1. Monique Honeyghan HCS Class of 2008 Professor Mark Borden Columbia University Chemical Engineering

2. Overview  Phospholipid Structures  Motivation  Procedure  Data  Conclusion  References  Acknowledgements

3. Phospholipid Structures Monomer SUV Bilayer MLV

4. Motivation  Prepare a protocol for a lab assignment designated for Columbia undergrad seniors majoring in Chemical Engineering  Create a solution of phospholipids and lysophospholipids  Run the lipid solution through the HPLC to identify and quantify both types of lipids for analysis  Future component of research involving phospholipids  Provide further understanding of lipids in relation to enzymatic digestion

5. Procedure  Preparation of lipids solution DSPC 1,2-Distearoyl-sn-Glycero-3-phosphocholine C 44 H 88 NO 8 P ○ stored in chloroform ○ Evaporation using nitrogen gas ○ Vacuum overnight ○ 10mg/ml Concentration vs. 15 mg/ml Concentration PBS – Pure Buffer Solution

6. Procedure  Sonication : sonic energy breaks up large vesicles into SUVs. Bath vs. Probe Clear, yet translucent (hazy blue) Heating Block above 60 o C

7. Procedure  Extrusion – mechanical energy in the form of force to push larger vesicles though small pores in order to help form uniformed vesicles. 11 passes 0.2 micron polycarbonate membrane Heat block above 60 o C * Bath sonicate stock sample to be extruded * Heat extruded samples Syringe filter (0.2 micron) vs. (0.45 micron) vs. no filtering Variable: hot bath vs. quenching with ice bath

8. Procedure  Testing on DLS machine Dynamic Light Scanner Size of liposomes Size distribution by volume

9. Procedure  Enzyme Reaction Phospholipase A 2 from bovine pancreas ○ 100 µL to 1mL lipid solution ○ 45 minutes on rotator*  Reactant: liposome (DSPC)  Products: lysophospholipid + fatty acid MLV Phospholipase A 2 SUV

10. Procedure  Extraction (Method by Folch et al ) Solvents - Chloroform: methanol in a 2:1 ratio ○ 0.6 mL per 1 ml reacted lipid solution Agitation to homogenize (3 minutes) Centrifugation (2000 rpm x 3 minutes) ○ Separates phases distinctly Upper: methanol, water, enzyme Lower: chloroform, PC and Lyso PC Remove upper phase Add 0.4 ml Filtered H 2 O Agitation to homogenize (1 minute) Centrifugation (2000 rpm x 3 minutes) Filtered using a 0.2 μm pore syringe filter Test upper phase on DLS* Test lipid phase (lower) on HPLC for efficiency of the Phospholipase A 2 enzyme

11. Procedure  HPLC - High Performance Liquid Chromatography – identify and quantify the phospholipids and lysophospholipids via an isocratic elution that is run through a silica column Buffer Components: Hexane: 2-Propanol: 25 mM Potassium Acetate: Acetonitrile: Glacial Acetic Acid 442: 490: 62: 25: 0.6 by volume 1. Run on buffer to establish a baseline 2. Run on PC and LysoPC to get readings (Control) 3. Inject samples of prepared lipid solution to see if the results match the standards

12. Procedure (HPLC Diagram)

13. Procedure (HPLC) Detector Injection site PUMPS Column Chamber r Buffer

14. Procedure (HPLC) Injection site Column Pump A Pump B

15. Data  Graph showing size distribution by volume of 10 mg/mL DSPC

16. Data  Graph showing size distribution by volume of 10 mg/mL DSPC

17. Data  Graph showing size distribution by volume of 15 mg/mL DSPC

18. Data  Graph showing size distribution by volume in 10 mg/mL enzyme extract (upper phase)

19. Data  Graph showing size distribution by volume of 10 mg/mL enzyme extract

20. Data  Graph showing size distribution by volume of 10 mg/mL enzyme extract

21. Results  Probe sonication is faster, but there is a risk to contaminating the sample with particles from the metal probe.  Hot bath is more efficient than quenching with cold water, however, if samples need to be cooled, it is better to do it rapidly.  Virtually no difference between syringe filtering and not doing so  Making 10 mg/ml samples is easier and more efficient than making 15 mg/ml samples.  HPLC has no final results. Progress with the reacted liposome samples shown by 2 peaks, which might correspond to the PC and Lyso-PC standards

22. Conclusion  Able to produce unilamellar vesicles averaging around 100 nm in diameter  Developed protocol so the undergrads can reproduce the experiment  Safety concerns established Working under a fume hood with chloroform and the HPLC buffer components Wear nitrile gloves when working with chloroform Wear heat resistant gloves when using Avanti Mini extruder on heat block

23. References  “Interfacial Enzyme Kinetcs at the Phospholipid/Water Interface: Practical Considerations “by Raymond A. Deems. www.ideallibrary.com  “Preparation of liposomes” and extrusion technique by Avanti Polar Lipids Inc. www. avantilipids.com  Phospholipase A 2 at the Bilayer Interface” by Fausto ramirez and Mahendra Kumar Jain  “Separation and Quantitation of Phospholipids and Lysophospholipids by High Performance Lipid Chromatography” by Edward Lesnefsky, Maria Stroll, Paul Minker and Charles Hoppel  “HPLC – High Performance Liquid Chromatography” and HPLC diagram by Resource Library of www.waters.com  “Folch ( et al) method of lipid extraction “ www.cyberlipid.org  Cuvette image www.krackeler.com/products/1092- Cuvettes/1071...  Image of DLS machine (Malvern Zetasizer) www.azon.com  Image of bath sonicator by all-spec.com  Multilamellar vessicle image by encapsula.com

24. Acknowledgements  Professor Borden  Cherry Chen  Melissa Moy  Borden Lab group  Dr. Sat Bhattacharya  Harlem Children Society  Dr. Zarou  Bronx Health Sciences High School  Family and Friends