Formulation of Phytochemicals in Lipid-Based Nanoparticles for Melanoma Treatment
Type of DegreePhD Dissertation
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Melanoma is one of the most common and aggressive types of cancer with high mortality. Current treatment is ineffective in preventing relapse of cancer. It also suffers from serious systemic side effects because of the non-selective action on normal cells. Lipid-based nanoparticles are a promising drug delivery system for chemotherapy due to their easy preparation and modification, biocompatibility, enhanced permeability and retention, and reduced toxicity. In this dissertation, different lipid-based nanoparticles that include proniosomes, microemulsions and liposomes were developed for treatment of melanoma. Topical/transdermal drug delivery for treatment of melanoma represents an attractive alternative to conventional systemic delivery methods because of lack invasiveness, easy targeted administration, good drug distribution and biostability with controlled drug release. One problem with topical delivery, however, is that the skin is highly impermeable membrane barrier which limits the number of drugs that could be delivered through the skin. In the past few decades, various approaches have been used to improve drug permeation across the skin with use of techniques such as microneedles, iontophoresis, sonophoresis, thermal ablation, chemical penetration enhancers, and special formulations. Formulation approaches such as microemulsions and proniosomes have been used to improve skin permeation for several drugs. In this work, we developed pharmacological formulations for three antioxidant compounds that include hydroxytyrosol, DAB, and hispolon. We also investigated the anticancer effects of these formulations in cell culture. Further, we investigated the anticancer effects of a formulation that involved a combination of hispolon and doxorubicin, a chemotherapeutic drug widely used in treatment of several common epithelial cancers. Hydroxytyrosol is a well-known anti-oxidant effective in melanoma. Proniosomes were prepared as transdermal delivery system for hydroxytyrosol by a coacervation method. The optimized formulation of 1% (w/w) hydroxytyrosol, 45% (w/w) span 60, 5% (w/w) cholesterol, 40% (w/w) ethanol and 9% (w/w) PBS showed the highest skin permeation compared to control. These proniosome formulations could be a promising system for transdermal delivery of hydroxytyrosol. Next, microemulsions were developed for enhanced transdermal delivery of DAB compound. Microemulsion formulations were selected based on the pseudo-ternary phase diagram and the pH of the selected formulations ranged from 5.26 to 6.5. Optimized formulations showed globule size of <50 nm and a polydispersity index of 0.31. There was a significant decrease in melanoma cell proliferation with all DAB microemulsions compared to the control. The IC50 for all the microemulsion formulations were 50-60-fold lower than free drug. The anti-cancer activity of hispolon has been reported in different types of cancer but not in melanoma. The cytotoxicity study of hispolon formulation in this study showed effective suppression of cell proliferation. We also showed that hispolon is a potent inducer of reactive oxygen species and modulator of several apoptotic genes. Hispolon treatments inhibited the expression of bcl-2 gene, an anti-apoptotic factor, and promoted the expression of Bax gene, a pro-apoptatic factor. It also enhanced expression of caspase enzymes (markers for apoptosis induction). It also inhibited complex I and IV. Complex I is required for cell survival while complex IV (or cytochrome c oxidase), catalyzes the final step in mitochondrial electron transfer chain, and is considered as one of the major regulation sites for oxidative phosphorylation. Hispolon also stimulated nitrite content and lipid peroxidation levels. All these processes are associated with cell death or apoptosis. The therapeutic use of doxorubicin is limited due to chronic doxorubicin induced cardiac toxicity and development of multidrug resistance in melanoma cells. To overcome these problems, we developed a combination therapy of doxorubicin and hispolon liposomes. The liposome formulations provided a mean diameter less than 100 nm with a narrow size distribution and 90% recovery for both drugs. Combination of doxorubicin liposomes with hispolon liposomes enhanced cell cytotoxicity and apoptosis more than either single treatment. The study of the doxorubicin and hispolon liposome combination provides a strong evidence for future potential use of this formulation for effective treatment of patients with melanoma.