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
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Date
04/07/2020Author
Ferrari, Giulia
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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.