1. AOCS 2014 MONIKA R. KULAK PROF. MILENA CORREDIG (SUPERVISOR) UNIVERSITY OF GUELPH, ON, CANADA Liposomal Nanoencapsulation of Bioactive Compounds and their Bioefficacy on Cancer Cells 2014-07-05 1

2. Outline 2014-07-05 2  Introduction  Purpose  Methods  Results  Stability Analysis  Cell Culture Analysis  Summary

3. Nanoencapsulation 2014-07-05 3 Protection of antioxidants from harsh environments Increased surface area enhance solubility Delay or inhibit the formation of undesirable odors/flavors Fathi, M.; Mozafari, M. R.; Mohebbi, M. Trends in food science & technology 2012.

4. Liposomes 2014-07-05 4 Colloidal structures formed via high energy input Closed, continuous vesicle structures  phospholipid bilayer in an aqueous environment Entrapment, delivery, and release of water/lipid soluble Mozafari, M. R.; Johnson, C.; Hatziantoniou, S.; Demetzos, C. J Liposome Res 2008, 18, 309–327.

5. Project 5 1) Synthesis and Characterization of Nanoliposomes of 3 varied bioactives over a period of time 2) Study the efficiency of curcumin bearing nanoliposomes in Caco-2 cells Encapsulate diverse bioactives within nanoliposomes for the purpose of enhancing uptake within the body Objectives Goal

6. 2014-07-05 6 Liposomal Synthesis & Analysis 1. Solvation 2. Encapsulation 3. Characterization (DLS, HPLC) Pic of DLS here

7. 2014-07-05 7 Table 1. Zeta potential Data (mV) Bioactive Beta- Carotene EGCGCurcumin Beta- carotene EGCGCurcumin Time PeriodFreshAged Molarity 0.0012-25.2 -26.48-24.6-24.4-25.32 0.0022-29.58-23.03-25.73-27.63-22.55-25.17 0.0032-28.07-23.7-24.7-26.4-23.3-24.1

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11. TEM 2014-07-05 11 0.2 um Blank Soy Soy B-Carotene Aged (10 days) Soy B-Carotene Fresh ( 24hrs<)

12. TEM 2014-07-05 12 100 nm MFGM SL Curcumin Soy SL Curcumin Soy DL Curcumin

13. Cell Culture Experimentation 2014-07-05 13 1. Caco-2 Cell Culture growth and preparation  (3 weeks, HBSS) 2. Sulforhodamine B (SRB) assay 3. Curcumin uptake on Caco-2confluent monolayers 4. All basolateral samples were collected after 4h incubation 5. Encapsulation Efficiency Measure with LC-MS

14. Trans-Epithelial Electrical Resistance Trial 2014-07-05 14

15. Cell Proliferation Over Time 2014-07-05 15

16. Summary of Research 2014-07-05 (1) High encapsulation efficiency within soy lecithin liposomes (2) Nanoscale particle distributions (3) Soy lecithin dispersions are more stable than MFGM (4) Physical characteristics of aged samples were comparable to their freshly made counterparts (5) In vitro study showed enhanced delivery of curcumin through cell monolayers. 16

17. Future Research 2014-07-05 pH variations Digestion Simulations Longer term stability and storage (+ 4 months) Food grade processing 17

18. 2014-07-05 Dr. Milena Corredig and her Lab Group Drs. Ibrahim Gulseren and Anilda Guri for their guidance and help University of Guelph TEM Imaging & CRIFS Cell Culture facilities Drs. Nissim Garti & Edgar J. Acosta for chairing the presentation 18 Acknowledgements

19. 2014-07-05 19 Questions? Thank you for your attention!

20. Transmission Electron Microscopy (TEM) 20 Electrons are transmitted through the sample Image formation Loss and scattering of electrons by individual parts of the specimen. Emergent electron beam is focused by objective lens. Fluorescent screen Objective lens Condenser lens Specimen (thin) Aperture Electron source Electron beam

21. 21 Scanning Electron Microscopy (SEM) Electrons in Electrons out Sample Objective lens Condenser Lens Detectors Electron Gun Electron beam Scan Coils Used to study surfaces of objects up to 10 5 magnification Electron beam scans surface

22. High Pressure Liquid Chromatography (HPLC) Pump Injector Column Detector Mobile Phases Gradient Controller

23. Liquid Chromatography-Gas Spectroscopy 2014-07-05 23

24. DLS 2014-07-05 24