Bactericidal Activity of Lipid-Shelled Nitric Oxide-Loaded Microbubbles

Abstract

The global pandemic of antibiotic resistance is an ever-burgeoning public health challenge, motivating the development of adjunct bactericidal therapies. Nitric oxide (NO) is a potent bioactive gas that induces a variety of therapeutic effects, including bactericidal and biofilm dispersion properties. The short half-life, high reactivity, and rapid diffusivity of NO make therapeutic delivery challenging. The goal of this work was to characterize NO-loaded microbubbles (MB) stabilized with a lipid shell and to assess the feasibility of antibacterial therapy in vitro. MB were loaded with either NO alone (NO-MB) or with NO and octafluoropropane (NO-OFP-MB) (9:1 v/v and 1:1 v/v). The size distribution and acoustic attenuation coefficient of NO-MB and NO-OFP-MB were measured. Ultrasound-triggered release of the encapsulated gas payload was demonstrated with 3-MHz pulsed Doppler ultrasound. An amperometric microelectrode sensor was used to measure NO concentration released from the MB and compared to an NO-OFP-saturated solution. The effect of NO delivery on the viability of planktonic (free living) Staphylococcus aureus (SA) USA 300, a methicillin-resistant strain, was evaluated in a 96 well-plate format. The co-encapsulation of NO with OFP increased the total volume and attenuation coefficient of MB. The NO-OFP-MB were destroyed with a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure (in situ MI of 1.34) but maintained their echogenicity when exposed to 0.02 MPa peak negative pressure (in situ MI of 0.01. The NO dose in NO-MB and NO-OFP-MB was more than 2-fold higher than the NO-OFPsaturated solution. Delivery of NO-OFP-MB increased bactericidal efficacy compared to the NO-OFP-saturated solution or air and OFP-loaded MB. These results suggest that encapsulation of NO with OFP in lipid-shelled MB enhances payload delivery. Furthermore, these studies demonstrate the feasibility and limitations of NO-OFP-MB for antibacterial applications.

Bactericidal and anti-biofilm effects of uncharged and cationic ultrasound-responsive nitric oxide microbubbles on Pseudomonas aeruginosa biofilms

“…Recent research has shown that release of NO from lipid-shelled MBs can occur rapidly within approximately 7 minutes of administration ( Lafond et al., 2020 ); it was therefore important that NOMBs were administered in the SAT immediately after production. Another important step taken in this research was to verify the inclusion of NO within the MB, this was achieved using a nitric oxide colorimetric assay (Abcam, plc.…”

“…The total NO concentration within a 1:5 diluted suspension of DBPC lipid-shelled NOMBs applied to biofilms in this research was 16.5 µM (SD ± 1.45 µM), which is well within the reported range of efficacy for NO mediated treatment of bacterial biofilms ( Barraud et al., 2015 ; Howlin et al., 2017 ). Lafond et al. (2020) , demonstrated quantification of NO concentration by amperometric sensor that showed an average concentration in the suspension of 5 mM in the similarly lipid-shelled Octafluoropropane-NO MB formulation utilised.…”

“…This previous work importantly showed the effect that different NO concentrations can have on bacterial activity; using concentrations of 0.175 – 2.622 µM a 50% decrease in bacterial viability was observed in planktonic suspensions of S. aureus , however, no discernible bactericidal activity was seen in planktonic suspensions of P. aeruginosa ( Choi et al., 2019 ). More recently the bactericidal activity of a lipid shelled MB co-loaded with NO and octafluoropropane has been demonstrated, whereby a clinical ultrasound scanner with an output of 2.48 MPa peak negative pressure was used to induce MB cavitation ( Lafond et al., 2020 ).…”

“…Despite recent research beginning to characterize the bactericidal efficacy of ultrasound responsive NO formulations in planktonic bacterial suspensions ( Lafond et al., 2020 ), to the best of our knowledge, there are currently no acoustically stimulated NOMBs that have been applied to the treatment of biofilms in vitro or in vivo. Consequently, there is currently no evidence on the efficacy of NOMBs as a means of inducing biofilm dispersal or potentiating bactericidal activity in biofilms.…”

Different Aspects of Using Ultrasound to Combat Microorganisms

“…By enclosing this gas in a shell of acoustically active microbubbles, it is possible to control the release of NO both spatially and temporally by using US. [11,84] Recently, Lafond et al [84] demonstrated the bactericidal efficacy of microbubbles against MRSA plankton when stabilized by a lipid membrane, and loaded with NO alone or combined with octafluoropropane.…”