1. Morphological Changes of Liposomes Induced by Melittin Jirun Sun Full Talk for Macromolecules Seminar J, Dufourcq et al., BBA 859, 1986, 33

2. Contents Background –Introduction of Liposomes –Introduction of Melittin Morphological changes of Liposomes with Melittin –Position of Melittin –Pore Formation in Liposomes –Morphological Changes of Liposomes

3. What Are Liposomes Artificial membrane vesicles. Drug delivery, chemical manufacturing, genetic engineering and so on http://www.ncnr.nist.gov/programs/reflect/rp/biology/cell_membrane.html

4. Each phospholipid includes  a polar region: glycerol, carbonyl of fatty acids, P i, & the polar head group (X)  2 non-polar hydrocarbon tails of fatty acids (R 1, R 2 ). Such an amphipathic lipid may be represented as at right. 3D picture of a Phospholipid

5. According to the head group X, there are several kinds of lipid. X=X= Choline (PC) Ethanolamine (PE) Glycerol (PG) Serine (PS) Inositol (PI) O-H Acid(PA)

6. The membrane lipid composition in an average mammalian cell Lipid% PC45-55 PE15-25 PI10-15 PS5-10 PA1-2 cholesterol10-20 Phosphatidylcholine (PC), or lecithin, with choline as polar head group. It is a common membrane lipid.

7. Some fatty acids and their common names: 14:0 myristic acid (DM); 16:0 palmitic acid (DP); 18:0 stearic acid (DS); 18:1 cis  9 oleic acid (DO) Double bonds in fatty acids usually have the cis configuration. Most naturally occurring fatty acids have an even number of carbon atoms. DOPC: two oleic acid tails and a Choline head group POPA: one P tail and one O tail, Acid head group

8. Liposomes Formed by Lipids http://www.nupedia.com/newsystem/upload_file/830/bilayer_micelle.png Bilayer Micelle Driven by hydrophilic and hydrophobic forces, the nonpolar tails of lipids (U) tend to cluster together, forming a lipid bilayer (1), a micelle (2). The polar heads (P) face the aqueous environment, Liposomes contain bilayers.

9. Size Determined by Methods Sonication: SUV Smaller than 100 nm diameter Extrusion: LUV (Size depends on the filters) 100 nm—1 µm diameter Evaporation: GUV Larger than 1 µm diameter MLV: Multilamellar vesicles SUV: Small unilamellar vesicles LUV: Large unilamellar vesicles GUV:Giant unilamellar vesicles http://www.avantilipids.com/PreparationOfLiposomes3Big.html

10. Preparation of Liposomes

11. Methods to Check the Morphology of liposomes Freeze-fracture electron microscope (FFEM) or electron microscope (EM) Light scattering (LS)

12. Freeze-fracture Electron Microscopy

13. Freeze-fracture electron Micrographs (a, b, c) and negative staining (d) of aqueous dispersions of DOPC/DOPA (80:20 mol%) after: 0(a); 10 (b and d) and 50 (c) cycles of freeze- thawing Eur Biophys J 2000 29; 184

14. Static Light Scattering Top view of the geometry around the sample cell q θ Photodetector qsqs Incident beam Test tube qiqi qiqi Index- matching liquid Unscattered beam Liposomes: DOPC 14 angles are checked R g = 519±3 Å

15. Dynamic Light Scattering (DLS) Stokes’ law Liposomes: DOPC Six angles, 30,40,50,60,70 and 90 degree were checked. R h = 500±7 Å G (2) (t) = = Intensity Measured by Autocorrelation function G (2) (t) = B(1 + f  g (1) (t)  2 ) The signal in the correlator is well approximated by g (1) (t) is a simple exponential g (1) (t) = e -  t

16. What do SLS and DLS tell us? RgRg RhRh If Rg/Rh ≈ 1 the thickness of a ball is about zero and then it more like a thin bubble. For liposomes, they are Unilamellar According to the DLS and SLS data in the previous two slides R g = 519±3 Å R h = 500±7 Å R g /R h = 1.04 That DOPC is Unilamellar

17. Phase Transition Temperature (T m ) Important when preparing the liposomes Important in the interactions of Melittin with liposomes http://www.virtuallaboratory.net/Biofundamentals/lectureNotes/Topic2-3_Membranes.htm

18. Melittin It is the main component (50-60%) in bee venom with 26 residues It has a very strong anti-inflammatory and anti-bacterial effect It may cause allergic reaction, which can act as a skin, eye or respiratory irritant. It is a cytolytic protein. It is an amphiphile protein. http://molvis.chem.indiana.edu/C581_F97/protein_projects/Melittin.html Tervilliger et.al., Nature, 1982, (299),371

19. The fact that melittin can act as a lytic agent is partly due to its detergent- like sequence. Leippe et al. PNAS, 88 (1991) 7659 ∂ -helical wheel of the first 20 residues. Hydrophilic Hydrophobic

20. Morphological Changes of Liposomes with Melittin A weapon of bees The most popular model for studying cytolytic activities Membrane fusion Drug releasing system Well understanding of the interaction will be helpful on Building new instruments And studying new amphiphile proteins

21. Points Melittin position in liposomes The existence of poles Morphology changes of liposomes –MLV or unilamellar –Ratio of lipid to melittin –Temperature –Time

22. Melittin in Membranes Simulation of melittin tetrameric aggregate The N-terminus and the C-terminus are denoted By N and C, respectively. The four helices are Labeled 1-4. Lin, et. al., Biophysical Journal, 78, 2000, 1714 Tervilliger et.al., Nature, 299, 1982, 371 Tetrameric aggregate End to end distance of melittin from crystal structure is about 3.6 nm

23. Models of Pores Barrel-stave model Toroidal model Biophysical Journal, 81, 2001, 1475 Toroidal model fits melittin better The difference is whether the water core is lined by both the peptides and the lipid head groups

24. Snapshot of the pore from top (A) and from side (B). Figures of Molecular Dynamics Simulation by Monte Carlo program Lin, et. al., Biophysical Journal, 78, 2000, 1714

25. Existence of Poles Prepare liposomes in the presence of fluorescent marker, like calcein Remove external calcein Measure background, set calcein releasing as zero Add melittin Measure the released calcein by spectrofluorimeter.

26. Leakage Experiments G. Anderluh et. al., JBC, 278, 2003, 45216 Permeabilzing capacity of melittin on MLV. The release of calcein from POPC/SMPC = 2:1 (mol/mol). Melittin Concentration 1 µm.

27. Morphology Changes of Liposomes Temperature effects (Gel Phase or liquid crystal phase) Unilamellar or multi-lamellar Ratio of lipid to melittin matters It is a dynamic process. Minor factors like buffer, pH values and so on That are combining results of all factors.

28. C. G. Morgan et. al., BBA 732 (1983)668 Fusion happens! Note for the cartoon: DPPC LUV L:M = 200 L:D = 200 No Fusion Melittin is Detergent-Like But They Are Not the Same Incubation: heat to T>Tm and hold there for a while and then cool down and measure at T<Tm.

29. Morphology Changes at Different Ratios Temp: 21 o C R h by checked by Quasi-elastic LS EPC: Egg PC EPC is in LC state at 21 o C Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33

30. A: Pure EPC SUV B: L:M = 200 C: L:M = 30 D: L:M = 5 Magnification: 50,000x Different L/M Ratios of EPC SUV Seen by FFEM From J, Dufourcq et al., BBA 859, 1986, 33

31. Different L/M Ratios of EPC MLV Seen by FFEM A: Pure EPC MLV B: L:M = 30 C: L:M = 15 Magnification: 50,000x From J, Dufourcq et al., BBA 859, 1986, 33

32. The Different Between MLV and SUV MLV + Melittin LUV+ MelittinTiny particles Fragmentation SUV + Melittin Fusion Vesicularisation J, Dufourcq et al., BBA 859, 1986, 33

33. Gel Phase or LC Phase LC phase: Morphology changes, Melittin binds to vesicles Gel phase: Only SUVs are morphologically affected Incubation: Morphology changes It is not reversible!

34. Morphological Changes at Different Temperatures Data digitalized according to: J, Dufourcq et al., BBA 859, 1986, 33 Liposome used DPPC MLV, Ratio of liposomes to melittin L:M=20 T m for DPPC is 41 o C Large particles scatter more!

35. Morphological Changes Detected by Freeze-fracture EM DPPC MLV DPPC: melittin = 30 A: temp 20 o C B: temp 50 o C C: temp 20 o C after incubation at 50 o C From J, Dufourcq et al., BBA 859, 1986, 33

36. It Is A Dynamic Process One of two “Delta Dawns” at LSU Size changing of liposome (DOPC LUV) by adding melittin checked by Rapid SLS using a commercially available, 18-detector instrument A touch is needed! Procedure: Measure background Normalize with Tol or Emulsion Measure sample Data processing Data from Dr Russo’s Lab agree with the results in literature: J, Dufourcq et al., BBA 859, 1986, 33

37. Conclusion –Liquid crystal state has more obvious morphology changes than gel state does. –The ratio of L/M matters –MLV, SUV behave differently with melittin –It is a dynamic process

38. Thanks