Liposome-Encapsulated ISMN: A Novel Nitric Oxide-Based Therapeutic Agent against Staphylococcus aureus Biofilms

Abstract

Background Staphylococcus aureus in its biofilm form has been associated with recalcitrant chronic rhinosinusitis with significant resistance to conventional therapies. This study aims to determine if liposomal-encapsulation of a precursor of the naturally occurring antimicrobial nitric oxide (NO) enhances its desired anti-biofilm effects against S. aureus, in the hope that improving its efficacy can provide an effective topical agent for future clinical use.Methodology S. aureus ATCC 25923 biofilms were grown in-vitro using the Minimum Biofilm Eradication Concentration (MBEC) device and exposed to 3 and 60 mg/mL of the NO donor isosorbide mononitrate (ISMN) encapsulated into different anionic liposomal formulations based on particle size (unilamellar ULV, multilamellar MLV) and lipid content (5 and 25 mM) at 24 h and 5 min exposure times. Biofilms were viewed using Live-Dead Baclight stain and confocal scanning laser microscopy and quantified using the software COMSTAT2.ResultsAt 3 and 60 mg/mL, ISMN-ULV liposomes had comparable and significant anti-biofilm effects compared to untreated control at 24 h exposure (p = 0.012 and 0.02 respectively). ULV blanks also had significant anti-biofilm effects at both 24 h and 5 min exposure (p = 0.02 and 0.047 respectively). At 5 min exposure, 60 mg/mL ISMN-MLV liposomes appeared to have greater anti-biofilm effects compared to pure ISMN or ULV particles. Increasing liposomal lipid content improved the anti-biofilm efficacy of both MLV and ULVs at 5 min exposure.ConclusionLiposome-encapsulated “nitric oxide” is highly effective in eradicating S. aureus biofilms in-vitro, giving great promise for use in the clinical setting to treat this burdensome infection. Further studies however are needed to assess its safety and efficacy in-vivo before clinical translation is attempted.

New insights into Legionella pneumophila biofilm regulation by c-di-GMP signaling

“…Among these signals, nitric oxide has been increasingly studied, especially because NO was shown to induce the dispersion of many pathogenic biolms (eg E. coli, Fusobacterium nucleatum, Staphylococcus aureus, and Vibrio cholerae) (Jardeleza et al 2014;Arora et al 2015;Barraud et al 2015), and to increase the sensitivity of bio lm bacteria to antibiotics (Barraud et al 2006;Barraud, Storey, et al 2009). However, bio lm formation by Azospirillum brazilense, Vibrio harveyi and Shewanella oneidensis appears to be induced by the addition of NO in the medium (Arruebarrena Di Palma et al 2013;Plate & Marletta 2012;Henares et al 2013).…”

Effects of isosorbide mononitrate loaded nanoparticles conjugated with anti‑Staphylococcus aureus α‑toxin on Staphylococcus aureus biofilms

“…Biofilm forming strain S. aureus ATCC25923 was used in this study. Bacterial cultures were established as previously described (3). Briefly, bacterial strains (frozen glycerol stock) were inoculated onto nutrient agar (Oxoid) plates and incubated overnight at 37˚C for recovery.…”

“…Chronic rhinosinusitis (CRS) is a common rhinology disease; the reported prevalence of Staphylococcus aureus (S. aureus) in CRS patients is 61% (1). Bacterial biofilms are considered as a common and important cause of persistent infections; biofilms require up to 1,000x higher antibiotic doses for effective treatment compared to planktonic cells, thus hindering eradication (2,3). Long-term use of antibiotics and emerging resistant bacteria pose a great threat to human health.…”

Future topical medications in chronic rhinosinusitis

“…Several studies have shown the potential antibiofilm therapeutic effect of nasal NO in CRS, possibly as a result of the formation of toxic acidified nistabutrite derivatives . Jardeleza et al designed a liposomal formulation of a NO donor, isosorbide mononitrate, and demonstrated antibiofilm safety and efficacy in vitro and in a sheep model. The results were further supported by identification of taste receptors, such as T2R38, that detect molecules generated by Gram‐negative bacteria and activate NO production, found to be inversely correlated with the formation of biofilms in CRS patients, and discussed later …”