Ultrasound-mediated therapies for the treatment of biofilms in chronic wound infections

Abstract

Bacterial biofilms are an ever growing concern for public health; featuring both inherited genetic resistance and a conferred innate tolerance to traditional antibiotic therapies. Consequently, there is a growing interest in novel methods of drug delivery, in order to increase the efficacy of antimicrobial agents. One such method is the use of acoustically activated microbubbles, which undergo volumetric oscillations and collapse upon exposure to an ultrasound field. This facilitates physical perturbation of the biofilm, and provides the means to control drug delivery both temporally and spatially. In line with current literature in this area, this report offers a rounded argument and research evidence to outline why ultrasound-responsive agents could be an integral part of advancing wound care. The fabrication of a stable ultrasound-responsive carrier of nitric oxide was an important first step in this research, this was achieved by exploring two primary lipid microbubble shell constituents: 1,2-distearoylphosphatidylcholine (DSPC) and 1,2-dibehenoyl-sn-glycero-3-phosphocholine (DBPC). It was successfully demonstrated that DBPC shelled microbubbles exhibited the greatest stability in ambient temperature over 60 minutes. Targeting biofilms to increase the efficacy of antimicrobial delivery and optimised local concentrations has proved challenging, due to the complex and heterogeneous nature of the biofilm extracellular matrix. This research assessed the utility of cationic microbubbles, as a means of non-selective electrostatic targeting of the anionic biofilm structure. Cationic microbubbles demonstrated the ability enhance microbubble-biofilm contact by up to 37%, with a binding strength comparable to that seen in specific ligand-receptor targeting of biofilm extracellular matrix constituents. The anti-biofilm and bactericidal efficacy of neutral and cationic airand nitric oxide-loaded microbubbles, were assessed both in terms of their passive nitric oxidemediated action and active response to ultrasound stimulation. To facilitate growth of biofilms with morphological and phenotypic characteristics of biofilms found in chronic wounds, a wound constituent media that featured key pathophysiological components of the in vivo wound environment was developed. It was successfully demonstrated that insonified DBPC nitric oxide microbubbles could elicit a clinically significant 99.9% (3-log reduction) in viable cells recovered from treated biofilms, by enhancing the efficacy of sub-inhibitory (4 µg/mL) concentrations of the antibiotic gentamicin. Moreover, the combination of the nitric oxide and the ultrasound mediated physical perturbation of biofilms by microbubbles, was also shown to elicit a significant 99.9% reduction in surface area covered by P. aeruginosa biofilms.

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