volume increase in Ch/EA20 group was 9 times lower than that in control groups at 3 weeks observation by measuring a caliper. No severe weight loss (>10% wt.) was observed from all groups. Histology observation indicated no evidence of severe toxicity surrounding the composite films. The high efficacy and low toxicity of the composite film was attributed to the slow release and localized effect of ellagic acid.

In order to further improve the delivery method and efficacy, chitosan based injectable hydrogel was developed for a local administration of ellagic acid to avoid surgical complications. Studies of the chitosan gel were performed with regard to chemical structure, surface morphology, viscoelasticity, release profile, and degradation behavior. Biocompatibility and anti-cancer activity on chitosan gel delivery system were examined. The results showed that the injectable chitosan liquid formulation underwent thermal gelation at body temperature via hydrophobic interactions using β-glycerophosphate salt (β-GP). Sol-gel transition was dependent on final pH values of the chitosan/β-GP solution and temperature. Dialysis of chitosan solution reduced the β-GP needed to reach pH 7.2, resulting in 4 times higher cell viability than undialyzed chitosan gel at 3 days culture. This result indicates improved biocompatibility of the delivery system. The chitosan/β-GP gels were enzymatically degradable for 3 weeks incubation and inhibited cancer cell growth in vitro in an ellagic acid concentration dependent manner. The significant inhibitory effect (p<0.05) was found in the gel containing 1% (w/v) of ellagic acid compared with other groups. Viability of U87 cells and C6 cells cultured on chitosan gels containing 1% (w/v) of ellagic acid were lower than the same cells on chitosan gels at 3 days incubation by 3.8 times and 6.5 times, respectively.

This research has demonstrated that the chitosan/ellagic acid delivery system is a promising biomaterial for a local cancer treatment. This study has also suggested a potential strategy with higher efficacy and lower toxicity to treat tumors by the combination of naturally based biopolymers such as chitosan and phenolic compounds such as ellagic acid. This study provides some rationale for further investigation of implantable polymeric delivery system.

DOI

10.21007/etd.cghs.2009.0163

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