Biomedical Engineering Reference
In-Depth Information
that does not separate for at least 30 min after sonication. The degree of
opalescence depends upon the solvent, phospholipid, and amount of aque-
ous phase in the preparation. The sonication temperature for diethyl ether
and most lipids is 0-5 °C unless otherwise stated.
(7)
Place the mixture on the rotary evaporator to evaporate the organic solvent
at 20-25 °C, rotating at approximately 200 rpm. As the solvent is removed,
the material first forms a viscous gel and subsequently (within 5-10 min)
becomes an aqueous suspension.
(8)
You may add water or buffer (optional) and evaporate for 15 min at 20 °C
to remove traces of solvent.
(9)
When lipid mixtures in the absence of cholesterol are used at low concen-
trations (<7.5 umol of lipid per milliliter of aqueous phase) the gel phase
may not be apparent since the system rapidly reverts to a lipid-in-water sus-
pension. In this case, either dialyze, pass through a Sepharose 4B column,
or centrifuge to remove nonencapsulated material and residual organic
solvent.
In general, a lipid preparation contains 33 µmol of phospholipid and 33 µmol of
cholesterol in 1.0 mL of aqueous phase (PBS) and 3 mL of solvent. These ratios
are optimal, but can be scaled down or up without any change in the character-
istics of the resulting vesicles. To make vesicles from Pal
2
PtdCho, an additional
3 mL chloroform or 0.8 mL of methanol is added and the vesicles are allowed to
remain at 45 °C for at least 30 min after evaporation of the solvent.
Detection of the amount of encapsulated small molecules such as sodium,
sucrose, or [
3
H]ara C can be achieved by dialyzing the vesicles overnight against
300 vol of PBS (three changes) at 4 °C. Column chromatography can be used
to separate encapsulated proteins from unencapsulated proteins (Sepharose 4B,
1.5 × 42 cm). Ultracentrifugation at 100,000
g
for 30 min can be used to separate
encapsulated [
3
H]poly(A) from unencapsulated material and resuspension of
the pelleted vesicles in buffer (twice); the unencapsulated poly(A) remains in
solution.
139
2.3.2 Polymeric NPs
142
Polymer-based nanoparticles (PNPs) are among the most advanced NMs when
it comes to their medical applications because these can effectively carry drugs,
proteins, and DNA to target cells and organs.
142
They are effective for cell
membrane permeation and they exhibit stability in the blood stream. They are
convenient materials for various unique molecular designs with many potential
medical applications.
143
Polymers are being studied to carry drugs (small molecules and/or proteins)
and to release these when the PNPs reach the destination.
144
Their long shelf life
and their minimal toxicity make them good candidates as delivery system for
drugs and proteins.
144,145
In aqueous solution, amphiphilic block or graft copo-
lymers assemble spontaneously into NPs.
142
Polymers, such as poly-lactic acid
