Nagy Sarah G, Metiva Joseph J, Schoenfisch Mark H
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599, United States.
Division of Pharmacoengineering and Molecular Pharmaceutics, UNC Eshelman School of Pharmacy, Chapel Hill, North Carolina 27599, United States.
ACS Appl Bio Mater. 2025 Jun 16;8(6):4829-4843. doi: 10.1021/acsabm.5c00218. Epub 2025 May 16.
Nontuberculosis mycobacteria (NTM) are ubiquitous, opportunistic pathogens that cause severe respiratory infection, primarily in elderly and immunocompromised populations. The second most prevalent NTM pathogen, , 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 revealed that lower p 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. 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.
非结核分枝杆菌（NTM）是普遍存在的机会致病菌，主要在老年人和免疫功能低下人群中引起严重的呼吸道感染。第二常见的NTM病原体因其生长速度快、细胞内存活力强和具有抗生素抗性，被认为是最难治疗的。因此，治疗方法稀少且通常无效，长期使用会导致抗生素抗性。一氧化氮（NO）是一种内源性产生的自由基，通过多种作用机制对病原体发挥抗菌作用，因此不太可能引发抗性。甲基三（二氮烯二醇）（MD3）是一种释放NO的小分子前药，能够持续释放NO，使其成为一种有吸引力的抗菌治疗候选药物；然而，其三个负电荷使其不太可能被细胞摄取。由于脂质体能够促进细胞摄取，将其用作MD3递送系统可能会进一步提高NO释放治疗细胞内NTM感染的效用。在此，对脂质体制剂进行了评估，考察其作为pH和缓冲液组成的函数，并针对MD3负载进行了优化，以实现杀菌水平NO的递送。对两种临床相关形态型的浮游菌研究表明，较低pH的脂质体系统具有更好的抗菌效果。预防和根除试验表明，与非脂质体MD3相比，脂质体MD3显著提高了生物膜抑制作用，并且能够在4 mg/mL时根除生物膜细菌。脂质体MD3和MD3在细胞内细菌载量方面有相似的降低，在相关浓度下至少实现了三个对数级的降低。24小时的荧光光谱表明，脂质体包封使膜不可渗透的荧光团的细胞内浓度增加了3.4倍。共聚焦显微镜用于可视化含有细胞内NO的细胞数量的增加以及细胞内NO的持续存在，证实脂质体MD3增加了小分子内化。
