Design of Luminescent Magnetic Nanostructures for Sensor, Drug Delivery and Bioimaging Applications

The present dissertation entitled, “Design of Luminescent Magnetic Nanostructures for Sensor, Drug delivery and Bioimaging Applications” is an embodiment of the investigations intended at developing simple inexpensive synthetic methods for producing luminescent carbon quantum dot and multifunctional magnetic luminescent nanostructures applicable for sensor and biomedical application. The in vitro applications of synthesized materials have also been investigated. The thesis is divided into two parts. The first part of the thesis includes the low cost synthetic route for fabrication of carbon quantum dots (CD) and their composites for sensing and bioimaging applications. Highly photoluminescent CDs with a quantum yield of 26% have been synthesized in one step by hydrothermal treatment of orange juice (Citrus nobilis deliciosa). Due to high photostability and low toxicity these CDs have been demonstrated as excellent probes in cellular imaging. These synthesized CD has been also used for the development of reusable novel magnetic silica/CD based hybrid nanostructure for monitoring and separation of fluoride ion through fluorescence sensing and external magnetic field respectively. The assay is based on the binding of fluoride ion into magnetic receptor substituting already bound CD. This method is highly sensitive, fast and selective for fluoride ion in aqueous solution having a linear response range of 1 to 20 μM (R2=0.992). The practical utility of the method is well tested with tap water and also extended for fluoride detection in cellular environment. Furthermore an easily separable sensitive CD based fluorescence glucose sensor comprising of CD deposited mesoporous silica nocomposite (m-SiO2-CD) and 3-aminophenylboronic acid (APBA) has been prepared. The observed fluorescence recovery of quenched APBA adsorbed m-SiO2-CD on addition of glucose is due to the formation of glucoboronate ester which could lift out the APBA from close vicinity of fluorophore. The boronic acid modified (m-SiO2-CD-APBA) fluorescent probe is also explored for targeted imaging of colon cancer cell overexpressed with sialyl Lewis A (sLea) receptors. To improve the luminescence properties of the CD, nitrogen, sulphur co-doped carbon quantum dot (NSCD) with an improved fluorescent quantum yield of 69% have been synthesized from single molecular precursor. The synthesized NSCD exhibits high selectivity and sensitivity towards mercury ion in aqueous environment. Due to high photostability, low toxicity and low detection limit as 0.05 nM, these NSCDs are demonstrated as excellent probes for the detection of Hg2+ in living cells.The second part of this thesis demonstrates the design and fabrication of multifunctional fluorescent magnetic nanostructures which are of special interest in cancer diagnostic and therapy. Multifunctional luminescent magnetic Fe3O4@mesoporous silica-YPO4:Tb core-shell nanoparticle has been prepared for the storage as well as controlled targeted release of 5-fluorouracil (5-FU). The hydrophobic anticancer drug 5-FU has been successfully loaded on the fluorescent magnetic nanoparticles via formation of 5-FU/β-cyclodextrin inclusion complex which favors more sustained release at lower pH owing to stability of inclusion complex. These findings show that the developed multifunctional nanocomposite can be potentially used in magnetically guided delivery of 5-FU. Furthermore, hierarchical theranostic hollow magnetic mesoporous spherical particles with fluorescent carbon encapsulated within mesoporous framework have been prepared by hydrothermal carbonization approach. These fluorescent magnetic nanoparticles have been conjugated with hydrophobic drug camptothecin and a molecular marker folic acid using appropriate surface chemistry to ensure the targeted specific delivery of the camptothecin. The drug conjugated hybrid nanoparticles inhibit cell growth through induction of apoptosis as demonstrated in HeLa cells. In addition to this, the particles show MR contrast behaviour by affecting the proton relaxation with transverse relaxivity (r2) 150.03 mM-1S-1

EVALUATION OF ANTIBACTERIAL AND HAEMOLYTIC ACTIVITY OF PHYTOCHEMICALS FROM FRESHWATER MICROALGA, EUGLENA VIRIDIS (EHREN)

Objective: The main aim of this research work was to evaluate the antibacterial and haemolytic activities of different extracts of Euglena viridis (E. viridis), a freshwater microalga.Methods: The solvent extraction has been followed by a preliminary screening of phytochemicals. The ethanolic extract, Eu(EtOH) was chromatographed on a silica gel column. The column was eluted with hexane and then with ethyl acetate/hexane mixtures of increasing polarity, 16 fractions (Ef1-Ef16) were collected and grouped according to their TLC (Thin layer chromatography). Antibacterial activities of different fractions of E. viridis against Pseudomonas putida (P. putida) ATCC49828, P. aeruginosa MTCC 35672, Aeromonas hydrophila (A. hydrophila) MTCC 646, ATCC 49140, eleven strains of Staphylococcus aureus (S. aureus) and thirteen strains of Flavobacterium columnare (F. columnare) was done using disc diffusion methods. Haemolytic activity was carried out by using blood agar plate method. The MIC (Minimum inhibitory concentration) values of active fractions were determined by the broth dilution method.Results: The results showed that the Eu(EtOH) poses significantly (p≤0.5) higher zone of inhibition (14.0±0.28, 13.5±0.28 mm) against FLV8 and FLV9 respectively. Three strains of Flavobacterium (FLV5, FLV6 and FLV10) were highly sensitive (zone size, 17 mm, 17.5 mm) towards 30% EA: Hex chromatographic eluents (Ef11) with lowest MIC values, e. i 60 µg and 30 µg respectively. Two chromatographic fractions, Ef11 and Ef13 were highly effective (zone size, 14.5 mm and 13.5 mm) against S. aureus (SA5) with lowest MIC value (60µg). Haemolytic activities of all the algal extracts were noticed that both Eu(EtOH) and methanolic extract, Eu(MeOH) of Euglena gives negative results.Conclusion: These findings suggest that the extract obtained from E. viridis have active substances contributing to the increasing antibacterial potential

Evaluation of the New Fluorescent Internal Tag (Soft Visible Implant Alphanumeric Tag) in the Freshwater Prawn, Macrobrachium rosenbergii

The new version of the polyester visible implant alphanumeric tag (VI Alpha tag; Northwest Marine Technology Inc., Shaw Island, Washington, USA) was evaluated in the giant river prawn (Macrobrachium rosenbergii) under laboratory conditions. The new VI Alpha tag is soft and fluorescent with an alphanumeric code designed to identify individual specimens. Two tag sizes - standard (1.0 x 2.5 mm) and large (1.5 x 3.5 mm) - were tested on juve- niles (standard size), subadults (standard size), and adults (large size) for 10 weeks. Retention, readability, and effects on growth and survival were evaluated. Final mean tag retention was 71.25±3.3% in juvenile prawns, 91.3%±2.73 in subadult prawns, and 59.0%±1.65 in adult prawns. Final tag readability was 100.0% in juveniles, 88.6±5.3 in subadults, and 77.8±4.8 in adults. Average daily growth (mg/day) and SGR (%/day) of tagged juvenile prawns (22.4±0.23 and 0.81±0.02, respectively) did not significantly differ (p>0.05) from that of the untagged control (20.5±1.47 and 0.83±0.04). Similar results were observed in subadult and adult M. rosenbergii. Mean final survival did not significantly differ (p>0.05) between tagged and untagged prawns. Results indicate that tagging M. rosenbergii with VI Alpha tags has no adverse effect on survival or growth and can therefore be effec- tively used to identify individuals in selective breeding programs

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Protective Effects of <i>Chlorella vulgaris</i> Supplemented Diet on Antibacterial Activity and Immune Responses in Rohu Fingerlings, <i>Labeo rohita</i> (Hamilton), Subjected to <i>Aeromonas hydrophila</i> Infection

The current study focuses on the antibacterial activity and potential efficiency of dietary supplements of Chlorella vulgaris on the immune response, improved growth performance, and disease resistance of Labeo rohita fingerlings against Aeromonas hydrophila infection. Crude ethanolic extract of Chlorella and partially purified fractions of the extract were tested against two selected fish pathogens using the disc diffusion method. A total number of 360 rohu fingerlings (25 ± 2 g) were allocated to 4 treatments for 90 days. They were fed with an experimental diet containing Chlorella powder (0, 0.1, 0.5, and 1.0 g Kg−1 of a basic diet). To evaluate the non-specific immunity parameters including serum bactericidal, lysozyme activity, superoxide anion production, and biochemical and haematological indices, the fish were sampled at day 30, 60, 90, and after bacterial challenge. Mortalities of the fish were observed over 10 days post challenge with A. hydrophila. The protein and globulin levels of the treatment group were significantly higher after being treated with Chlorella than those of the control group. The total blood-cell count and haemoglobin content were also increased in the algal-diet-treated group. Among all the experimental diets, the 0.5 g Kg−1Chlorella fed group of fish showed significantly (p −1 diet fed group on day 30. The Chlorella treated group exhibited a better growth performance of the fish. The maximum survivability (86.5%) was recorded in the 1.0 g Kg−1 diet fed group at the end of the 10-day fish exposure to A. hydrophila. These results suggest that the optimum dietary Chlorella supplementation could be 0.5–1.0 g Kg−1 of the diet, which stimulates immunity and protects L. rohita from A. hydrophila infection

Antibacterial activity of Curcuma longa on fish microbial pathogens

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