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Technical
Information
Preparation of
Liposomes
1. Mechanism of Vesicle
Formation Liposomes (lipid vesicles) are formed when
thin lipid films or lipid cakes are hydrated and stacks of liquid
crystalline bilayers become fluid and swell. The hydrated lipid
sheets detach during agitation and self-close to form large,
multilamellar vesicles (LMV) which prevents interaction of water
with the hydrocarbon core of the bilayer at the edges. Once these
particles have formed, reducing the size of the particle requires
energy input in the form of sonic energy (sonication) or mechanical
energy (extrusion).
2. Method of Liposome
Preparation Properties of lipid formulations can vary
depending on the composition (cationic, anionic, neutral lipid
species), however, the same preparation method can be used for all
lipid vesicles regardless of composition. The general elements of
the procedure involve preparation of the lipid for hydration,
hydration with agitation, and sizing to a homogeneous distribution
of vesicles. a. Preparation of lipid for hydration: When
preparing liposomes with mixed lipid
composition, the lipids must first be dissolved and mixed in an
organic solvent to assure a homogeneous mixture of lipids. Usually
this process is carried out using chloroform or chloroform:methanol
mixtures. The intent is to obtain a clear lipid solution for
complete mixing of lipids. Typically lipid solutions are prepared at
10-20mg lipid/ml organic solvent, although higher concentrations may
be used if the lipid solubility and mixing are acceptable. Once the
lipids are thoroughly mixed in the organic solvent, the solvent is
removed to yield a lipid film. For small volumes of organic solvent
(<1mL), the solvent may be evaporated using a dry nitrogen or
argon stream in a fume hood. For larger volumes, the organic solvent
should be removed by rotary evaporation yielding a thin lipid film
on the sides of a round bottom flask. The lipid film is thoroughly
dried to remove residual organic solvent by placing the vial or
flask on a vacuum pump overnight. If the use of chloroform is
objectionable, an alternative is to dissolve the lipid(s) in
tertiary butanol or cyclohexane. The lipid solution is transferred
to containers and frozen by placing the containers on a block of dry
ice or swirling the container in a dry ice-acetone or alcohol
(ethanol or methanol) bath. Care should be taken when using the bath
procedure that the container can withstand sudden temperature
changes without cracking. After freezing completely, the frozen
lipid cake is placed on a vacuum pump and lyophilized until dry (1-3
days depending on volume). The thickness of the lipid cake should be
no more than the diameter of the container being used for
lyophilization.Dry lipid films or cakes can be removed from the
vacuum pump, the container close tightly and taped, and stored
frozen until ready to hydrate. b. Hydration of lipid
film/cake: Hydration of the dry lipid film/cake is accomplished
simply by adding an aqueous medium to the container of dry lipid and
agitating. The temperature of the hydrating medium should be above
the gel-liquid crystal transition temperature (Tc or Tm) of the
lipid with the highest Tc before adding to the dry lipid. After
addition of the hydrating medium, the lipid suspension should be
maintained above the Tc during the hydration period. For high
transition lipids, this is easily accomplished by transferring the
lipid suspension to a round bottom flask and placing the flask on a
rotory evaporation system without a vacuum. Spinning the round
bottom flask in the warm water bath maintained at a temperature
above the Tc of the lipid suspension allows the lipid to hydrate in
its fluid phase with adequate agitation. Hydration time may differ
slightly among lipid species and structure, however, a hydration
time of 1 hour with vigorous shaking, mixing, or stirring is highly
recommended. It is also believed that allowing the vesicle
suspension to stand overnight (aging) prior to downsizing makes the
sizing process easier and improves the homogeneity of the size
distribution. Aging is not recommended for high transition lipids as
lipid hydrolysis increases with elevated temperatures. The hydration
medium is generally determined by the application of the lipid
vesicles. Suitable hydration media include distilled water, buffer
solutions, saline, and nonelectrolytes such as sugar solutions.
Physiological osmolality (290 mOsm/kg) is recommended for in vivo
applications. Generally accepted solutions with meet these
conditions are 0.9% saline, 5% dextrose, and 10% sucrose. During
hydration some lipids form complexes unique to their structure.
Highly charged lipids have been observed to form a viscous gel when
hydrated with low ionic strength solutions. The problem can be
alleviated by addition of salt or by downsizing the lipid
suspension. Poorly hydrating lipids such as phosphatidylethanolamine
have a tendency to self aggregate upon hydration. Lipid vesicles
containing more than 60 mol% phosphatidylethanolamine form particles
having a small
hydration layer
surrounding the vesicle. As particles approach one another there is
no hydration repulsion to repel the approaching particle and the two
membranes fall into an energy well where they adhere and form
aggregates. The aggregates settle out of solution as large
floculates which will disperse on agitation but reform upon sitting.
The product of hydration is a large, multilamellar vesicle (LMV)
analogous in structure to an onion, with each lipid bilayer
separated by a water layer. The spacing between lipid layers is
dictated by composition with poly hydrating layers being closer
together than highly charged layers which separate based on
electrostatic repulsion. Once a stable, hydrated LMV suspension has
been produced, the particles can be downsized by a variety of
techniques, including sonication or extrusion. c. Sizing of
lipid suspension: i. Sonication*:
Disruption of LMV suspensions using sonic energy (sonication)
typically produces small, unilamellar vesicles (SUV) with diameters
in the range of 15-50nm. The most common instrumentation for
preparation of sonicated particles are bath and probe tip
sonicators. Cup-horn sonicators, although less widely used, have
successfully produced SUV. Probe tip sonicators deliver high en-ergy
input to the lipid suspension but suffer from overheating of the
lipid suspension causing degradation. Sonication tips also tend to
release titanium particles into the lipid suspension which must be
removed by centrifugation prior to use. For these reasons, bath
sonicators are the most widely used instrumentation for preparation
of SUV. Sonication of an LMV dispersion is accomplished by placing a
test tube containing the suspension in a bath sonicator (or placing
the tip of the sonicator in the test tube) and sonicating for 5-10
minutes above the Tc of the lipid. The lipid suspension should begin
to clarify to yield a slightly hazy transparent solution. The haze
is due to light scattering induced by residual large particles
remaining in the suspension. These particles can be removed by
centrifugation to yield a clear suspension of SUV. Mean size and
distribution is influenced by composition and concentration,
temperature, sonication time and power, volume, and sonicator
tuning. Since it is nearly impossible to reproduce the conditions of
sonication, size variation between batches produced at different
times is not uncommon. Also, due to the high degree of curvature of
these membranes, SUV are inherently unstable and will spontaneously
fuse to form larger vesicles when stored below their phase
transition temperature.
ii. Extrusion**:
Lipid extrusion
is a technique in which a lipid suspension is forced through a
polycarbonate filter with a defined pore size to yield particles
having a diameter near the pore size of the filter used. Prior to
extrusion through the final pore size, LMV suspensions are disrupted
either by several freeze-thaw cycles or by prefiltering the
suspension through a larger pore size (typically 0.2µm-1.0µm). This
method helps prevent the membranes from fouling and improves the
homogeneity of the size distribution of the final suspension. As
with all procedures for downsizing LMV dispersions, the extrusion
should be done at a temperature above the Tc of the lipid. Attempts
to extrude below the Tc will be unsuccessful as the membrane has
atendency to foul with rigid membranes which cannot pass through the
pores. Extrusion through filters with 100nm pores typically yields
large, unilamellar vesicles (LUV) with a mean diameter of 120-140nm.
Mean particle size also depends on lipid composition and is quite
reproducible from batch to batch.
The three illustrations above are excerpted from the
book 'Liposomes in Gene Delivery' by Danilo D. Lasic, published 1997
by CRC Press LLC. Avanti thanks the publisher for kind permission to
reproduce these drawings.