Alginate modified-PLGA nanoparticles entrapping amikacin and moxifloxacin as a novel host-directed therapy for multidrug-resistant tuberculosis

Abstract

Polymeric nanoparticles have been widely investigated as a controlled release drug delivery platform for the treatment of tuberculosis (TB). These nanoparticles are readily internalised into macrophages and leads to high intracellular drug concentration. In this study two anti-TB drugs, amikacin and moxifloxacin were encapsulated into PLGA nanoparticles. The novelty of this work includes: (1) the efficient encapsulation of two hydrophilic second-line anti-TB drugs, and (2) intra-macrophage delivery of this combination potentially for rapid treatment of multi-drug resistant TB (MDR-TB). Two water-oil-water (w/o/w) emulsion strategies were employed in this study to produce: (1) alginate coated PLGA nanoparticles, and (2) alginate entrapped PLGA nanoparticles. The average particle size and polydispersity index (PDI) of the alginate coated PLGA nanoparticles were found to be unfavourably high with values of 640 ± 32 nm and 0.63 ± 0.09, respectively. In contrast, the alginate entrapped PLGA nanoparticles were within the desired particle size range of 312–365 nm and the PDI was 0.14– 0.31, and therefore were chosen for subsequent studies. Alginate entrapped nanoparticles exhibited an entrapment of moxifloxacin in the range of 10.1–18.7 μg/mg and an entrapment of amikacin in the range of 15–17.4 μg/mg. On the contrary, non-modified PLGA nanoparticles exhibited a significantly less amikacin loading of 6.2 μg/mg polymer and 4.2 μg/mg of moxifloxacin. The release of alginate modified PLGA nanoparticles showed slower release in comparison with the non-modified PLGA nanoparticles. The alginate entrapped PLGA nanoparticles did not show significant difference from the alginate coated PLGA nanoparticles in the release pattern. To study the macrophage uptake efficiency, both formulations of the alginate entrapped nanoparticle; non-calcium formulation and calcium, and alginate coated PLGA nanoparticles (that includes no calcium) formulation was loaded with coumarin-6 as a marker, seeded to THP-1 derived macrophages infected with Mycobacterium tuberculosis (H37Ra), and examined by confocal microscopy. Both formulations of alginate entrapped particles (calcium and non-calcium) were readily internalised into the macrophages and highly concentrated in the perinuclear region, and the non-calcium alginate entrapped formulation showed a maximum internalisation. Furthermore, the anti-mycobacterial activity of the dually entrapped drug-loaded particles (moxifloxacin and amikacin) was evaluated using M. tuberculosis-infected macrophages, which revealed an enhanced inhibition of viable bacterial count compared to single drug loaded nanoparticle formulations, and untreated cells. The dually entrapped nanoparticles showed bacterial viability of 0.6% relative to the untreated group, compared to 6.49% for amikacin alone nanoparticles and 3.27% for moxifloxacin alone nanoparticles. In conclusion, the amikacin-moxifloxacin alginate entrapped PLGA nanoparticles are promising for further in vivo studies.