Film-forming Agents as Potential Barriers to Fungal Skin Infections

Abstract

Fungal skin infections caused by dermatophytes or yeasts (e.g. Candida species) are one of the most common causes of human diseases. Dermatophytosis is caused by fungi that can degrade keratin, infecting the keratinised tissues such as skin, hair and nails. Although the infections are mild and rarely life-threatening, they are highly recurrent, and their incidence has increased continuously. Simultaneously, there is a growing resistance to antifungals, exposing, in particular, immunocompromised patients to higher levels of risk. This thesis is devoted to the study of polymers as physical barriers that can prevent and treat the early stages of skin infections and avoid the development of antifungal resistance and cross-contamination. To do this, a series of studies were undertaken to identify the ideal antifungal polymer.

The first part of the thesis aimed to develop an ex vivo porcine skin model to study dermatophyte infections. The explanted porcine skin was sterilised by chlorine gas to remove contamination with other microbes. To characterise the model, adherence of Trichophyton rubrum was examined via adherence assays to the ex vivo skin, and this was compared to an in vitro model using HaCaT cells. Subsequently, the pathophysiology investigated by electron microscopy analysis. It was shown that the ex vivo model is particularly appropriate to the study the early stages of the fungal skin infection and to test novel antifungal regimes.

In the second part of the thesis, two cationic polymers, ABIL T Quat 60 and Eudragit E100 were identified for their potential as barriers to skin infections caused by T. rubrum, Trichophyton interdigitale and Candida albicans. Both polymers were found to be fungistatic agents that can prevent and treat fungal skin infections in the porcine skin model. The results suggest that the mechanism of action of ABIL T Quat 60 is based on its ability to bind to the negatively charged fungal cell wall, followed by impeding the fungi to acquire crucial nutrients such as essential metal ions and carbohydrates. As for Eudragit E100, this polymer might bind to the fungal cell wall as well, but its antifungal activity was affected by the environmental pH.

The third section aimed to evaluate the performance of these two polymers to form films in vivo, and the patient acceptability regarding these films. Following application as sprays to the arms of healthy subjects, the washability, drying time and stickiness of the films were tested for topical use. The same in vivo study characterised the tolerability of the films, in terms of the transepidermal water loss and skin redness. The results suggested the films to be well-tolerated and non-toxic, with no signs of skin irritation, barrier damage or skin occlusion.

Apart from the antifungal activity of polymers, their antibacterial activities against methicillin-sensitive/-resistant Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were examined, to determine potential use to prevent wound infection. Only ABIL T Quat 60 showed an antibiofilm activity against S. aureus and E. coli but, surprisingly, stimulated the biofilm formation of P. aeruginosa.

In addition to the two polymers, the antifungal activities against the \textit{T. rubrum} of three common formulation ingredients, ethylenediaminetetraacetic acid, cetrimonium chloride and polyoxyethylene (10) tridecyl ether were established through a novel phenol red viability assay. Cetrimonium chloride was identified as fungicidal, whereas the other two compounds were fungistatic.
The phenol red viability assay was further developed and enabled the study of the antifungal susceptibility in a mildly acidic environment optimised for dermatophytes growth, and to resolve technical challenges caused by the absorption of stains used in other types of assays by polymers.

Date of Award	13 May 2020
Original language	English
Awarding Institution	University of Bath
Supervisor	Albert Bolhuis (Supervisor) & Begona Delgado-Charro (Supervisor)