Garima Pant

Inhalable Particles Containing Rapamycin for Induction of Autophagy in Macrophages Infected with Mycobacterium Tuberculosis

We investigated whether particles suitable for delivery to alveolar macrophages may provide a means of targeting rapamycin, an inducer of autophagy, to alveolar macrophages as a host-directed antituberculosis agent. Inhalable particles were prepared by spray-drying and characterized using laser scattering and electron microscopy. Their aerodynamic diameter was calculated from bulk and tapped densities. In vitro drug release was studied in PBS containing 1% SDS. In vitro uptake of particles by THP-1 derived macrophages was studied by flow cytometry. Cytotoxicity of the particles toward macrophages and their efficacy against intracellular Mycobacterium tuberculosis were studied using a methyltetrazolium assay and counting bacterial colonies obtained when cell lysates were plated on agar. The encapsulation efficiency was 88.8 ± 1.13% and drug content 22 ± 4% w/w. The particles had a median diameter of 2.88 ± 0.8 μm and appeared as collapsed spheres. Their calculated aerodynamic diameter was about 1 μm. In vitro drug release from the particles was first-order and extended beyond 10 days. Flow cytometry indicated that the particles were taken up by macrophages within 3 h. Macrophages exposed to the particles or rapamycin in solution at a concentration of 100 μg/mL over a 24 h period maintained 79.37 ± 0.72% and 58.33 ± 1.39% viability, respectively. Efficacy studies concluded that particles were more effective in clearing intracellular mycobacteria than rapamycin in solution. It was concluded that the preparation was suitable for formulating as a dry powder inhalation to test efficacy of inhaled, macrophage-targeted rapamycin against TB.

Plumbagin, a plant-derived naphthoquinone metabolite induces mitochondria mediated apoptosis-like cell death in Leishmania donovani: an ultrastructural and physiological study.

Naphthoquinones are known to exhibit a broad range of biological activities against microbes, cancer and parasitic diseases and have been widely used in Indian traditional medicine. Plumbagin is a plant-derived naphthoquinone metabolite (5-hydroxy-2-methyl-1,4-naphthoquinone) reported to inhibit trypanothione reductase, the principal enzyme and a validated drug target involved in detoxification of oxidative stress in Leishmania. Here, we report the mechanistic aspects of cell death induced by plumbagin including physiological effects in the promastigote form and ultrastructural alterations in both promastigote and amastigote forms of Leishmania donovani which till now remained largely unknown. Our observations show that oxidative stress induced by plumbagin resulted in depolarization of the mitochondrial membrane, depletion in ATP levels, elevation of cytosolic calcium, increase in caspase 3/7-like protease activity and lipid peroxidation in promastigotes. Apoptosis-like cell death induction post plumbagin treatment was confirmed by biochemical assays like Annexin V/FITC staining, TUNEL as well as morphological and ultrastructural studies. These findings collectively highlight the mode of action and importance of oxidative stress inducing agents in effectively killing both forms of the Leishmania parasite and opens up the possibility of exploring plumbagin and its derivatives as promising candidates in the chemotherapy of Leishmaniasis.

Cholesterol oxidase production from entrapped cells of Streptomyces sp.

Production of cholesterol oxidase (COD) under batch conditions through Ca‐alginate immobilized cells of Streptomyces sp. was investigated. The process was studied for optimal immobilization conditions, beads operational stability and comparisons were made with the COD production via free cells. Influence of Na‐alginate concentration (1–5 g L−1) and initial biomass loading on enzyme production were studied. Effects of initial pH of the production medium, temperature, shaker speed, as well as reuse of beads on the COD production were also investigated. It was observed that COD production with immobilized cells (5.6 U ml−1) was higher in comparison to free cells (4.5 U ml−1) under optimized conditions. The maximum COD production by free cells was observed with initial pH 7.0, rpm 200 after 96 h of incubation while immobilized cells sustain a broad pH range 6.0–9.0, rpm 300 for maximum production after 72 h. The immobilized and free cells produced maximum COD in the culture incubated at 37 and 30 °C, respectively. Other parameters bead size and Na‐alginate concentration found to be optimum with 1.5 mm and 4% w/v, respectively. Scanning electron microscope study of the immobilized cells indicated that the cells in Ca‐alginate beads remained in normal shape with no alterations in the morphology.

Biofilm inhibition and anti-Candida activity of a cationic lipo-benzamide molecule with twin-nonyl chain

A series of cationic lipo-benzamide compounds with varying lengths of hydrocarbon chains (C2M-C18M) were evaluated for anti-Candida activity. Four compounds harbouring 8-11 hydrocarbon chains demonstrated concentration-dependent inhibition of fungal cell growth with Minimum Inhibitory Concentration (MIC) of ≤6.2 µg ml-1. The most active compound (C9M) inhibited growth of both Candida albicans and non-albicans strains and is equally active against pairs of azole sensitive and resistant clinical isolates of C. albicans. Compound C9M also inhibited different stages of Candida biofilms. Scanning Electron Microscopy (SEM) of Candida cells after C9M treatment was also done and no significant cell lysis was observed. Hemolysis assay was performed and only 2.5% haemolysis was observed at MIC concentration.

Chain-length-specific anti-Candida activity of cationic lipo-oxazoles: a new class of quaternary ammonium compounds

PURPOSE Candida species have become resistant to commonly used anti-fungal drugs like fluconazole and echinocandins. In our screen, a series of quaternary ammonium compounds (QACs) emerged as an alternative treatment choice for drug-resistant Candida infections. METHODOLOGY Medium alkyl chain cationic lipo-oxazoles comprising six to thirteen twin carbon chains and a quaternary ammonium unit were synthesized and evaluated for their in vitro anti-Candida and biofilm inhibition activity. SEM was performed to visualize membrane distortion.Results/Key findings. Heptyl and octyl chain analogues (5c, 6b and 6c) showed promising anti-fungal activity. Compound 5c was active against both fluconazole-sensitive and resistant clinical isolates of Candida albicans as well as non-albicans Candida strains. 5c also inhibited the adhesion of C. albicans cells to a polystyrene surface and restricted biofilm formation. SEM further confirmed Candida cell membrane distortion by 5c. CONCLUSION A novel class of QACs, called cationic lipo-oxazoles, was tested and found to exhibit anti-fungal activity against planktonic cells as well as biofilms of Candida.