1. Preparation and Multilayered Aggregation of Uniform Colloidal Cholesterol Particles Vuk Uskoković, Egon Matijević Center for Advanced Materials Processing Clarkson University, Potsdam, NY, USA

4. Results of a typical precipitation of cholesterol

5. Procedure for the Preparation of Uniform Cholesterol Particles: After dissolving cholesterol in 1-propanol, water was added to supersaturate the solution. After 10 min, a few drops of the suspension were deposited onto an SEM sample carrier, dried in air and analyzed. Throughout the entire procedure, the suspension was kept undisturbed, i.e. without any agitation.

9. Laser Diffraction Analysis:

10. X-Ray Diffraction pattern d = 33.4 Å (bilayer reflection) d = 16.7 Å (monolayer reflection) 1.7 nm

11. Zeta Potential measurement:

12. DSC analysis: melting re-crystallization

13. Studied effects: Solvent effect Concentration effect pH effect Time effect Temperature effect Ionic strength effect

14. Solvent effect

15. 2-propanol instead of 1-propanol

16. Methanol instead of 1-propanol

17. Ethanol instead of 1-propanol

18. Acetone instead of 1-propanol

19. Tech. grade isopropanol (90% purity) instead of 1-propanol

20. Concentration Effects

21. 1.25 times higher solvent/non-solvent ratio

22. 2.7 times lower solvent/non-solvent ratio

23. 4 times lower concentration of cholesterol

24. 4 times higher concentration of cholesterol

25. pH effect

26. pH = 7.7 (in comparison with pH = 6.3 in the “standard” procedure)

27. pH = 1.8 (isoelectric point )

28. pH = 1.3 (positively charged particles)

29. Aging effects

30. Particles sampled out from the “standard” procedure after 10 seconds of aging

31. Particles sampled out after 3 min of aging time

32. Particles sampled out after 5 min of aging time

33. Particles aged for one month at atmospheric conditions

36. Particles aged for four months at atmospheric conditions

38. Temperature effects

39. Particles aged for 2 days at 34 o C

40. Particles aged for 2 days at room T

41. Particles aged for 15 min at 37 o C

42. Particles precipitated at 31 o C and aged for 5h at 37 o C

43. Effect of the addition of salt

44. Particles obtained with the addition of 0.5 M of NaCl

45. Particles obtained with the addition of 0.5 M of NaCl, aged for 2.5 h

47. Particles obtained with the addition of 0.05 M of BaCl2, aged for 2.5 h

48. Particles obtained with the addition of NaCl in c = 0.5 M after 10 min aging, aged for 2.5 h

49. Particles coated with silica

51. žžžžžž

52. Precipitation by solvent/non-solvent evaporation Particles prepared by evaporation of the unsaturated “standard” cholesterol solution

55. Conclusions/Achievements 1. The first reported method for preparation of uniform cholesterol particles and their stable dispersions, that can be used as models for the further aggregation and deposition studies

56. “Amazingly small means leading to extraordinarily satisfying results” Ernst Fritz Schumacher, Small is Beautiful, 1973.

57. 2. Surface charge effects evidenced as crucial in stabilization of cholesterol dispersions 3. Increases in ionic strength (NaCl concentration) proven as leading to an increased rate of aggregation of cholesterol platelets. The direct link between artherosclerosis and high blood pressure as two major health threats in the developed world evidenced for the first time at the physicochemical level.

58. Preparation and Multilayered Aggregation of Uniform Colloidal Cholesterol Particles Vuk Uskoković, Egon Matijević Center for Advanced Materials Processing Clarkson University, Potsdam, NY, USA

59. Phase diagram of cholesterol in Bile (to be healthy, the composition of this 3-component system has to correspond to the micellar zone) Nick Myant (Cholesterol Metabolism, Ldl, and the Ldl Receptor)