Aptamer-Loaded Cubosome Lipid Nanoparticles for the Treatment of Tuberculosis.

Tuberculosis (TB), particularly multidrug-resistant TB (MDR-TB), remains a critical global health challenge, necessitating innovative approaches to enhance treatment efficacy and overcome resistance mechanisms. Aptamers, short, single-stranded nucleic acids, are under active investigation for both diagnosis and treatment of TB due to their high specificity, low immunogenicity, and strong binding affinity to molecular targets. A single stranded DNA (SSDA) aptamer was recently designed as an inhibitor for Malate Synthase (MS) of Mycobacterium tuberculosis (MTB). MS, also known as GlcB, is a key enzyme in the glyoxylate pathway, which is crucial for the persistent infection of MTB. Moreover, when it is present on the cell surface it acts like an adhesin and helps bacteria to invade the host cell. However, the clinical translation of aptamers is hindered by challenges such as rapid degradation in vivo and poor cellular membrane penetration. This study is the first to investigate the use of cubosome lipid nanoparticles for the delivery of an aptamer therapeutic, specifically the MS10-Trunc (5-GGTGGTGGTGG-3') SSDA sequence which was loaded in monoolein (MO)-based cationic cubosomes for delivery to MTB-H37Ra cells. Notably, the use of cationic cubosomes lowered the minimum inhibitory concentration (MIC) of the MS10-Trunc SSDA by ∼3.5 fold against actively replicating MTB-H37Ra and shortened the bacterial lifecycle from 5 to 3 days, compared to the unencapsulated aptamer. Mechanistic insights from confocal fluorescence microscopy and total internal reflection fluorescence microscopy (TIRFM) demonstrated efficient uptake of MS10-Trunc SSDA-loaded cubosomes by bacteria and MTB model membranes, likely resulting in the observed improvement in efficacy. This innovative approach, combining the high specificity of MS10-Trunc SSDA with the drug delivery advantages of cubosomes, advances research in aptamer-based drug delivery systems and introduces a new strategic approach to personalized TB therapies, including against MDR-TB.