Nitric Oxide-Releasing Liposomes for Treatment of Mycobacterium abscessus Infections.

Abstract

Nontuberculosis mycobacteria (NTM) are ubiquitous, opportunistic pathogens that cause severe respiratory infection, primarily in elderly and immunocompromised populations. The second most prevalent NTM pathogen, Mycobacterium abscessus, is considered the most refractory due to its fast growth rate, intracellular survivability, and antibiotic resistance. Treatments are thus sparse and generally ineffective, promoting antibiotic resistance upon chronic use. Nitric oxide (NO) is an endogenously produced free radical that exerts antimicrobial effects against pathogens via several mechanisms of action, and as such, it is unlikely to elicit resistance. Methyl tris diazeniumdiolate (MD3) is a small-molecule NO-releasing prodrug that is capable of sustained NO release, making it an attractive candidate as an antimicrobial therapeutic; however, its triple negative charge makes cellular uptake unlikely. As liposomes enable cellular uptake, their use as an MD3 delivery system may further enhance the utility of NO release for treating intracellular NTM infections. Herein, liposomal formulations were evaluated as a function of pH and buffer composition and optimized for MD3 loading to enable the delivery of bactericidal levels of NO. Planktonic studies with two clinically relevant morphotypes of M. abscessus revealed that lower pK_a liposomal systems employ a better antimicrobial efficacy. Prevention and eradication assays revealed that liposomal MD3 significantly improves biofilm inhibition compared to nonliposomal MD3 and was capable of eradicating biofilm bacteria at 4 mg mL^-1. Liposomal MD3 and MD3 had similar reductions in intracellular bacterial load, achieving at least a three-log reduction at relevant concentrations. Fluorescence spectroscopy over 24 h demonstrated that liposomal encapsulation increased the intracellular concentration of a membrane-impermeable fluorophore by 3.4-fold. Confocal microscopy was used to visualize the increase in the number of cells containing intracellular NO and the sustained presence of NO within the cell, confirming that liposomal MD3 increases small-molecule internalization.