Multilamellar liposomes having improved trapping efficiencies

A multilamellar vesicle dispersed in an aqueous phase comprising an aqueous medium, a lipid concentration of at least about 50 mg/ml and a trapping efficiency of at least about 40 percent. The vesicle can be prepared by dispersing the lipid in an aqueous phase to form a multilamellar vesicle, rapidly freezing the multilamellar vesicle to obtain a frozen lipid-aqueous medium mixture, and warming the mixture to obtain a frozen and thawed multilamellar vesicle dispersed in an aqueous phase.

BACKGROUND OF THE INVENTION 
The present invention is directed to liposomes. More particularly, the 
present invention is directed to multilamellar vesicles having improved 
trapping efficiency and equilibrium transbilayer solute distribution. 
Liposomes of the type which are multilamellar vesicles (MLV's) are usually 
formed by mechanical dispersion of dried lipid in an aqueous buffer. It is 
commonly assumed that this procedure results in an equilibrium 
interlamellar distribution of solutes present in the buffer. However, it 
has been demonstrated that the trapped buffer may have reduced solute 
concentrations resulting in osmotic imbalances between exterior and 
interior environments. Gruner et al., Biochemistry, 24, 2833-2842 (1985). 
These osmotic imbalances can lead to membrane potentials, transbilayer pH 
gradients and deformations due to osmotic forces. Equilibrium solute 
distributions can be achieved by techniques involving dispersion of the 
lipid in mixtures of organic solvent and aqueous buffer, where the organic 
solvent is subsequently removed under reduced pressure. 
Alternative procedures have been described in U.S. Pat. No. 4,522,803 and 
copending U.S. patent applications Ser. Nos. 476,496 and 521,176, and 
incorporated herein by reference, to prepare liposomes having properties 
different than MLV's. 
The low trapping volume and trapping efficiency of MLV systems have 
presented difficulties in employing these systems in applications such as 
drug delivery. As a result, alternative procedures have been developed to 
prepare liposomal systems which involve the use of organic solvents or 
detergents as solubilizing agents. Organic solvents and detergents are 
undesirable ingredients in drug delivery systems. 
It would, therefore,-be desirable to obtain a multilamellar vesicle having 
high trapping efficiency, high trapped volumes and equilibrium 
transbilayer solute distribution prepared in the absence of organic 
solvents or detergents. 
SUMMARY OF THE INVENTION 
We have prepared a new multilamellar vesicle dispersed in an aqueous phase 
comprising an aqueous medium, a trapping efficiency of at least 40 percent 
and a lipid concentration of at least about 50 mg/ml. Preferably, the 
trapping efficiency is at least about 50 percent and the lipid 
concentration is at least about 100 mg/ml, more preferably between about 
100 and 1000 mg/ml, and still more preferably between about 100 and 400 
mg/ml. 
The vesicles of the present invention have an interlamellar equal solute 
distribution. The vesicle can contain a bioactive agent. The lipid can 
comprise phospholipid such as phosphatidylcholine, and may additionally 
comprise a sterol such as cholesterol. The vesicle can be concentrated by 
removal of all or part of the aqueous phase. 
The multilamellar vesicles of the present invention can be prepared by 
dispersing (a) a lipid in an aqueous phase to form a multilamellar 
vesicle, (b) rapidly cooling the multilamellar vesicle to obtain a frozen 
lipid-aqueous medium mixture, and (c) warming the mixture. The rapid 
cooling step preferably employed liquid nitrogen. Preferably, steps (b) 
and (c) are performed at least about five times. 
In addition, the resulting multilamellar vesicles of the present invention 
can be filtered through polycarbonate filters to obtain a resulting 
vesicle dispersed in an aqueous phase comprising a lipid concentration of 
at least about 5 mg/ml of aqueous phase, a mean diameter of less than 
about 100 nanometers and a trapping efficiency of at least about 10%. 
Preferably the lipid concentration is at least about 100 mg/ml, more 
preferably about 100-400 mg/ml. The trapping efficiency is preferably 
about 40%. Preferably the resulting vesicles have a uniform size 
distribution.

DETAILED DESCRIPTION OF THE INVENTION 
The following definitions will be employed: 
liposome--any structure composed of lipid bilayers that enclose a volume. 
multilamellar vesicles (MLVs)--liposomes containing multiple lipid bilayers 
forming two or more shells. 
FATMLV--A MLV which has been subject to at least one freeze-thaw cycle. 
lipid--an agent exhibiting amphipathic characteristics causing it to 
spontaneously adopt an organized structure in water wherein the 
hydrophobic portion of the molecule is sequestered away from the aqueous 
phase. 
freeze-thaw-cycle--cooling a liposome below the freezing point of the 
aqueous solvent contained within the liposome, then warming to a 
temperature whereby the aqueous medium or phase is melted. 
trapping efficiency or encapsulation efficiency--the fraction of an aqueous 
phase sequestered by liposome bilayers when a lipid is dispersed in the 
aqueous phase; given as the percent of the original volume of the aqueous 
phase. 
captured volume or trapped volume--the volume enclosed by a given amount of 
lipid with units of liters entrapped per mole of total lipid. 
lipid concentration--the amount of lipid added per ml of aqueous phase; the 
units are generally mg/ml. 
Multilamellar vesicles (MLV's) can be prepared by a number of methods. In 
one process, one or more selected lipids are deposited on the inside walls 
of a suitable vessel by dissolving the lipids in an organic solvent such 
as chloroform and then evaporating the organic solvent, adding an aqueous 
phase which is to be encapsulated to the vessel, allowing the aqueous 
phase to hydrate the lipid, and mechanically agitating, for example, 
swirling or vortexing, the resulting lipid suspension to produce the 
desired liposomes. 
Alternatively, one or more selected lipids can be dispersed by employing 
mechanical agitation in an aqueous phase to produce MLV's. The process 
requires about 1-10 minutes at a temperature above the gel/liquid 
crystalline transition temperature. 
The organic solvent can contain a bioactive agent such as a drug, 
preferably a bioactive agent which is both soluble in the organic solvent 
and is lipophilic. 
The aqueous medium is that enclosed by lipid bilayers. Generally the 
aqueous medium will have the same constituents as the aqueous phase, 
although the amounts may be different. Although the following discussion 
is directed to an aqueous medium, it clearly also applies to the aqueous 
phase. The aqueous medium can be for example, water or water containing 
dissolved salt or buffer. The aqueous medium may contain a bioactive 
agent, preferably a bioactive agent which is water soluble. Water soluble 
bioactive agents which can be incorporated into FATMLV's of the present 
invention include antibacterial aminoglycosides such as tobramycin. 
Pilocarpine can be incorporated for ocular administration for treating 
glaucoma. 
In the MLV the bioactive agent generally partitions between the aqueous and 
lipid portions of the liposome depending on the agent's lipophilic and 
hydrophilic character. 
In addition, the MLV's produced by the procedures previously described have 
low trapped volumes and corresponding low trapping efficiencies, which 
causes the loss of valuable solutes in the aqueous solvent and the added 
cost of recycling the untrapped aqueous phase. 
The lipids which can be employed in the present invention include 
cholesteryl hemisuccinate and salts thereof, tocopheryl hemisuccinate and 
salts thereof, a glycolipid, a phospholipid such as phosphatidylcholine 
(PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), 
phosphatidylglycerol (PG), phosphatidic acid (PA), phosphatatidylinositol 
(PI), sphingomylin (SPM), and the like, alone or in combination. The 
phospholipids can be synthetic or derived from natural sources such as egg 
or soy. Sterols such as cholesterol can be combined with the 
phospholipids. The phospholipids employed and the amount of sterol present 
depends on a number of factors such as lipophilicity of any added 
bioactive agent and the required properties of the liposome. These factors 
are well known to those skilled in the art. 
The lipid concentration for the present invention is at least about 50 
mg/ml. At lower concentrations, multilamellar vesicles of the present 
invention having a high trapping efficiency are difficult or impossible to 
form. A preferred lipid concentration is between about 100 and 1000 mg/ml, 
more preferably 100-600 mg/ml, and still more preferably 100-400 mg/ml. 
A high trapping efficiency results in a large fraction of the aqueous phase 
being entrapped in the liposome. In the present invention, the liposomes 
have a trapped efficiency of at least about 40 percent, preferably at 
least 50 percent and, more preferably about 50-90 percent. Generally, a 
lipid concentration of at least about 100 mg/ml is needed to obtain a 
trapping efficiency of at least about 50 percent. 
The dispersed lipid mixture results in predominately MLV's. These MLV's 
have been shown to be in osmotic imbalance when a solute is present in the 
aqueous medium prior to addition of the lipid. For example, when Mn.sup.2+ 
ion from MnCl.sub.2 is present in an aqueous buffer, non-equilibrium 
transmembrane distributions of the paramagnetic Mn.sup.2+ ion in the 
MLV's can be observed by NMR spectroscopy. Mn.sup.2+ is a "broadening" 
agent which quenches the .sup.31 P NMR signal arising from phospholipids 
with which the ion comes in proximity. When there are asymmetric 
tribilayer concentrations of Mn.sup.2+ in the MLV, the .sup.31 P NMR 
resomme arising from the phospholipids on either side of the bilayers are 
not equally quenched. 
The freeze-thaw cycle of the present invention requires rapid freezing of 
the dispersed lipid medium mixture and then warming the frozen mixture in 
a constant temperature bath, to a temperature which will cause the aqueous 
phase to melt. The temperature employed is generally above the transition 
temperature for the gel-liquid crystalline transition. A constant 
temperature bath of about 25.degree.-50.degree. C., preferably about 
40.degree. C., is generally effective. 
Liquid nitrogen baths have been found to be particularly effective for the 
freezing step. A comparison was made of the FATMLV's prepared by placing 
the mixture in dry ice/ethanol and liquid nitrogen baths. For FATMLV's 
prepared by freezing liquid nitrogen, the trapping efficiency and the 
trapped volume was at least about 1.5-fold greater then when dry 
ice/ethanol was employed. At higher lipid concentrations, trapping 
efficiencies at least four-fold greater can be achieved. FATMLV's prepared 
using a dry ice/ethanol bath do not have the properties of those of the 
present invention. Strauss, "Freezing and Thawing of Liposome 
Suspensions," in Liposome Technology, Ed. G. Gregoriadis, Chapter 15, CRC 
Press, Inc., Boca Raton, FL, 1984, discusses the rapid vs. slow cooling of 
liposome preparations and the relevant portions are incorporated herein by 
reference. 
The number of freeze-thaw cycles affects the properties of the resulting 
FATMLV. Generally, three or more freeze-thaw cycles are required to obtain 
an equilibrium interlamellar osmotic balance. The balance can be observed 
by following the effect of the number of freeze-thaw cycles upon the 
distribution of Mn.sup.2+ as measured by .sup.31 P NMR when the FATMLV 
includes phospholipid-containing bilayers. 
When a lipid containing phospholipid solubilized in an aqueous phase by 
means of a detergent such as Triton X-100, the .sup.31 P NMR spectrum in 
the presence of Mn.sup.2+ shows all the phosphorous to be equally 
quenched. Essentially the same spectrum is observed for a FATMLV after 
three cycles. 
About five freeze-thaw cycles in liquid nitrogen and a 40.degree. C. 
constant temperature bath, result in FATMLV's of the present invention. 
After five freeze-thaw cycles the morphology of the FATMLV changes. Before 
freeze-thawing, the MLV'S exhibit the tightly packed "onion skin" 
arrangements of concentric bilayers normally associated with multilamellar 
liposomes. After five freeze-thaw cycles, a new structure is observed by 
freeze-fraction electron micrographs. The interlamellar spacing is 
increased by up to about five fold and closed lamellar systems can be 
intercalated between bilayers. The tightly packed arrangement is 
substantially absent. 
The FATMLV's after at least about five cycles have significantly greater 
trapped volume and trapping efficiencies. The trapped volume per umol 
phospholipid can be determined using .sup.22 Na.sup.+ as an aqueous 
marker. At 100 mg/ml phosphatidylcholine, more than an order of magnitude 
increase in trapped volume is observed after five freeze-thaw cycles 
compared to non-freeze-thawed MLV's. After about eight freeze-thaw cycles 
additional cycles do not result in any further significant changes in 
trapped volume. 
The freeze-thaw protocol of the present invention is effective for various 
lipid compositions. For example, for a lipid whose components are egg PC 
containing between 0 and 50 mole percent cholesterol, substantially the 
same results with regard to trapped volume were obtained after five 
freeze-thaw cycles utilizing liquid nitrogen and 40.degree. C. water 
constant temperature bath. 
The fraction of lipid in the outer bilayer in FATMLV's of the present 
invention is less than about 35 percent. When the lipid is egg PC at a 
lipid concentration of 50 mg/ml the fraction of lipid in the outer bilayer 
is about 32-34 percent. The fraction of lipid in the outer bilayer 
decreases with increasing lipid concentration, but is generally larger 
than that of MLV's or FATMLV's of the prior art. For egg PC (100 mg/ml) 
the outer bilayer fraction is about 26-30 percent, 200 mg/ml is about 
13-17 percent, and 400 mg/ml is about 13-17 percent. Comparisons should be 
made for the same ingredients and formulations. 
The aqueous phase of the dispersions of the present invention vesicle can 
be removed to form a concentrated FATMLV dispersion. Methods of removal 
include diahysis, centrifugation, dehydration and lyophilyation. 
Dehydration is described in a copending application filed June 26, 1985, 
M.B. Bally et. al., "Encapslation of Antineoplastic Agents in Liposomes," 
Ser. No. 749,161 and in a copending application filed July 26, 1985, A.S. 
Janoff et al., "Dehydrated Liposomes", Ser. No. 759,419, relevant portions 
of which are incorporated herein by reference. 
Variable size vesicles can be prepared by a rapid extrusion technique where 
lipid dispersions are passed under moderate pressure, e.g., nitrogen at 
pressures up to about 800 psi, through polycarbonate filters whose pore 
size can be varied from 30-800 nm or more. Methods relating to rapid 
extrusion techniques through polycarbonate filters and the use of the 
methods to obtain liposomes of uniform size distribution are described in 
the following copending application, relevant portions of which are 
inforporate herein by reference: Cullis et al., Ser. No. 22,690, filed 
June 20, 1984; Cullis et al., Ser. No. 622,502, filed June 20, 1984; and 
Cullis et al., "Extrusion Technique for Producing Unilamellar Vesicles", 
filed Oct. 16, 1985, Docket No. LIP-101 Div. Unilamellar vesicles with a 
large, e.g., (151 nm) mean diameter can be produced if FATMLV's of the 
present invention are passed through 200 nm pore size filters. This 
freeze-thaw process also increased the aqueous trapped volume of vesicles 
1.5 to 3.0 times when filters with pore sizes ranging from 100 to 400 nm 
were utilized. Increasing the lipid concentration to 400 mg/ml of aqueous 
buffer for these systems increased the trapping efficiency levels to 80 
and 50 percent for vesicle produced employing 400 and 100 nm pore size 
filters, respectively. Freeze fracture electron microscopy revealed that 
vesicles produced at the very high lipid concentrations exhibited 
negligible alterations in size distribution or extent of multilamellarity 
as compared to systems produced at lower lipid levels. These results 
suggest that the freeze-thaw extrusion protocol offers a very general and 
versatile method for producing vesicles of variable sizes and has several 
advantages that were unavailable with previous procedures. Sized vesicles 
containing drugs or other bioactive agents are useful for treating 
illnesses in mammals including humans. For example, sized vesicles 
containing pilocarpine may be used to treat glaucoma and those containing 
tobramycin have reduced toxicity. 
A variety of unilamellar and multilamellar vesicles of differing sizes can 
be generated by extrusion of MLV's through polycarbonate filters with 
different pore sizes. It is convenient to introduce the general term 
"VET's" to indicate "vesicles by extrusion techniques" with a numerical 
subscript to indicate the pore size employed. Thus a VET.sub.50 system 
indicates vesicles extruded through filters with 50 nm pore size whereas a 
VET.sub.400 system indicates extrusion through 400 nm pore size filters. 
Once the lamellarity and size of these systems has been determined, this 
nomenclature can be further refined to indicate the large or small 
character of unilamellar systems or the multilamellarity of other (e.g., 
large unilamellar vesicles by extrusion through 100 nm 
filters--LUVET.sub.100 's; small unilamellar vesicles by extrusion through 
50 nm filters--SUVET.sub.50 's; or multilamellar vesicles obtained by 
extrusion through 400 nm filters--MLVET.sub.400 's). An arbitrary decision 
to designate unilamellar systems extruded through 100 nm or larger pores 
as "large" has been made, whereas systems extruded through 50 nm or 
smaller pores are indicated as "small". 
The influence of external Mn.sup.2+ on the .sup.31 P NMP signal intensity 
of EPC MLV systems (100 mg/ml) extruded through polycarbonate filters with 
pore sizes in the range 30 nm to 400 nm is illustrated in FIG. 6A. The 
signal intensity of the VET.sub.100 systems decreases to 50%, indicating 
unilamellar character, after 8 passes through two stacked filters. Only 4 
passes were found to be required to obtain predominantly unilamellar 
character when the lipid concentration is about 50 mg/ml or less. The 
VET.sub.50 and VET.sub.30 systems obtained on extrusion through the 50 and 
30 nm pore size filters exhibit residual .sup.31 P NMR intensities of 48% 
and 44% respectively (after 8 passes) consistent with a unilamellar 
population of smaller vesicles. 
The MLV systems extruded through the 200 nm and 400 nm filters retain 
multilamellar character as the residual .sup.31 P NMR signal intensities 
are 70 and 78% respectively. If it is assumed that the multilamellar 
vesicle population contain only two bilayers which are tightly packed 
(e.g., separated by 10 nm or less), then this would indicate that 
approximately 20% of the VET.sub.200 systems are unilamellar and 
approximately 80% bilamellar. If the average number of lamellar is more 
than two, the proportion of unilamellar vesicle will be higher. Similar 
calculations for the VET.sub.400 systems are consistent with nearly all 
the vesicles exhibiting bilamellar character, or with a small proportion 
of unilamellar vesicles which will increase for higher proportions of 
multilamellar vesicles with three or more bilayers. 
FATMLVs exhibit significantly larger interlamellar spacings and much larger 
trapped volumes. It may therefore be expected that VET.sub.400 and 
VET.sub.200 systems prepared from FATMLV's should exhibit somewhat higher 
unilamellar character due to the reduced fraction of tightly packed 
lamellae in the FATMLV precursors. This appears to be the case as 
illustrated in FIG. 6B, where it is shown that the residual .sup.31 P NMR 
intensities (after addition of Mn.sup.2+) for the VET.sub.400 and 
VET.sub.200 systems are 68% and 56% respectively. This would correspond to 
an average of approximately 20% unilamellar and 80% containing two 
(tightly packed) lamellae for the VET.sub.400 systems and substantial 
population of unilamellar vesicles (at least 75%) for the VET.sub.200 
systems when prepared from FATMLV's. It may also be noted that fewer 
passes are required to generate the unilamellar LUVET.sub.100 system from 
FATMLV's than from non-freeze thawed MLV's. Similar effects were observed 
for the SUVET.sub.50 and SUVET.sub.30 preparations. 
Assuming a similar size distribution, the increased unilamellar character 
of the VET.sub.400 and VET.sub.200 systems prepared from FATMLV's suggests 
that the trapped volume (expressed as liters trapped/ mol phospholipid) of 
freeze-thawed, sized vesicles should be significantly increased. That this 
is the case is illustrated in FIG. 7, where the trapped volume of the 
vesicles prepared from FATMLV's increases from 1 1/mol to 3.6 1/ mol 
phospholipid as the pore size is increased from 30 to 400 nm. This 
contrasts strongly with the trapped volumes of the VET's prepared from MLV 
precursors, which increase by 20% or less for the same range of pore 
sizes. It is interesting to note that whereas the trapped volumes of the 
SUVET.sub.30 and SUVET.sub.50 vesicles are the same for FATMLV and MLV 
precursors, the trapped volumes of the LUVET.sub.100 systems increase by 
50% (to 1.5 l/mol) when FATMLV's are employed. Comparable results were 
observed when .sup.14 C-inulin was used as the aqueous trap marker rather 
than .sup.22 Na. Given that the .sup. 31 P NMR data indicate unilamellar 
character for FATMLV and MLV systems extruded through filters with 100 nm 
pore size or less, it would not be expected that the trapped volumes 
observed should be sensitive to the freeze-thaw procedure. 
The size distributions calculated by the procedure of van Venetie et al 
(see Methods and Materials) are given in Table 3 and reveal relatively 
homogeneous vesicle populations whose mean sizes are either somewhat 
smaller than the filter pore size (e.g., the VET.sub.400 and VET.sub.200 
systems) or of the same size or larger than the filter pore size (e.g., 
the SUVET.sub.50 and SUVET.sub.30 systems). The size of the VET.sub.200 
vesicles is likely related do the fact that the actual pore sizes of the 
Nucleopore membranes are approximately 10% smaller than specified, whereas 
the sizes of the SUVET.sub.50 and SUVET.sub.30 systems may reflect a 
limiting size of the VET systems. 
The vesicle sizes determined by light scattering techniques are also given 
in Table 3. 
The results indicate that homogeneously-sized vesicles of SUV, LUV or MLV 
character can be readily generated by extruding MLV's or FATMLV's through 
polycarbonate filters of appropriate pore size. 
In FIG. 8, trapping efficiencies of 81%, 56% and 50% are obtained for 
FATMLV's (prepared at 400 mg/ml) extruded through 400 nm, 100 nm and 50 nm 
filters respectively. These trapping efficiencies are clearly remarkable, 
particularly for the smaller VET.sub.100 and VET.sub.50 systems. 
Freeze-fracture micrographs of VET.sub.100 systems obtained at 400 mg/ml 
and then diluted to 100 mg/ml show that the size distribution is similar 
to that observed for the LUVET.sub.100 systems prepared at 100 mg/ml and 
the low frequency of cross-fractures supports a unilamellar character. 
When injected directly into the bloodstream, small vesicles are generally 
less leaky and remain in the blood for a longer time than larger systems. 
MATERIALS AND METHODS 
Egg phosphatidylcholine (EPC) was purified from hen egg yolks according to 
established procedures (see Singleton et. al., Journal of the American Oil 
Chemical Society 42, 53 (1965) ) and was chromatographically pure. .sup.22 
NaCl and .sup.14 C-inulin were obtained from NEN Canada, Quebec. 
Extrusion of the MLV or FATMLV preparations through two (stacked) 
polycarbonate filters of the various pore sizes (30-400 nm) was performed 
employing nitrogen pressures of up to 800 psi. .sup.31 P NMR spectra of 
egg PC liposomes were obtained employing a Bruker WP-200 spectrometer 
operating at 81.0 MHz. Free induction decays corresponding to 1000 
transients were accumulated utilizing 15 usec 90.degree. radio frequency 
pulse, gated proton decoupling and a 20 KHz sweep width. An exponential 
multiplication corresponding to a 40 Hz line broadening was applied prior 
to Fourier transformation. Signal intensities were determined by cutting 
and weighing spectra. 
The size distributions of the extruded liposomal systems were determined by 
freeze-fracture microscopy and quasi-elastic light scattering. Vesicle 
preparations to be used for freeze-fracture were mixed with glycerol (25% 
by volume) and frozen in a Freon slush. Samples were fractured and 
replicas obtained employing a Balzers BAF 400D apparatus, and micrographs 
of the replicas were produced using a Phillips' 400 electron microscope. 
Vesicle size distributions were estimated by measuring the diameter of 
fractured vesicles exhibiting 50% shadowing according to the procedure of 
van Venetie et al, Journal of Microscopy, 118, 401-408 (1980). Size 
distributions determined by quasi-elastic light scattering (QELS) analysis 
was performed utilizing a Nicomp Model 200 Laser Particle Sizer with a 5 
milliwatt Helium-Neon Laser at an exciting wavelength of 632.8 nm. QELS, 
also referred to as dynamic light scattering or photon correlation 
spectroscopy, employs digital autocorrelation to analyze the fluctuations 
in scattered light intensity generated by the diffusion of vesicles in 
solution. The measured diffusion coefficient is used to obtain the average 
hydrodynamic radius and hence the mean diameter of the vesicles. 
Vesicle trapped volumes were determined as follows: Phospholipid vesicles 
were hydrated and dispersed in the presence of tracer amounts of .sup.22 
NaCl or .sup.14 C-inulin (1 uCi/ml). Subsequent to the extrusion process 
the vesicles were diluted to 100 mg/ml (when necessary) and passed down a 
Sephadex 650 or Ultrogel (LKB AcA-34) column to remove untrapped .sup.22 
Na+ or .sup.14 C-inulin, respectively Aliquots of the vesicle-containing 
fraction were assayed for lipid phosphorus (15) and monitored for .sup.22 
Na+ utilizing a Beckman 8000 gamma counter or .sup.14 C-inulin using a 
Phillips' PW-4700 liquid scintillation counter. Trapped volumes were 
calculated and expressed as ul of aqueous trapped volume per umol of 
phospholipid. Trapping efficiencies were calculated as the dpm/umol 
phospholipid after gel filtration divided by the dpm/umol phospholipid 
before the gel filtration step. 
EXAMPLE 1 
FIGS. 1 (A) and (B) show the 81.0 MHz .sup.31 P NMR spectra of egg 
phosphatidylcholine multilamellar vesicles of the prior art dispersed (A) 
in the absence of Mn.sup.2+ and (B) in the presence of 0.5 mM Mn.sup.2+. 
The spectra of (C) was obtained from the MLV's dispersed in the presence 
of 0.5 mM Mn.sup.2+ which were subsequently subjected to 5 freeze-thaw 
cycles employing liquid nitrogen and a 40.degree. C. water constant 
temperature bath in order to obtain vesicles of the present invention. The 
phosphatidylcholine was isolated from egg yolks employing standard 
procedures and was more than 99% pure as indicated by thin layer 
chromatography. The MLV's were prepared by adding 2 ml of buffer (150 mM 
sodium chloride, 20 mM Hepes, pH 7.5) to 200 mg of lipid. This dispersion 
was vortexed intermittently (2 min. vortexing, 3 min. interval) over 20 
min. The .sup.31 P NMR spectra were collected at 20.degree. C. employing a 
Bruker WP 200 spectrometer utilizing a 20 KHz sweep width, 2 sec 
interpulse delay and broad band proton decoupling. 
EXAMPLE 2 
The influence of the number of liquid nitrogen 40.degree. C. water constant 
temperature bath freeze-thaw cycles on the .sup.31 P NMR signal intensity 
of egg phosphatidylcholine multilamellar vesicles prepared in the presence 
of 0.5 mM Mn.sup.2+ (.circle.) or 5 mM Mn.sup.2+ (.circle.) are shown in 
FIG. 2. The samples were prepared using the materials and procedures of 
Example 1. The signal intensities were obtained by cutting and weighing 
the normalized spectra. The arrow indicates the signal intensity obtained 
after addition of sufficient aqueous Triton X-100 (10%, wt per vol) to 
solubilize the sample. 
EXAMPLE 3 
Freeze fracture electron micrographs of MLV's before (A) and after (B) five 
liquid nitrogen 40.degree. C. water constant temperature bath freeze-thaw 
cycles are shown in FIG. 3. The phospholipid concentration was 100 mg/ml. 
The samples were prepared using the procedures and materials of Example 1. 
The arrow indicates the direction of shadowing and the bar represents 140 
nm. 
COMATIVE EXAMPLE 1 
Two vesicle preparation procedures were employed. The first, as described 
in Example 1, entailed dispersion of dry egg phosphatidylcholine (PC) in 
20 mM Hepes, 150 mM sodium chloride (pH 7.5) by vortex mixing followed by 
five freeze-thaw cycles employing liquid nitrogen and a 40.degree. C. 
water constant temperature bath. 
The second procedure was that of Westman et al., Biochemica et Biophysica 
Acta, 685, 315-328 (1982), which utilized egg PC dried down to a thin film 
on the walls of a glass test tube as the starting material. To the thin 
film was added a sufficient amount of an unbuffered 0.1M sodium chloride 
solution containing 0, 2 or 4 mg tetracaine/ml to yield a final lipid 
concentration of 50 mg/ml. The lipid is then dispersed by vortex mixing. 
The pH of the sample is then adjusted to 7.5 using sodium hydroxide or 
hydrochloric acid followed by five freeze-thaw cycles employing 
ethanol/dry ice and a 40.degree. water constant temperature bath. 
Trapped volumes and trapping efficiencies were determined by including 
.sup.22 Na.sup.+ (1 uCi/ml) in the aqueous phase in which the lipid was 
dispersed. After the multilamellar vesicles were formed, aliquots were 
assayed for lipid phosphorus and .sup.22 Na.sup.+ untrapped .sup.22 
Na.sup.+ was removed by washing with .sup.22 Na.sup.+ -free buffer 
employing low speed centrifugation. This procedure was repeated until 
supernatant counts were reduced to background levels. Aliquots of the 
pellet were then assayed for .sup.22 Na.sup.+ and lipid phosphorous. 
Trapping efficiencies were calculated as the ratio of the cpm per umol 
lipid after and before removal of untrapped .sup.22 Na.sup.+. Standard 
deviations, when given, were calculated from results obtained from 3 
samples. 
.sup.31 Phosphorus-NMR spectra were collected at 20.degree. C. employing a 
Bruker WP200 spectrometer utilizing a 10 KHz sweep width, 2 sec interpulse 
delay and broad band proton decoupling. Peak intensities were obtained by 
cutting and weighing before and after addition of MnCl.sub.2 in sufficient 
amounts to totally quench the externally exposed phospholipid signal. 
Freeze-fracture replicas were prepared by mixing vesicle preparations 
(adjusted to 50 mg/ml) with glycerol (25% by volume), freezing samples in 
a freon slush and fracturing them employing a Balzers BAF 400D apparatus. 
Micrographs were obtained by using a Phillips 400 electron microscope. 
Table 1 summarizes the trapped volume, trapping efficiency and .sup.31 
P-NMR characteristics for MLV's and FATMLV's produced at varying 
concentrations by the procedures of the present invention and those 
produced by the Westman, et. al. procedure at 50 mg egg PC/ml in the 
presence of 0, 2 and 4 mg tetracaine/ml. Five freeze-thaw cycles employing 
liquid nitrogen dramatically increases the trapped volume and 
corresponding trapping efficiency of FATMLV preparations as compared to 
the non-freeze-thawed counterpart. This trapped volume increase is greater 
than 10-fold for FATMLVs prepared at 50 and 100 mg egg PC/ml while 
FATMLV's prepared st 200 and 400 mg egg PC/ml exhibit increases of 6.5- 
and 3.7-fold, respectively. The less dramatic trapped volume increases 
observed for FATMLV systems prepare at 200 and 400 mg egg PC/ml are likely 
due to the limited availability of aqueous phase where 76.7 and 88.6% of 
the sample volume, respectively, is contained within the FATMLVs. The 
freeze-thaw protocol employed by Westman et al. also increases the trapped 
volume of FATMLVs but not to the extent observed for our systems (1.92, 
2.23 and 3.44 ul/umol egg PC in the presence of 0, 2 and 4 mg 
tetracaine/ml, respectively, compared with 5.02 ul/umol egg PC with our 
preparation). Table 1 also demonstrates that the trapping efficiencies of 
MLV's and Westman FATMLV's are significantly lower than those observed for 
FATMLV's of the present invention. 
.sup.31 P-NMR and freeze-fracture electron microscopy results corroborate 
the relationship of trapped volumes amoung the various vesicle 
preparations. The proportion of lipid in the outermost bilayer of vesicles 
can be determined by multiplying the percent .sup.31 P-NMR signal removed 
with Mn.sup.2+ addition by a factor of 2. As demonstrated in FIG. 4, 
FATMLV's produced by our procedure contain approximately 33% of the lipid 
in the outermost bilayer, a factor of 2 and 3 greater than Westman 
FATMLV's and standard MLVs, respectively. This result indicates that MLVs 
and Westman vesicles contain more lamellae per vesicle than do FATMLV's of 
the present invention produced at 50 mg/ml and is consistent with the 
lower trapped volumes observed for the prior systems. 
In summary, the FATMLVs produced according to the Westman procedure and 
FATMLVs prepared by our protocol exhibit three major differences in 
physical characteristics. First, ours exhibit a 1.4- to 2.2-fold greater 
trapped volume than Westman's. Second, our FATMLVs display greater 
trapping efficiencies than do Westman's when prepared at 50 mg egg PC/ml 
and trapping efficiencies approaching 90% can be achieved by increasing 
the lipid concentration. Third, our FATMLVs contain approximately 2-fold 
more lipid in the outermost bilayer than do Westman's. 
COMATIVE EXAMPLE 2 
Results were compared using liquid nitrogen and dry ice/ethanol (ETOH) in 
the freezing and thawing of multilamellar vesicles. 
Egg PC MLV's were prepared at a concentration of 50 mg/ml in 20 mM Hepes, 
150 mM NaCl (pH 7.5) by vortex mixing for approximately 5 min. in the 
presence of .sup.3 H-inulin as an aqueous marker. In the cases where lipid 
was used as a film, 50 mg egg PC was dissolved in 0.25 ml chloroform, the 
chloroform was removed with a stream of nitrogen and residual solvent was 
removed by high vacuum. The MLV's were subsequently frozen and thawed five 
times employing either a dry ice/ethanol bath or liquid nitrogen as 
indicated and a 400C. water constant temperature bath. Aliquots were then 
taken and analyzed for lipid-phosphorus and radioactivity. Free 
(unentrapped) .sup.3 H-inulin was removed by washing the vesicles 
employing low speed centrifugation until supernatant counts were reduced 
to background levels. Aliquots were again taken and analyzed for lipid 
phosphorus and radioactivity. Trapping efficiencies were calculated as the 
dpm/umol egg PC after removal of free inulin divided by the dpm/umol egg 
PC obtained before the washing procedure. Results are shown in Table 2. 
EXAMPLE 4 
FIG. 4 shows the effects of the number of freeze-thaw cycles on the trapped 
volume of FATMLV's of the present invention. MLV's were prepared from 
powdered egg PC in 20 mM Hepes, 150 mM sodium chloride (ph 7.5) as 
described in Example 1. The samples were frozen and thawed the indicated 
number of times employing liquid nitrogen and a 40.degree. C. water 
constant temperature bath. The samples (50 mg egg PC/ml) were assayed for 
lipid phosphous and radioactivity before and after removal of unentrapped 
.sup.3 H-inulin as described in Comparative Example 2. Vesicle trapped 
volume increased dramatically between zero and eight freeze thaw cycles 
from 0.5 to 8 ul/m mole egg PC, respectively. Increasing the number of 
freeze-thaw cycles from 8 to 20 resulted in no significant increase in 
FATMLV trapped volume. 
EXAMPLE 5 
FIG. 5 shows the effect of cholesterol content on the trapped volume of 
FATMLV's of the present invention. Varying amounts of egg PC and 
cholesterol as indicated in FIG. 6 were dissolved in 0.5 ml chloroform and 
dried to a thin film on the walls of glass test tubes utilizing a stream 
of nitrogen and high vacuum. When no cholesterol was present, the 
concentration of egg PC was 50 mg/ml. MLV's were then prepared in the 
presences of 20 mM Hepes, 150 mM sodium chloride (pH 7.5) containing 
.sup.3 H-inulin as described in Example 1. FATMLV's were produced by 
freeze thawing the MLV's five times employing liquid nitrogen and a 
40.degree. C. constant temperature bath. Removal of unentrapped .sup.3 
H-inulin and determination of vesicle trapped volumes were accomplished as 
described in Comparative Example 2. 
EXAMPLE 6 
FATMLV's using tobramycin phosphate (100 mg/ml), egg PC (300 mg/ml), at a 
pH of less than 3, preferably between 2 and 3, resulted in a 43.1% 
trapping efficiency. Trapping efficiencies can be increased by reducing 
the tobramycin phosphate concentration. Tobramycin does not act as an 
ideal aqueous marker because tobramycin interacts with the PC headgroup. 
The procedures of Example 1 were employed. An aqueous solution (1 ml of 
tobramycin adjusted to a pH of 2.0 with phosphoric acid was added to 300 
mg of egg PC and dispersed with vortexing. The samples were freeze-thawed 
five times; sized to 400 nm with five passes through two stacked 400 nm 
pore-sized polycarbonate filters at a pressure of 200 p.s.i., 
freeze-thawed an additional two times and extruded an additional five 
times through two stacked polycarbonate filters having a 400 nm pore size. 
A sized tobramycin-liposome composition resulted. 
EXAMPLE 7 
FATMLV's were prepared from egg PC (400 mg/ml), pilocarpine hydrochloride 
(40 mg/ml, pH=4.1) according to the procedure of Example 1. The FATMLV had 
a trapping efficiency of 88.6 percent and a trapped volume of 1.77 ul/umol 
phospholipid. Homogeneously sized vesicles were prepared by extruding the 
FATMLV ten times through two stacked polycarbonate filters with a 50 nm 
pore size. Sized pilocarpine-containing vesicles resulted. 
TABLE 1 
__________________________________________________________________________ 
Physical Characteristics of MLV's, Westman FATMLV's and FATMLV's 
of the Present Invention 
% .sup.31 P NMR 
Trapping 
Signal 
Lipid Concentration 
Trapped Volume 
Efficiency 
Removed 
Sample (mg/ml) (ul/umol lipid) 
(%) with Mn.sup.2+ 
__________________________________________________________________________ 
MLV 100 0.47 0.03 
5.8 5.0 
Westman FATMLV 
50 1.92 0.5 12.0 14.0 
(0 mg tetracaine) 
Westman FATMLV 
50 2.23 13.9 18.5 
(2 mg tetracaine) 
Westman FATMLV 
50 3.44 21.5 18.8 
(4 mg tetracaine) 
FATMLV 50 5.02 .+-. 0.04 
31.3 16.6 
FATMLV 100 5.27 .+-. 0.17 
65.9 14.7 
FATMLV 200 3.07 .+-. 0.05 
76.7 7.4 
FATMLV 400 1.77 .+-. 0.09 
88.6 7.2 
__________________________________________________________________________ 
TABLE 2 
______________________________________ 
Trapped Trapping 
Volume Efficiency 
Sample Freezing Media 
(ul/umol EPC) 
(%) 
______________________________________ 
EPC (powder) 
dry ice/ETOH 
3.44 21.5 
EPC (film) 
dry ice/ETOH 
3.62 22.6 
EPC (powder) 
liquid nitrogen 
6.81 42.5 
EPC (film) 
liquid nitrogen 
5.54 34.6 
______________________________________ 
TABLE 3 
______________________________________ 
Size Distributions of Extruded Vesicles.sup.a 
Mean Diameter .+-. S.D. (nm) 
Freeze-fracture 
Quasielastic 
Filter pore size (nm) 
electron microscopy.sup.b 
light scattering.sup.c 
______________________________________ 
400 243 .+-. 91 N.D..sup.d 
200 151 .+-. 36 179.9 .+-. 55 
100 103 .+-. 20 138.7 .+-. 36 
50 68 .+-. 19 73.8 .+-. 18 
30 56 .+-. 17 63.1 .+-. 17 
______________________________________ 
.sup.a MLV's (100 mg EPC/ml) were frozen and thawed 5 times prior to the 
extrusion process. These FATMLV's were then passed 20 times through 2 
(stacked) polycarbonate filters of the indicated pore size. 
.sup.b Size distribution analysis of vesicle employing freezefracture 
electron microscopy were completed as described in Materials and Methods. 
Mean diameters and S.D. were determined by measuring &gt;150 vesicles. 
.sup.c Size distribution analysis of vesicles employing quasielastic ligh 
scattering were completed as described in Materials and Methods. 
.sup.d Statistical analysis of the data yielding low fit error could not 
be accomplished. A mean vesicle diameter value is therefore not reported.