Evaluation of the bioactivity and stability of gold nanoparticles conjugated with Os-C(W5) against Candida albicans biofilms

Abstract

The increased prevalence of antimicrobial resistance has led to the need for new antimicrobial drugs with novel modes of action. Antimicrobial peptides (AMPs) are cationic peptides that, in contrast to traditional antimicrobials targeting specific cellular components, exhibit varied mechanisms of action by engaging in a non-specific manner to disrupt the integrity of microbial membranes, thereby diminishing the probability of resistance development. Although promising antimicrobial agents, the main challenges are low activity in physiological environments and loss of activity in the presence of proteases. These limitations can be overcome with several different approaches including amidation, tryptophan (Trp) tagging, inclusion of unnatural amino acids, and various formulation strategies. Os-C is a cationic AMP, an analogue derived from a defensin identified in the midgut of the tick Ornithodoros savigny. Despite Os-C exhibiting noteworthy antibacterial and antifungal properties, Os-C exhibited reduced activity in physiological environments, impeding potential clinical application. The attachment of five Trp residues to the terminal ends of Os-C led to analogues (W5)Os-C and Os-C(W5), which exhibit antimicrobial activity in physiological environments. To further develop these analogues for antifungal treatment it was necessary to further reduce their susceptibility to proteases. Recent studies have shown that the presence of Trp residues on the N-terminal of a peptide reduces gold (III) chloride, resulting in the formation of gold nanoparticles (GNPs) coated with peptide. This method offers the advantage of easy synthesis without the use of toxic chemicals or the formation of toxic by-products. Therefore, it is ideal for physiological applications. The aim of this study was to synthesise and characterise (W5)Os-C and Os-C(W5) conjugated onto GNPs. For the more stable peptide-coated GNPs, then to determine the antifungal activity against C. albicans biofilms, cytotoxicity against human epidermal cells and nitic oxide (NO) anti-inflammatory properties. Both Os-C(W5)@GNPs and (W5)Os-C@GNPs were successfully synthesised. Os-C(W5)@GNP proved to be the most stable, and further studies were conducted with this analogue. Os-C(W5)@GNPs had a quasi-spherical structure with an average diameter of 14.62 ± 0.31 nm. High-magnification TEM imaging revealed a distinct corona, affirming the presence of peptides on the surface of the GNPs, quantified at 2.4 ± 0.15 nm. Approximately,174.96 of peptides were estimated to be bound per nm2. Subsequent circular dichroism studies revealed that Os-C(W5) retained a disordered secondary structure when bound to the GNP surface. As this was the first study in which AMPs were bound to GNPs using this method of non-covalent conjugation, it was necessary to determine if the antibiofilm activity of Os-C(W5) is v retained. The activity of Os-C(W5)@GNP against C. albicans was assessed using the biofilm inhibition (50% biofilm inhibitory activity (BIC50)), eradication (50% biofilm eradication activity (BEC50)), and degradation (50% biofilm degradation activity (BDC50)) assays, with miconazole as a drug control and Tyr@GNP as a GNP control. Compared to Os-C(W5), the BIC50 for cell viability and biomass of Os-C(W5)@GNP was similar. The BIC50 for cell viability was 7.40 ± 0.42 μM for Os-C(W5) and 8.93 ± 1.24 μM for Os-C(W5)@GNP. The respective BIC50 values for biomass were 7.07 ± 0.57 μM and 5.99 ± 2.28 μM. Light microscopy of stained biofilms revealed a less dense biofilm with some budding hyphae. The GNP control, Tyr@GNP, displayed no biofilm inhibitory activity, suggesting that released Os-C(W5) rather the GNPs inhibited the development of C. albicans biofilms. Determining the BEC50 for Os-C(W5)@GNP was not feasible since the GNPs reached their maximum concentration at 37.5 μM, which was still below the anticipated BEC50. Nevertheless, notable effects were observed at this highest concentration, including, 50% inhibition of cell viability and 15% inhibition of biomass. The BDC50 for Os-C(W5)@GNP was 45.7 ± 5.9 μM and >150 μM for cell viability and biomass respectively, indicating that Os-C(W5)@GNPs was cell targeting rather than targeting the biofilm synthesis pathway. Os-C(W5)@GNPs showed no cytotoxicity after evaluated in the HaCat cell line when compared with melittin, a cytolytic AMP. Os-C(W5)@GNPs maintained biofilm inhibitory activity in artificial saliva, a physiological salt solution lacking the biomolecules present in human saliva. A lack of activity was observed in human saliva, potentially due to the presence of salivary proteins, proteases, and mucin. Previous studies have shown that Os-C and related analogues, such as Os, are multifunctional and possess anti-inflammatory activity, which is related to the ability of these AMPs to scavenge NO. In contrast to glutathione and Os, Os-C had reduced activity. Trp tagging and subsequent binding to GNPs did not increase NO scavenging activity. To conclude, Os-C(W5)@GNP localises a high concentration of Os-C(W5) onto GNPs, via non-covalent bonding. Compared with Os-C(W5), biofilm inhibitory activity was retained while biofilm eradication and degradation activity were minimal. Os-C(W5)@GNP was not cytotoxic at the concentrations evaluated and lacked NO scavenging activity.