Antimicrobial efficacy of tobramycin polymeric nanoparticles for Pseudomonas aeruginosa infections in cystic fibrosis: Formulation, characterisation and functionalisation with dornase alfa (DNase)

Abstract

Inhaled antibiotics, such as tobramycin, for the treatment of Pseudomonas aeruginosa pulmonary infections are associated with the increase in life expectancy seen in cystic fibrosis (CF) patients over recent years. However, the effectiveness of this aminoglycoside is still limited by its inability to penetrate the thick DNA-rich mucus in the lungs of these patients, leading to low antibiotic exposure to resident bacteria. In this study, we created novel polymeric nanoparticle (NP) delivery vehicles for tobramycin. Using isothermal titration calorimetry, we showed that tobramycin binds with alginate polymer and, by exploiting this interaction, optimised the production of tobramycin alginate/chitosan NPs. It was established that NP antimicrobial activity against P. aeruginosa PA01 was equivalent to unencapsulated tobramycin (minimum inhibitory concentration 0.625mg/L). Galleria mellonella was employed as an in vivo model for P. aeruginosa infection. Survival rates of 90% were observed following injection of NPs, inferring low NP toxicity. After infection with P. aeruginosa, we showed that a lethal inoculum was effectively cleared by tobramycin NPs in a dose dependent manner. Crucially, a treatment with NPs prior to infection provided a longer window of antibiotic protection, doubling survival rates from 40% with free tobramycin to 80% with NP treatment. Tobramycin NPs were then functionalised with dornase alfa (recombinant human deoxyribonuclease I, DNase), demonstrating DNA degradation and improved NP penetration of CF sputum. Following incubation with CF sputum, tobramycin NPs both with and without DNase functionalisation, exhibited anti-pseudomonal effects. Overall, this work demonstrates the production of effective antimicrobial NPs, which may have clinical utility as mucus-penetrating tobramycin delivery vehicles, combining two widely used CF therapeutics into a single NP formulation. This nano-antibiotic represents a strategy to overcome the mucus barrier, increase local drug concentrations, avoid systemic adverse effects and improve outcomes for pulmonary infections in CF.

Targeting microbial biofilms: current and prospective therapeutic strategies

“…Nanoparticles functionalized with biofilm EPS matrix-digesting enzymes (DNase) and loaded with ciprofloxacin eradicated established P. aeruginosa biofilms without cytotoxicity against macrophages 49 . Likewise, tobramycin alginate-chitosan nanoparticles functionalised by conjugation to dornase alfa (DNase) demonstrated better biofilm penetration and DNA degradation in sputum from patient with cystic fibrosis and increased protection against bacteria in an invertebrate infection model 148 . Furthermore, nanoparticles that were designed to release and activate NO in situ had antifungal and antibacterial effects, inhibited biofilm formation and promoted the degradation of the EPS matrix in vitro and in vivo 149,150 .…”

Pseudomonas Aeruginosa : Arsenal of Resistance Mechanisms, Decades of Changing Resistance Profiles, and Future Antimicrobial Therapies

“…There have been increasing reports on the incorporation of antibiotics into nanocomposites and nanodelivery systems. Recently, DNase tobramycin polymeric nanoparticles have shown improved drug delivery and antimicrobial activity against P. aeruginosa [212]. Despite progress being made, nanoantimicrobial agents are not yet used in human clinical applications.…”

“…Discussion on some of the newer antibiotics at different developmental and approval stages can be located elsewhere [216,217]. Numerous studies reported the effectiveness of nanoantimicrobial agents against Gram-negative bacteria including P. aeruginosa [212,[218][219][220][221][222][223][224]. …”

Biofunctionalization of surfaces using polyelectrolyte multilayers

“…One possible approach is the use of specific PEMs that allow versatile and tunable release of drugs like antibacterial substances and make them additionally attractive as coatings for implantable devices [62].…”