Alternative therapeutic options for cutaneous melanoma using nanoparticles for enhanced delivery

Abstract

The research provided in this thesis highlights the benefits of using a nano-delivery system to improve the PDT effect as an anticancer mechanism. PDT is a non-invasive therapy for a variety of superficial lesions, including those of the skin, head, and neck. The current treatment and management options for cutaneous melanoma, as well as advances made using either a photosensitiser (PS) or peptide-based compound, are discussed in Chapter 1. The chapter also emphasises the use of nanoparticles, particularly polymersomes (Ps) and liposomes (Ls), to improve the delivery of these molecules to tumour cells. Chapter 2 discusses the formulation of the novel PS-peptide conjugate, RB-K2. RB-K2 was synthesised conjugating C-terminus of photosensitiser rose bengal (RB) with and N-terminus of peptide, C(KLAKLAK)2 (K2), which produces novel synthesised drug RB-K2. RB-K2 was formulated into polymer-based Ps and lipid-based Ls to provide an enhanced PDT effect of RB-K2. The Ps formulation of RB-K2 (Ps-RB-K2) was seen to be 2.25 times more effective when compared to RB-K2 alone at concentrations of 0.1μM and 1 minute light activation, this was true across multiple cell lines. The Ls formulation of RB-K2 (Ls-RB-K2) was seen to be ~50% less effective than the Ps formulation at 0.1μM and again with 1 minute light activation. The third chapter focuses on understanding the mechanism behind the improved efficacy of RB-K2 using nanoparticles (NP). In unencapsulated and encapsulated NP form, the individual PS and peptide parts of RB-K2 were investigated. The PS (RB) as well as a C8 derivative of rose bengal, rose bengal octanoic acid (RB-OA), the peptide C(KLAKLAK)2 (K2) and a C10 derivative of K2 (K2C10) were synthesised and investigated individually and encapsulated within NP form. To gain a better knowledge of the mechanism of the original drug RB-K2, other Ps formulations containing the peptide and the PS independently in a single formulation were also included, and for comparison, the shorter chain peptide RB-CKLAKLAK (RB-K1) which contained half amount of lysine, leucine and alanine compared to (KLAKLAK)2, was investigated. The encapsulation of the PS (RB) into a Ps formulation enhanced its PDT efficacy by 1.9 times, a further anchoring of K2C10 on the outside of the Ps increased the toxicity further by 13.1±4.7% as Ps-RB+K2C10 (10:1) when treated with 5μM with 2 min light. RB-OA (1.0μM) encapsulated within a Ps with 1 min light caused cell death of 74.4±8.6% and 58.1±3.6% respectively compared to RB-K1 and RB-K2 which display almost complete cell destruction (i.e., >95%) at the same concentration and conditions. RB-K1 has a similar potential as RB-K2 but it failed to form stable polymersomal formulations but might be more suitable for other types of formulation. The peptide, K2, on the other hand caused cytotoxicity of 41.2±1.7% at 5.0 μM in B16 cells and its NP and C10 derivative showed similar degrees of toxicity. At last, none of the formulation were seen to be as potent as Ps-RB-K2 or the original drug RB-K2 and we established that both RB and K2 need to be bonded to produce the desirable PDT effect. Chapter 4 examines the potential translation of the increased PDT effect by nanoparticle encapsulation from the in-vitro experimentation into an in-vivo tumour model in mice bearing B16 melanoma tumours. Using intralesional administration of the compounds, mice bearing dorsal tumours of B16 melanoma, were treated with either polymersomes encapsulating RB-K2 (Ps[10]-RB-K2) or RB-K2 in free drug form with light activation using white light LEDs. The tumours treated with the polymersomal formulation grew 5.26 times slower than RB-K2 treated tumours, accumulating 2.77 times more drug within the tumours in under 2 hours, and causing a 1.76-fold increase in immunogenic response in tumour tissue. According to in-vitro experiments, the Ps formulation enhances RB-K2 absorption by 2.5 times in melanoma cells, resulting in a 63% increase in apoptosis. As indicated by evidence of both fluorescent studies and the singlet oxygen assay using DPBF for quantitatively determining singlet oxygen produced, the Ps also prevents the free drug from self-quenching and aggregating, both of which result in a decrease in PDT activity. Overall, the results indicate that encapsulating RB-K2 in Ps potentially lead to a significant increase in PDT activity.

Date of Award	May 2022
Original language	English
Sponsors	Department for the Economy
Supervisor	Bridgeen Callan (Supervisor), Anthony McHale (Supervisor) & John Callan (Supervisor)