Dual bioresponsive antibiotic and quorum sensing inhibitor combination nanoparticles for treatment of Pseudomonas aeruginosa biofilms in vitro and ex vivo.

Nishant Singh,Paul Williams,Patricia F Monteiro,Manuel Romero,Morgan R Alexander,Cameron Alexander,Alessandra Travanut,Kim R Hardie,Elena Jordana-Lluch

doi:10.1039/c9bm00773c

Abstract

Many debilitating infections result from persistent microbial biofilms that do not respond to conventional antibiotic regimens. A potential method to treat such chronic infections is to combine agents which interfere with bacterial biofilm development together with an antibiotic in a single formulation. Here, we explore the use of a new bioresponsive polymer formulation derived from specifically modified alginate nanoparticles (NPs) in order to deliver ciprofloxacin (CIP) in combination with the quorum sensing inhibitor (QSI) 3-amino-7-chloro-2-nonylquinazolin-4(3H)-one (ACNQ) to mature Pseudomonas aeruginosa biofilms. The alginate NPs were engineered to incorporate a pH-responsive linker between the polysaccharide backbone and the QSI, and to encapsulate CIP via charge-charge interactions of the positively-charged drug with the carboxyl residues of the alginate matrix. In this way, a dual-action release of antibiotic and QSI was designed for the low-pH regions of a biofilm, involving cleavage of the QSI-linker to the alginate matrix and reduced charge-charge interactions between CIP and the polysaccharide as the alginate carboxyl side-chains protonated. When tested in a biofilm model the concomitant release of CIP + QSI from the pH-responsive nanoparticles significantly reduced the viability of the biofilm compared with CIP treatment alone. In addition, the alginate NPs were shown to penetrate deeply into P. aeruginosa biofilms, which we attribute in part to the charges of the NPs and the release of the QSI agent. Finally, we tested the formulation in both a 2D keratinocyte and a 3D ex vivo skin infection model. The dual-action bio-responsive QSI and CIP release nanoparticles effectively cleared the infection in the latter, suggesting considerable promise for combination therapeutics which prevent biofilm formation as well as effectively killing mature P. aeruginosa biofilms.

Highlights

The increasing problems of multi-antibiotic resistance urgently require alternative therapeutic options.[1,2,3] One of the main issues associated with indiscriminate antibiotic use is the activation of defence mechanisms in pathogens,[4] and as a survival strategy, bacteria form biofilms.[5]
The design principles of the combination therapeutic were firstly that it needed to be derived from a simple, readily accessible polymer already used in human clinical applications
In this study we have shown that alginate biopolymers can be modified to contain a responsive linker to the quorum sensing inhibitor (QSI) ACNQ, and induced to self-assemble in the presence of an antibiotic, ciprofloxacin, to generate well-defined nanoparticles

Summary

Introduction

The increasing problems of multi-antibiotic resistance urgently require alternative therapeutic options.[1,2,3] One of the main issues associated with indiscriminate antibiotic use is the activation of defence mechanisms in pathogens,[4] and as a survival strategy, bacteria form biofilms.[5]. Biofilms are refractory to clearance by the host immune system and so elicit a number

Methods

Results

Conclusion