Aptamer-nanosilver conjugates and their antibacterial activity

Abstract

The rise of antibiotic resistance in bacteria constitutes one of the greatest emerging threats to human health that requires the urgent development of new drugs and treatment approaches. One of these problematic bacterial species is Pseudomonas aeruginosa which was ranked second on the list of drug-resistant bacteria that pose the greatest risk to human health by the World Health Organisation in 2017. P. aeruginosa is an opportunistic pathogen that has intrinsic multi-drug resistance, readily forms biofilms, secretes numerous virulence factors and can evade antibiotic therapy through the induction of the persister phenotype. The objective of this thesis was to develop a new, novel therapeutic to combat P. aeruginosa infections. This was achieved through the development of DNA aptamers that bind specifically to P. aeruginosa and the exploration of their use as antibacterial agents. It was hypothesised that the P. aeruginosa aptamers may exert intrinsic antibacterial activity, or could be used as targeting moieties to deliver therapeutics to the bacteria.

Aptamers were generated using a cell-SELEX method, and were specific to P. aeruginosa, but had no antibacterial activity. As an alternative approach, the aptamers were conjugated to DNA scaffolded silver nanoclusters. While the use of silver for medicinal purposes is ancient and well established in modern medicine, its use as an antibacterial is limited by its therapeutic index. It was reasoned that the targeting of nanosilver to the pathogens using DNA aptamers could potentially increase the utility of silver as an antibacterial agent. Aptamer-silver nanoclusters exhibited enhanced antibacterial activity compared to the equivalent untargeted silver nanoclusters. They had a rapid, dose-dependent antibacterial activity against planktonic and biofilm cells, and extended the median survival time in a Galleria mellonella model of infection.

During the aptamer-silver nanocluster investigation it was discovered that the addition of excess silver ions to the DNA scaffolds used to generate silver nanoclusters was facilitating folding of the DNA into complex secondary structures called i-motifs. The silver ions were unexpectedly stably associated with the DNA upon removal of the excess free silver. This facilitated the development and characterisation of aptamer conjugated silver i-motifs which exhibited stronger antibacterial activity than silver nanoclusters at equivalent concentrations, likely due to the delivery of Ag+ rather than Ag0. Aptamer silver i-motifs had a rapid mechanism of action, and caused a significant reduction in the biofilm biomass and viability. Strong synergistic activity with common antibiotics was exhibited, and silver i-motifs without aptamers also displayed strong antibacterial activity against a range of different bacterial species, Gram-positive and Gram-negative. These findings indicate that aptamer-silver i-motifs are effective novel antibacterial agents. The rapid onset of killing occurs independently of bacterial replication, as such, they may be effective for treating non-replicative and slow-dividing cells such as persister cells that cause the recalcitrance of disease and lead to antibiotic tolerance and resistance. Potential resistance to these compounds may not occur as they are not substrates for multidrug resistance efflux pumps and silver ions have multiple toxic mechanisms of action. Due to their synergistic activity with common antibiotics, they could be used as adjuvants to antibiotic therapy to increase the efficacy and repurposing of antibiotics once thought ineffective due to acquired resistance.

Aptamer silver i-motifs as antibacterials is a highly novel and significant discovery, and has not been reported previously in the literature. This thesis highlights the potential utility of aptamer silver i-motifs in the treatment of P. aeruginosa infections, with the recommendation for further development as a therapeutic to treat chronic infections in cystic fibrosis patients.