Biomedical Engineering Reference
In-Depth Information
of the lipid in the internal lamellae, and the close apposition of the adjacent
concentric bilayers restricts the internal water space. In single-compartment
vesicles formed during SUV, the ratio of surface area to encapsulated volume is
larger resulting in only a small aqueous volume per mole of lipid.
Various methods for the synthesis of MLVs and SUVs have been described.
One of these is the ethanol injection method that produces vesicles of about the
same size as SUV, while the ether infusion technique produces large unilamel-
lar vesicles with high captured volumes per mole of lipid low encapsulation
efficiency.
139
Szoka's group produced vesicles with the following desirable properties: (i)
the ability to entrap a large percentage of the aqueous material presented; (ii) a
high aqueous space-to-lipid ratio; and (iii) widely variable chemistry of the lipid
components.
139
They developed conditions that achieve a high percentage of
encapsulation and vesicles with a large aqueous space-to-lipid ratio in a method
they called the reverse-phase evaporation technique.
140
2.3.1.1 Liposome Synthesis
There are a number of published protocols for the synthesis of different lipo-
some NMs. In this topic, we focused on the protocol pioneered by the group of
Papahadjopoulos.
139-141
In their process, cholesterol, palmitic acid, phosphati-
dylcholine, (PtdCho), phosphatidylglycerol (PtdGro), and phosphatidylserine
(PtdSer) used were isolated from egg yolk and bovine brain,
140
while phospha-
tidic acid and dipalmitoyl phosphatidylcholine (Pal
2
PtdCho) were synthesized
as reported.
140
These were purified with silicic acid columns and tested by thin-
layer chromatography to check the purity
141
before storage in chloroform in
sealed ampules under nitrogen at −50 °C until use.
Protocol for preparation of reverse-phase evaporation vesicles is given
below:
139
(1)
Prepare a mixture of several phospholipids, either pure or mixed with other
lipids such as cholesterol, long-chain alcohols, etc., can be used with simi-
lar results.
(2)
Add the lipid mixture to a 50-mL round-bottom flask with a long exten-
sion neck, and remove the solvent by rotary evaporation under reduced
pressure.
(3)
Purge the system with nitrogen and redissolve the lipids in the organic
phase to form the reversed phase vesicles using diethyl ether and isopropyl
ether or halothane and trifluorotrichloroethane.
(4)
When the lipid has low solubility in ether, add chloroform or methanol to
increase their solubility (Table X from Szoka and Papahadjopoulos
139
).
(5)
Add the aqueous phase and keep the system continuously under nitrogen.
(6)
Briefly sonicate the resulting two-phase system (2-5 min) in a bath-type
sonicator (Lab Supply T-80-80-IRS) until the mixture becomes either
a clear one-phase dispersion or a homogeneous opalescent dispersion
