Preparation and Multilayered Aggregation of Uniform Colloidal

Cholesterol Particles

Vuk Uskoković, Egon Matijević

Center for Advanced Materials Processing

Clarkson University, Potsdam, NY, USA

Results of a typical precipitation of cholesterol

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.

Laser Diffraction Analysis:

X-Ray Diffraction pattern

d = 33.4 Å (bilayer reflection)

d = 16.7 Å (monolayer reflection)

1.7 nm

Zeta Potential measurement:

DSC analysis:

melting

re-crystallization

Studied effects:

• Solvent effect

• Concentration effect

• pH effect

• Time effect

• Temperature effect

• Ionic strength effect

Solvent effect

2-propanol instead of 1-propanol

Methanol instead of 1-propanol

Ethanol instead of 1-propanol

Acetone instead of 1-propanol

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

Concentration Effects

1.25 times higher solvent/non-solvent ratio

2.7 times lower solvent/non-solvent ratio

4 times lower concentration of cholesterol

4 times higher concentration of cholesterol

pH effect

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

pH = 1.8 (isoelectric point)

pH = 1.3 (positively charged particles)

Aging effects

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

Particles sampled out after 3 min of aging time

Particles sampled out after 5 min of aging time

Particles aged for one month at atmospheric conditions

Particles aged for one month at atmospheric conditions

Particles aged for one month at atmospheric conditions

Particles aged for four months at atmospheric conditions

Particles aged for four months at atmospheric conditions

Temperature effects

Particles aged for 2 days at 34 oC

Particles aged for 2 days at room T

Particles aged for 15 min at 37 oC

Particles precipitated at 31 oC and aged for 5h at 37 oC

Effect of the addition of salt

Particles obtained with the addition of 0.5 M of NaCl

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

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

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

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

Particles coated with silica

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Precipitation by solvent/non-solvent evaporation

• Particles prepared by evaporation of the unsaturated “standard” cholesterol solution

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

“Amazingly small means leading to extraordinarily satisfying results”

Ernst Fritz Schumacher, Small is Beautiful, 1973.

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.

Preparation and Multilayered Aggregation of Uniform Colloidal

Cholesterol Particles

Vuk Uskoković, Egon Matijević

Center for Advanced Materials Processing

Clarkson University, Potsdam, NY, USA

Bile Salt

PhosphatidylcholineC

hole

ster

ol

100

80

60

40

20

100 80 60 40 20 (mol %)

20

40

60

80

100

micellar zone

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)