Preparation of polysaccharide-based nanoemulsions to reduce melanoma drug resistance and inflammation markers

Melanoma is the most aggressive type of skin cancer and various treatments have been investigated to treat this disease. However, an intrinsic issue in the clinical setting is the development of multidrug resistance. So far no single approach has emerged that may overcome such drug resistance by drug delivery. Alongside this, inflammation is an important marker of melanoma relating both to the incidence and progression of the disease, with upregulation in COX-2 and iNOS reported as playing a role in melanoma progression.

The work embodied in this thesis describes the development, optimisation, and characterisation of alginate, chitosan, and pullulan nano-emulsions as a delivery platform to target melanoma. In this study, a novel nano-emulsion delivery system was designed and optimised using a series of experimental steps including, in vitro drug release, cell viability (MTT), Cellular apoptosis ELISA, confocal and fluorescent microscopy, PCR, and Western blot to address melanoma chemo-resistance, and COX-2 and iNOS expression.

Chitosan-pullulan, pullulan-alginate, and alginate-chitosan polymer blends were prepared as nano-emulsions. A comparative analysis of their effect on melanoma (A375) and keratinocyte (HaCaT) cells was conducted. The “pullulan-chitosan” formulation was taken forward with folate-modification and used to target A375 cells. Subsequently, the melanocortin type 1 and 3 receptor agonists BMS-470539-dihydrochloride and [DTrp8]-γ-MSH were delivered to A375 cells using folate-modified pullulan-chitosan nano-emulsion, to target regulation of iNOS and COX-2 as important markers in melanoma progression.

Folate-modified pullulan-chitosan nano-emulsion enabled the delivery of doxorubicin and dacarbazine to A375 cells and diminished cell viability to 14 and 48% for doxorubicin and dacarbazine loaded nano-emulsions, respectively. Furthermore, BMS470539-dihydrochloride [DTrp8]-γ-MSH loaded nano-emulsions were able to decrease the regulation of iNOS and
COX-2 to zero, after 72 hours.

Optimal delivery of therapeutics has been successfully applied to address chemo-resistance and enhanced apoptosis induction to melanoma cells. A novel optimal pullulan-chitosan nanoemulsion delivery system was developed for malignant melanoma. The nano-emulsion provided a controlled delivery of the chemotherapeutics to defeat chemo-resistance.
Furthermore, the anti-inflammatory compound loaded pullulan-chitosan nano-emulsion decreased the iNOS and COX-2 expression in the A375 cell. This indicates that the compounds are more effective while delivered intracellularly, as opposed to the attachment to surface melanocortin receptors. This novel approach can be a promising tool to be used alongside treatments to prevent melanoma progression in its early stages.