Despite progress in tuberculosis (TB) treatment, emergence of multi-drug resistant (MDR) and extensive-drug resistant (XDR) strains of Mycobacterium tuberculosis still remains a global health challenge. Among the various mechanisms responsible for drug resistance, decreased intracellular drug levels due to combination of lowered influx of drugs into the cells (decreased membrane permeability) and increased efflux of drugs outside the cells (over-expression of efflux pumps) plays a very important role in increasing the tolerance of Mycobacterium towards the anti-TB drugs. Thus, development of novel strategies to enhance this effective intracellular drug concentration is crucial. In our earlier work, we demonstrated that nanoparticles can be used to enhance the efficacy of anti-TB drugs to target intrinsic resistance in Mycobacterium smegmatis, a model mycobacteria. In the present work, we demonstrate for the first time that nanoparticles can be used to overcome the acquired drug resistance in mycobacteria, such as in clinical isolates. To mimic the situation of clinical drug resistance, we have generated in vitro hyper-resistant mutant strains of M. smegmatis, which confers a very high level of resistance to anti-TB drugs compared to the wild type strain.
Polyacrylic acid coated magnetite nanoparticles (PAA-MNP) were used in combination with anti-TB drugs to enhance the drug efficacy in hyper-resistant mutants of M. smegmatis. PAA-MNP’s synthesized by thermal decomposition route formed a stable dispersion with an average size of 9 nm. These nanoparticles were found to interact with the mutant strains of M. smegmatis via surface attachment and internalization as revealed by Transmission Electron Microscopy (TEM) and Inductively Coupled Plasma−Atomic Emission Spectroscopy (ICP-AES), respectively. Next, we have investigated the effect of these coated nanoparticles alone, drug alone and a combination of coated nanoparticles with drugs on growth profile of the mutants. These nanoparticles were non-toxic to mutants when used alone, thus making them a superior candidate for therapeutic use. A synergistic enhancement in growth inhibition of mutants, upto 16-folds was observed using anti-TB drugs (rifampicin or isoniazid or norfloxacin) in combination with PAA-MNP in comparison to free drug alone.
In order to mechanistically understand the role of coated magnetite nanoparticles in overcoming drug resistance, we have performed extensive biochemical analysis and drug transport studies in the hyper-resistant mutants. As the alteration of drug transport due to permeability barrier and active efflux of drugs plays a major role in development of drug resistance, we have measured the intracellular drug levels in mutants in presence of drug alone and drug in combination with nanoparticles. PAA-MNP’s were able to enhance the intracellular levels of various anti-TB drugs in mutants by atleast 4-folds when compared to free drug. Furthermore, the real-time transport kinetics of fluorescent tracers revealed that PAA-MNP enhances the drug uptake by increasing the influx of drugs into the cells while simultaneously inhibiting efflux of the drug out of cells, thereby enhancing the intracellular drug concentration.
Altogether, these results suggest that coated magnetite nanoparticles can act as a new class of permeability enhancers and efflux inhibitors, which in combination with anti-TB drugs, can effectively target drug resistant mycobacteria.