The Development and evaaluation of photo-antimicrobial isoalloxazine dyes towards infection control

Abstract

In today’s world, antimicrobial resistance is one of the biggest global health issues that mankind is facing. This most effective way to ensure a wound does not become infected is through cleanliness and continued disinfection of the wound site.

There is a lack of new antimicrobial drugs coming to the market due to economical and clinical reasons, this is evident in Lord O’Neill’s 2016 report and is addressed by Professor Dame Sally Davies in Parliament, (“We have reached a critical point and must act now on a global scale to slow down antimicrobial resistance”2). Prescription drugs have led to this epidemic that was highlighted by O’Neill.

The latest report (2016) by O’Neill states, it is critical to improve sanitation and hygiene, refrain from overusing antibiotics in agriculture and the environment as well as introducing rapid diagnostics and vaccines3. This is leading to the need for photodynamic antimicrobial chemotherapy (PACT) that involves the use of a reactive oxygen species (ROS), photosensitiser, and light to cause microbial death. PACT is a treatment for resistant and non-resistant pathogens that is included in the treatment of multidrug resistant infections. The approach is to use novel antimicrobial drugs topically, avoiding systemic photo-toxicity, thus leading itself towards topical infection control.

Herein, we report the development of a range of novel photosensitisers based on the second generation photodynamic therapeutic dyes (PDT) that are based on the tricyclic isoalloxazine structure of riboflavin, vitamin B2. Photosensitisers were synthesised using similar strategies to the isoalloxazine for a number of reasons:
e.g. photoactivity and capability of degradation. In order to investigate which photosensitisers gave the highest reactive oxygen yield, functional group changes were made on the N-phenyl ring by substituting a range of electron withdrawing/donating substituents at different positions (ortho, meta, para). The free amide moiety was used to attach the photosensitiser to a solid support that would act as proof of principal of a photosensitiser attached to a bandage.

These dyes show a phototherapeutic response via a Type I and II mechanism upon illumination by light of a selected wavelength. The mechanisms produce highly toxic oxygen-species, such as radical production via Type I pathway and singlet oxygen generation by Type II, thus causing terminal damage to microbes in a short time period. The synthesised photosensitisers are illuminated using blue light (440 - 490 nm) and white light in order to monitor and compare the singlet oxygen and radical yields generated as they absorb approximately at 440 nm, thus blue light being ideal for irradiation. The outstanding singlet oxygen result generated by compound 12c of 172% and a radical production by 11c of 227% show promising generators of cytotoxic species, resulting in microbial death.

The synthesised photosensitisers have been tested against two opportunistic microbes (Gram positive and Gram negative bacteria; Staphylococcus aureus (S. aureus), and Escherichia coli (E.coli). They have proven to be problematic from its presence within the healthcare system especially when found on surgical site infections. From the statistics generated for the National Health Service (NHS) in the UK we can see that 52.4 % of S. aureus, and 43.1 % E.coli originates within the hospital environment.

Antimicrobial activity was observed for several compounds under different light regimes on and off the solid support. As a result, the best observed MIC value of 0.25 mM/mL was achieved for S. aureus in darkness and in blue light without the polymer support. Additionally, when these compounds were linked to a polymer support (mimicking a bandage), antimicrobial activity was retained when irradiated using blue light at
1.0 mM/mL. These results show potential towards the next generation of antimicrobial disinfection agents.

In time, these compounds could be integrated into the healthcare system for use as a new generation of self- cleaning bandages towards post-operative wound disinfection rather than employing front line antimicrobials. This is a moot subject under review in parliament and former UK prime minister has highlighted the concern. In a statement recently released, he states “If we fail to act, we are looking at an almost unthinkable scenario where antibiotics no longer work and we are cast back into the dark ages of medicine" – David Cameron, UK Prime Minister2.