(158s) Zinc Oxide Nanoparticles with an Anti-Tubercular Drug for Overcoming Mycobacterial Drug Resistance

Tuberculosis, caused by Mycobacterium tuberculosis, is one of the top ten causes of death worldwide. With the emergence of multi-drug resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains, the disease has reached serious proportions. The intrinsic drug resistance of mycobacteria, due to mycolic acid-rich cell wall and active efflux pumps, is one of the major limitations in tuberculosis treatment. These factors are responsible for low intracellular drug concentration. In addition to intrinsic drug resistance, MDR and XDR strains have also developed acquired drug resistance towards the first-line anti-TB drugs in current use. In this aspect, nanoparticles can be a useful tool for overcoming bacterial drug resistance. Metal or metal oxide nanoparticles such as zinc oxide, silver, titanium dioxide, copper are themselves, anti-microbial agents. Therefore, if combined with other anti-microbial drugs, they can demonstrate an additive or synergistic inhibitory effect. In this study, we present a nanoparticle-drug combination for enhancing the effectiveness of existing anti-tubercular drugs. To this end, zinc oxide nanoparticles (ZnO NPs) were synthesized and evaluated their antibacterial effect against mycobacteria, alone as well as in combination with the first-line anti-TB drug, rifampicin.

The ZnO NPs were synthesized using precipitation in the liquid phase. The particle size of ZnO NPs, as determined by transmission electron microscopy images, was found to be 11 nm. X-ray diffraction pattern indicated the formation of the hexagonal wurtzite phase. These ZnO NPs themselves were not inhibitory against wild-type (WT) Mycobacterium smegmatis up to a concentration as high as 256 µg/ml. However, a sub-inhibitory concentration of 32 µg/ml of ZnO NPs, was able to decrease the minimum inhibitory concentration (MIC) of rifampicin by four-fold against WT M. smegmatis. The fractional inhibitory concentration index (FICI) for the combination was found to be 0.375, which proves the synergistic interaction of the combination.

Further, to understand the mechanism of synergistic action, the effect of the NP and drug combination on various physiological parameters was investigated. ZnO NPs and rifampicin combination was observed to enhance the cell membrane permeability, which increased with time. The membrane permeability changes due to interaction with ZnO NPs with the bacterial cell membrane. The morphology of the bacteria, as observed under Cryo-SEM, displayed dramatic changes in bacterial morphology after 4 h of treatment with ZnO NPs alone as well as in combination with rifampicin. However, cells treated with ZnO NPs alone exhibited normal morphology after 12 h, suggesting eventual recovery of the cell over a longer time duration. However, in the case of the combination, it was observed to have extensive damage to the bacterial cells. These morphological translations are well correlated with membrane permeability data. Consequently, we have seen increased rifampicin uptake in a bacterial cell after treatment with a combination of ZnO NPs and rifampicin, due to underlying higher membrane permeability, which eventually results in the loss of cell viability.

Moreover, it is interesting to note that several studies have reported reactive oxygen species (ROS) production to be the primary mechanism for the anti-bacterial action of ZnO NPs. However, in our work, we have observed higher ROS production in the presence of ZnO NPs alone. In contrast, for the combination of ZnO NPs and rifampicin, ROS production was much lower compared to the corresponding concentration of ZnO NPs. Hence, we can conclude that the low ROS production did not affect the killing of the bacteria, but the combined impact of membrane damage and anti-mycobacterial activity of rifampicin explained the synergistic activity for the ZnO NPs and rifampicin combination.

Finally, we have also evaluated the combination of ZnO NPs and rifampicin against laboratory-generated drug-resistant strains of M. smegmatis as well as M. bovis BCG, an attenuated strain of M. bovis and a close relative of the virulent strain, M. tuberculosis. The laboratory-generated drug-resistant strains of M. smegmatis exhibit a MDR phenotype with two to four-fold resistance towards rifampicin. Interestingly, the combination also exhibited a synergistic interaction against all strains. Hence, with the detailed mechanistic study of the combination of ZnO NPs and rifampicin, we propose that this developed combination system can be promising for overcoming mycobacterial drug resistance. Further, we are working on mannose functionalized solid lipid nanoparticles (SLN) for delivering ZnO NPs and rifampicin to specifically target bacteria residing in macrophages.

Keywords: Drug resistance, zinc oxide nanoparticle, Mycobacterium, rifampicin, membrane permeability