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Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

bWith the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G 1 -phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.

Biocompatible and degradable dual‐delivery gel systems based on hyperbranched dendritic−linear−dendritic copolymers (HBDLDs) is herein conceptualized and accomplished via thiol‐ene click chemistry. The elasticity of the hydrogels is tunable by varying the lengths of PEG (2, 6, 10 kDa) or the dry weight percentages (20, 30, 40 wt%), and are found to range from 2–14.7 kPa, comparable to human skin. The co‐delivery of antibiotics is achieved, where the hydrophilic drug novobiocin sodium salt (NB) is entrapped within the hydrophilic hydrogel, while the hydrophobic antibiotic ciprofloxacin (CIP) is encapsulated within the dendritic nanogels (DNGs) with hydrophobic cores (DNGs‐CIP). The DNGs‐CIP with drug loading capacity of 2.83 wt% are then physically entrapped within the hybrid hydrogels through UV curing. The hybrid hydrogels enable the quick release of NB and prolonged released of CIP. In vitro cell infection assays showed that the antibiotic‐loaded hybrid hydrogels are able to treat bacterial infections with significant bacterial reduction. Hybrid hydrogel band aids are fabricated and exhibited better antibacterial activity compared with commercial antimicrobial band aids. Remarkably, most hydrogels and hybrid hydrogels show enhanced human dermal cell proliferation and could be degraded into non‐toxic constituents, showing great promise as wound dressing materials.