THE DEVELOPMENT AND EVALUATION OF SYSTEMIC DRUG DELIVERY SYSTEMS FOR C6-CERAMIDE AS A BREAST CANCER CHEMOTHERAPEUTIC AGENT
Open Access
- Author:
- Stover, Thomas Christopher
- Graduate Program:
- Pharmacology
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- November 16, 2005
- Committee Members:
- Mark Kester, Committee Chair/Co-Chair
Jong Kak Yun, Committee Member
Charles D Smith, Committee Member
Melvin Lee Billingsley, Committee Member
Tao Lu Lowe, Committee Member - Keywords:
- tumor
breast cancer
dendrimers
liposomes
drug delivery
Ceramide
pharmacokinetics - Abstract:
- Ceramide is a bioactive sphingolipid-derived second messenger that has been demonstrated to induce apoptosis and cell cycle arrest in various cancer cell culture systems. An extensive amount of work has been performed in elucidating both the biochemical and biophysical mechanisms of ceramide-induced apoptosis and cell cycle arrest. Interestingly, the administration of several anti-mitogenic cytokines and pro-apoptotic chemotherapeutic agents has been shown to result in the intracellular accumulation of ceramide. Moreover, exogenous short-chain ceramide analogues, such as C6-ceramide (C6), have been observed to act synergistically with various chemotherapeutic agents to induce cancer cell apoptosis. Although in vitro tumor cell culture models have illuminated the potential therapeutic utility of C6, in vivo delivery is impeded by the extreme hydrophobicity and physical-chemical properties of these bioactive lipids. Effective intravenous drug delivery systems are essential in order to realize the true therapeutic value of exogenous ceramide treatment. Therefore, we investigated the utilization and applicability of liposomal and polymeric nanoparticulate methodologies. Initial work focused on the design and optimization of ceramide-incorporated liposomal vehicles to augment ceramide delivery to MDA-MB-231 breast adenocarcinoma cells, a highly aggressive, metastatic cell-line. We designed conventional, cationic, and pegylated drug delivery vesicles to efficaciously deliver ceramide to MDA-MB-231 cells. In vitro pharmacokinetic analysis demonstrated that liposomal ceramide delivery resulted in significantly greater accumulation of ceramide compared to free administration. These formulations significantly limited MDA-MB-231 cell proliferation, inhibited phosphorylated Akt levels, and stimulated Caspase-3/7 activity more effectively than non-liposomal ceramide. Due to the enhanced in vitro efficacy of pegylated liposomal-C6 formulations, we next investigated the effectiveness of the pegylated formulations in a Balb/C mouse solid tumor model of breast adenocarcinoma. Liposmal-C6 was found to induce significant apoptosis in murine 410.4 mammary adenocarcinoma cells, whereas no cytotoxicity was observed in normal murine HC11 mammary epithelial cells, signifying that normal, healthy mammary cells may be less sensitive to ceramide treatment. It has been shown that different cell types can metabolize exogenous ceramide as varying rates, resulting in ceramide insensitivity. Over a three-week treatment period, the systemic intravenous delivery of this formulation elicited an approximate 80% reduction in 410.4-derived tumor size when compared to empty “ghost” liposomal treatment. Importantly, the total body weights of treated mice did not significantly differ over the treatment period, suggesting limited systemic toxicity. Histological analyses of solid tumors from liposomal-treated mice demonstrated a marked increase in the presence of apoptotic cells, with a concomitant decrease in cellular proliferation and in the development of a microvessel network. The biophysical mechanisms underlying liposomal-C6-induced cancer cell cytoxicity and apoptosis include the accumulation of ceramide within caveolae and mitochondrial. Furthermore, a pharmacokinetic analysis of systemic liposomal-C6 delivery demonstrated that the pegylated liposomal formulation follows first-order kinetics in the blood with a half-life of nearly 11 hours and achieved a steady-state concentration in tumor tissue above the in vitro IC50 of liposomal-C6. With the advent of multi-functional polymeric drug delivery, additional efforts were undertaken to develop a drug delivery strategy to specifically target solid tumor tissue. We evaluated the feasibility of delivering C6 in thermo-responsive, biodegradeable dendritic nanoparticles. These dendrimers are comprised of thermo-responsive poly(N-isopropylacrylamide), biodegradable poly(L-lactic acid), and dendritic poly(L-lysine) and have a lower critical solution temperature of 34°C. Temperature-sensitive uptake of fluorescein isothiocyanate-labeled dendrimers was confirmed by confocal microscopy and quantified by flow cytometry in MDA-MB-231 cells. Upon the confirmation that the dendrimers can be efficiently loaded with ceramide, in vitro drug release assays were performed to validate the utilization of temperature-dependant drug delivery. Importantly, the dendrimers did not show any cytotoxicity in MDA-MB-231 tumor cells at concentrations of up to 300 mg/ml, and the treatment of MDA-MB-231 cells with C6-loaded dendrimers resulted in the significant growth inhibition. This novel formulation of C6-loaded dendrimers has the potential to thermally target solid tumor tissue in vivo. Together, these results indicate the bioactive ceramide analogue, C6, can be successfully incorporated into both pegylated liposomal vehicles and dendritic thermo-responsive nanoparticulates. The enhanced in vitro and in vivo efficacy observed with liposomal delivery suggests that ceramide has utility as an anticancer agent. Whether or not ceramide has potential as a first-line therapy remains unknown, but it has significant potential as an adjunctive therapy as a synergistic pro-apoptotic agent. The optimization of targeted ceramide-loaded vehicles would serve to further advance the clinical utility of ceramide treatment in cancer therapy. The development of ceramide-loaded thermo-responsive dendrimers exemplifies this strategy due to the potential to target solid tumor tissue.