Evaluation of the efficacy of bleomycin encapsulated within liposomes as novel, topical treatment for skin cancer in veterinary species and its potential for human medicine: an in vitro, ex vivo and in vivo study

Abstract

Bleomycin is a potent anticancer agent that is able to induce single and double stranded breaks in DNA. It is effective against several types of cancer, including cutaneous skin tumours, in both human and veterinary medicine. However, due to its molecular characteristics, including polar charge and high molecular weight, it is unable to effectively penetrate the skin barrier and freely cross the plasma membrane. Bleomycin can be administered systemically but is known to carry severe side effects such as fatal pulmonary fibrosis, which is the most concerning one and represents the main limiting factor to its potential use in clinical applications. Furthermore, several studies claim that bleomycin causes cell death in a cell-type dependent manner, although to date, the actual mechanism of action is not fully elucidated. Advances in cancer therapy aim to minimise the side effects of treatments and improve the quality of life of patients. Within the context of developing minimally invasive treatments, liposomes have become highly valued for their ability to deliver payload drugs to the target tumour tissues and to be applied topically therefore decreasing systemic toxicity. Bleosome is a novel formulation of bleomycin where the cytotoxic agent bleomycin has been encapsulated in ultra-deformable (UD) liposomes. In this study, we evaluated Bleosome as a topical, non-invasive treatment for non-melanoma skin cancer (NMSC). This PhD project also aimed to evaluate the mechanism of action and efficacy of Bleosome ex vivo, in vitro and in vivo. We hypothesised that encapsulation of bleomycin within nanoparticles, namely UD liposomes, enhances the penetration of the drug through the skin, and we tested this in three model systems: canine, equine, and human skin explants. Firstly, we optimised an imaging technique that allowed us to directly visualise the liposomes using the transmission electron microscope, and effectively fluorescently labelled bleomycin, prior to encapsulation within liposomes, to visualise penetration through skin and equine patient-derived sarcoids, using multiphoton microscopy. We concluded that lipid nanoparticles could act as penetration enhancers, carrying the entrapped bleomycin through the channels of the outermost stratum corneum and subsequently releasing the drug and allowing it to penetrate deeper in the skin. At the cellular level, we determined the effect of Bleosome on cell viability on a panel of canine, feline and human cancer cell lines in vitro. Using live cell imaging, we showed that Bleosome is taken up by cancer cells more efficiently than free bleomycin. We also compared the variation in protein and gene expression in two canine cancer cell lines after 8 and 24 hours post-Bleosome treatment. Preliminary data showed that the response to Bleosome is cell type dependent, and the transcriptomic profiling of a canine cancer cell line treated with a time course of Bleosome revealed that these specific cells are unable to repair the DNA lesions, produced by Bleosome treatment. Lastly, we administered Bleosome to equine patients bearing cutaneous sarcoids, in an adjuvant setting, following laser excision. After surgical excision of the sarcoids, Bleosome was applied topically on the site of the scar in 12 horses, twice a day, for a mean length of 4 weeks. To date, after an overall follow-up of 12 months, 5 patients were in complete remission (41.6%), 4 experienced recurrences (33.3%), and 3 failed to follow up (25%). The treatment was overall very well tolerated and the study was still on going at the time of writing. Overall, the findings of my PhD provide compelling evidence of the mechanism of action, efficacy and clinical benefit of Bleosome as topical, non-invasive treatment for NMSC.