Synthesis and Spectral Characterisation of Fabricated Cerium-Doped Magnesium Oxide Nanoparticles: Evaluation of the Antimicrobial Potential and Its Membranolytic Activity through Large Unilamellar Vesicles.

Considerable attention has been given to Magnesium oxide nanoparticles lately due to their antimicrobial potential, low toxicity to humans, high thermal stability, biocompatibility, and low cost of production. However, their successful transformation into sustainable drugs is limited due to their low membrane permeability, which reduces their bioavailability in target cells. Herein we propose Cerium-doped magnesium oxide nanoparticles (MgOCeNPs) as a powerful solution to above mentioned limitations and are compared with MgO NPs for their membrane permeability and antimicrobial activity. Both pure and Ce-doped were characterized by various spectroscopic and microscopic techniques, in which an X-ray diffraction (XRD) examination reveals the lattice patterns for doped nanoparticles. Furthermore, Atomic Force Microscopy (AFM) revealed the three-dimensional (3D) structure and height of the nanoparticle. The crystal structure (FCC) of MgO did not change with Ce doping. However, microstructural properties like lattice parameter, crystallite size and biological activity of MgO significantly changed with Ce doping. In order to evaluate the antimicrobial potential of MgOCeNPs in comparison to MgO NPs and to understand the underlying mechanisms, the antibacterial activity was investigated against human pathogenic bacteria E. coli and P. aeruginosa, and antifungal activity against THY-1, a fungal strain. MgOCeNPs were studied by several methods, which resulted in a strong antibacterial and antifungal activity in the form of an elevated zone of inhibition, reduced growth curve, lower minimum inhibitory concentration (MIC80) and enhanced cytotoxicity in both bacterial and fungal strain as compared to MgO nanoparticles. The study of the growth curve showed early and prolonged stationary phase and early decline log phase. Both bacterial and fungal strains showed dose-dependent cytotoxicity with enhancement in intracellular reactive oxygen species (ROS) generation and formation of pores in the membrane when interacting with egg-phosphatidylcholine model Large Unilamellar Vesicles (LUVs). The proposed mechanism of MgOCeNPs toxicity evidently is membranolytic activity and induction of ROS production, which may cause oxidative stress-mediated cytotoxicity. These results confirmed that MgOCeNPs are a novel and very potent antimicrobial agent with a great promise of controlling and treating other microbes.

Evaluation of antimicrobial, anticancer potential and Flippase induced leakage in model membrane of Centella asiatica fabricated MgONPs.

The use of chemically synthesized nanoparticles and crude plant extracts as antimicrobial -anticancer agents have many limitations. In this study, we have used Centella asiatica extract (CaE) having relatively less explored but tremendous medicinal properties, as reducing and stabilizing agents to green synthesize magnesium oxide nanoparticles (MgONPs) using magnesium nitrate. In comparison to the bulk material, capabilities of Ca-MgONPs as an improved antibacterial, antifungal, and anticancer agent in human prostatic carcinoma cells (PC3), as well as membranolytic capability in model cell membrane, were studied. The phyto-functionalized Ca-MgONPs were characterized using UV-Visible spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Energy Dispersive X-Ray Spectroscopy (EDX), X-ray Diffraction (XRD), Fourier Transform Infra-Red Spectroscopy (FT-IR) and Atomic Force Microscopy (AFM). Observation of characteristic peaks by spectroscopic and microscopic analysis confirmed the synthesis of Ca-MgONPs. The Ca-MgONPs showed broad spectrum of bactericidal activity against both gram-positive and gram-negative bacteria and fungicidal activity against two species of the Candida fungus. The Ca-MgONPs also exhibited dose-dependent and selective inhibition of proliferating PC3 cells with IC50 of 123.65 ± 4.82 µg/mL at 24 h, however, without having any cytotoxicity toward non-cancerous HEK293 cells. Further studies aimed at understanding the probable mechanism of toxicity of Ca-MgONPs in PC3 cells, the results indicated a significant reduction in cell migration capacities, increment in cytosolic ROS, loss of mitochondrial transmembrane potential, DNA damage and S-phase cell cycle arrest. Ca-MgONPs also induced pore formation in a synthetic large unilamellar vesicle. Thus, Ca-MgONPs might be useful in the effective management of several human pathogens of concern and some more cancer types.

Rational design, chemical synthesis and cellular evaluation of novel 1,3-diynyl derivatives of noscapine as potent tubulin binding anticancer agents.

We present a new class of derivatives of noscapine, 1,3-diynyl-noscapinoids of an antitussive plant alkaloid, noscapine based on our in silico efforts that binds tubulin and displays anticancer activity against a panel of breast cancer cells. Structure-activity analyses pointed the C-9 position of the isoquinoline ring which was modified by coupling of 1,3-diynyl structural motifs to rationally design and screened a series of novel 1,3-diynyl-noscapinoids (20-22) with robust binding affinity with tubulin. The selected 1,3-diynyl-noscapinoids, 20-22 revealed improved predicted binding energy of -6.568 kcal/mol for 20, -7.367 kcal/mol for 21 and -7.922 kcal/mol for 22, respectively in comparison to the lead molecule (-5.246 kcal/mol). These novel derivatives were chemically synthesized and validated their anticancer activity based on cellular studies using two human breast adenocarcinoma, MCF-7 and MDAMB-231, as well as with a panel of primary breast cancer cells isolated from patients. Interestingly, all these derivatives inhibited cellular proliferation in all the cancer cells that ranged between 6.2 to 38.9 µM, which is 6.7 to 1.5 fold lower than that of noscapine. Unlike previously reported derivatives of noscapine that arrests cells in the S-phase, these novel derivatives effectively inhibit proliferation of cancer cells, arrests cell cycle in the G2/M-phase followed by apoptosis and appearance of apoptotic cells. Thus, we conclude that 1,3-diynyl-noscapinoids have great potential to be a novel therapeutic agent for breast cancers.

In Vivo Assessment of Immunogenicity and Toxicity of the Bacteriocin TSU4 in BALB/c Mice.

Bacteriocin TSU4 is a novel antimicrobial peptide isolated from Catla catla gut isolate Lactobacillus animalis TSU4. It has been reported for its potential antimicrobial activity against fish pathogens and food spoilage bacteria. In vivo safety evaluation is necessary to determine its immunogenicity, toxicity, and importance in real-life applications. The present study was designed to evaluate the immunogenicity, acute and sub-chronic toxicity of bacteriocin TSU4 in BALB/c mice to ensure its safety in industrial application. Male BALB/c mice were administered intraperitoneally for immunogenicity assessment, by oral gavage with 50, 100, and 200 mg/kg/body weight for acute test and 0.5 mg/kg/day dose of bacteriocin TSU4 for sub-chronic toxicity test. Neither mortality nor any infections were observed during experimental period. There was no major increase in antibody titer during the immunogenicity test, and no mortality was observed during acute or sub-chronic toxicity tests. The LD50 value of bacteriocin TSU4 was found to be higher than 200 ± 0.45 mg/kg. No significant change in the serum biochemical markers, histopathological analysis and visual observation in spleen sizes was observed. These findings revealed that bacteriocin TSU4 is a non-immunogenic, safe, non-toxic, and could be a potential candidate for industrial applications in food preservation and aquaculture industries.

Purification and Molecular Characterization of the Novel Highly Potent Bacteriocin TSU4 Produced by Lactobacillus animalis TSU4.

Bacterial infections causing fish diseases and spoilage during fish food processing and storage are major concerns in aquaculture. Use of bacteriocins has recently been considered as an effective strategy for prevention of bacterial infections. A novel bacteriocin produced by Catla catla gut isolates, Lactobacillus animalis TSU4, designated as bacteriocin TSU4 was purified to homogeneity by a three-step protocol. The molecular mass of bacteriocin TSU4 was 4117 Da determined by Q-TOF LC/MS analysis. Its isoelectric point was ~9. Secondary conformation obtained by circular dichroism spectroscopy showed molecular conformation with significant proportions of the structure in α-helix (23.7 %) and ß-sheets (17.1 %). N-terminal sequencing was carried out by the Edman degradation method; partial sequence identified was NH2-SMSGFSKPHD. Bacteriocin TSU4 exhibited a wide range of antimicrobial activity, pH and thermal stability. It showed a bacteriocidal mode of action against the indicator strain Aeromonas hydrophila MTCC 646. Bacteriocin TSU4 is the first reported bacteriocin produced by fish isolate Lactobacillus animalis. The characterization of bacteriocin TSU4 suggested that it is a novel bacteriocin with potential value against infections of bacteria such as A. hydrophila MTCC 646 and Pseudomonas aeruginosa MTCC 1688 and application to prevent spoilage during food preservation.

In Vitro Evaluation of Probiotic Properties of Lactic Acid Bacteria from the Gut of Labeo rohita and Catla catla.

We report the evaluation of probiotic properties of potent lactic acid bacteria (LAB) from the gut of freshwater fishes, Labeo rohita and Catla catla, for eventually developing probiotic strains for the prevention of bacterial infections in aquaculture and food preservation. Five different LAB strains were isolated and characterized for their probiotic properties. Based on physiological, morphological and biochemical characteristics, three isolates from Labeo rohita and two from Catla catla were identified as putative probiotics and were denoted as LR11, LR14 and LR16 and CC3 and CC4, respectively. Isolates CC3 and CC4 were acid (pH 2.5) and bile salt (0.3% oxygall) tolerant and exhibited strong antibacterial activities against all pathogens including Aeromonas hydrophila. In addition, all LAB isolates were susceptible to tested antibiotics, except CC3 and CC4 which were vancomycin resistant. Furthermore, the isolates CC3 and CC4 showed significantly higher in vitro cell surface properties, i.e., hydrophobicity, auto- and co-aggregation. Biochemical tests, PCR detection and 16S rRNA sequence analysis established that LR11, LR14, LR16, CC3 and CC4 are Enterococcus avium TSU11, Enterococcus pseudoavium TSU14, Enterococcus raffinosus TSU16, Lactobacillus gasseri TSU3 and Lactobacillus animalis TSU4, respectively. Studies revealed that, Lactobacillus gasseri TSU3 and Lactobacillus animalis TSU4 are ideal probiotic candidates for its use in aquaculture and require further exploratory in vivo evaluation and safety studies.