Method for making liposomes of enhanced entrapping capacity toward foreign substances to be encapsulated

Liposome vesicles are prepared containing water or very dilute solutions encapsulated therein. These "empty" liposomes are suspended in a carrier liquid containing, dispersed therein, substances of interest to be loaded into the vesicles and incubated for a period of time at temperatures above the lipids transition temperature, whereby loading by transmembrane permeation occurs in high yields.

The present invention concerns liposomes with improved trans-membrane 
loading capacity. It also concerns a method for making such liposomes and 
the loading thereof with substances of interest. 
As is well known, liposomes consist of small vesicles bounded by a membrane 
wall of lameliar lipids surrounding a core filled with an entrapped 
aqueous liquid phase. Liposome vesicles can be loaded with foreign 
substances like drugs and may thereafter be used to selectively deliver 
such encapsulated drugs to selected organs in the body. Liposomes, when in 
suspension in aqueous carriers, are particularly suitable to deliver drugs 
to patients by parenteral, peroral, topical and inhalation routes. 
Liposome drug formulations may improve treatment efficiency, provide 
prolonged drug release and therapeutic activity, increase the therapeutic 
ratio and may reduce the overall amount of drugs needed for treating a 
given kind of ailment or disorder. For a review, see Liposomes as Drug 
Carriers by G. Gregoriadis, Wiley & Sons, New-York (1988). 
Many methods exist for preparing liposomes and loading them with foreign 
substances of interest, most of which methods involve forming the liposome 
vesicles within an aqueous carrier liquid containing said substances 
distributed therein. During liposome formation, a portion of said carrier 
liquid becomes entrapped within the vesicles, together of course, with a 
small amount of the desired substances to be encapsulated. This technique 
is called "passive entrappment". The efficiency of loading liposomes with 
passively entrapped aqueous phases is often quite low because it strongly 
depends on the nature of the carrier phase and, particularly, the 
concentration of the substances dissolved therein which may affect the 
yield of liposome formation, However, for drug delivery purposes, the 
loading efficiency (which is generally defined as the weight of material 
entrapped over the total weight of material involved in entrappment) is 
usually not critical because the non-entrapped material can generally be 
recovered and reused afterwards; hence, the important factor is rather the 
ratio of useful entrapped material versus the weight of the lipids used 
for entrappment, i.e., the lipids involved in forming the liposomes 
membrane. Clearly, minimizing lipid dead-weight upon injection or 
otherwise, i.e. keeping the weight of vector drug carriers administered to 
patients to the lowest possible level for a given amount of 
therapeutically active species is a strong asset in the development of new 
pharmaceuticals or diagnostic reagents. Now, obviously, the ratio of the 
weight of encapsulated material over the weight of encapsulating lipids is 
in direct relation with the so-called captured volume, i.e. the volume of 
the aqueous phase entrapped in the liposomes core per weight of liposome 
lipids (.mu.l/mg). 
In a classical passive entrappment method described by BANGHAM et al., (J. 
Mol. Biol. 12, (1965), 238), the aqueous phase containing the compound of 
interest is put into contact with a film of dried phospholipids deposited 
on the walls of a reaction vessel. Upon agitation by mechanical means, 
swelling of the lipids will occur and multilamellar vesicles (MLV) will 
form. The captured volume of MLV's is low, typically near 2 to 4 .mu.l/mg 
of lipids. By sonication, the MLV's can be converted to small unilamellar 
vesicles (SUV) whose captured volume is even smaller, e.g., near 0.5-1 
.mu.l/mg. Other methods of preparation giving liposomes with larger 
captured volume have been described, particularly large unilamellar 
vesicles (LUV). For instance, DEAMER & BANGHAM (Biochim. Biophys. Acta 
443, (1976), 629) have described a method in which membrane forming lipids 
are dissolved in ether and, instead of first evaporating the ether to form 
a thin film on a surface, this film being thereafter put into contact with 
an aqueous phase to be encapsulated, the ether solution is directly 
injected into said aqueous phase and the ether is evaporated afterwards, 
whereby liposomes with captured volumes of 14 .mu.l/mg were obtained. Also 
the Reverse Phase Evaporation (REV) method described by SZOKA & 
PAPAHADJOPOULOS (P.N.A.S. 75, (1978), 4194) in which a solution of lipids 
in a water insoluble organic solvent is emulsified in an aqueous carrier 
phase and the organic solvent is subsequently removed under reduced 
pressure, gave liposomes with captured volumes of 8-15 .mu.l/mg of lipids. 
Improved passive entrappment has been achieved by subjecting liposomes to 
successive dehydration and rehydration treatment, or freezing and thawing; 
dehydration was carried out by evaporation or freeze-drying. This 
technique is disclosed for example by KIRBY & GREGORIADIS (Biotechnology, 
November 1984, 979-984). Also, SHEW & DEAMER (Biochim. et Biophys. Acta 
816 (1985), 1-8) indicate that liposomes prepared by sonication are mixed 
in aqueous solution with the solute to be encapsulated, and the mixture is 
dried under nitrogen in a rotating flask. Upon rehydration, large 
liposomes are produced in which a significant fraction of the solute has 
been encapsulated. 
Further attempts to increase the amount of substance entrapped in liposomes 
by using higher concentrations thereof in the carrier liquid have been 
brought about with little success. Indeed, as said before, the captured 
volume often decreases at high solute concentrations in the carrier phase 
which indicates that the presence of the substances to be entrapped in 
high concentrations has a detrimental effect on captured volumes. For 
instance, SZOKA et al.(loc.cit.) have reported a progressive decrease in 
the entrappment of cytosine arabinoside with increasing concentrations of 
NaCl in the carrier liquid. A similar situation is described in 
WO-A-89/11272 (MINCHEY et al.) according to which a drastic decrease in 
cephalosporin entrappment yield occurs with increasing the drug 
concentration in the carrier liquid. 
According to another route for filling liposomes with foreign non-lipidic 
substances, conditions are provided under which such substances can 
penetrate into the vesicle core through its walls; this technique, called 
"transmembrane loading", involves internalizing the substances to be 
encapsulated into the liposome vesicles after the latter have been formed. 
Normally, the crossing over of the lipid membrane by foreign substances 
(particularly ionic) is difficult because the incoming substances are 
repelled by the polar groups of said lipids. However this effect can be 
minimized by incorporating "shield" carriers to the lipid membrane. For 
instance, liposomes can be loaded with cations at room temperature when 
the lipid membrane contains a lipophilic carrier such as acetylacetone 
(BEAUMIER et al., J. Nucl. Med. 32 (1982) 810). Otherwise, foreign 
substances may be internalized into liposomes by osmotically controlled 
permeation through the lipidic membrane wall. For instance, the uptake of 
foreign substances by the liposomes can be promoted by a transmembrane 
ionic gradient, e.g. a Na.sup.+ /K.sup.+ gradient as disclosed in J. 
Biol. Chem. 260 (1985), 802-808. A pH gradient is also effective for 
promoting transmembrane loading as mentioned in Biochim. Biophys. Acta 857 
(1986), 123-126, WO-A-89/04656 and PCT/US85/01501. However, this technique 
is limited to some specific categories of drugs, more particularly weak 
bases, as acknowledged in Chem. Phys. Lipids 53 (1990), 37. Furthermore, 
making liposomes in a carrier phase of pH different from that of the core 
phase is difficult and, in addition, too low or too high a pH may cause 
membrane damage due to premature hydrolysis of the lipids. 
In EP-A-361.894 (THE HEBREW UNIVERSITY), there is disclosed a technique in 
which amphipatic drugs are loaded into liposomic vesicles by transmembrane 
internalization under the control of a post-generated pH gradient. The key 
feature of this technique depends on the leakage of ammonia (NH.sub.3) 
from the core of liposome vesicles loaded with an aqueous solution of an 
ammonium compound and placed in an ammonium-free carrier medium. Leakage 
of NH.sub.3 from NH.sub.4.sup.+ releases a proton with consecutive 
lowering of the pH of the entrapped liquid and consecutive establishment 
of a pH gradient across the liposome membrane, i.e. the carrier liquid 
becomes alkaline relative to the internal content of the liposome core. 
When an amphipatic compound (e.g. a drug with a deprotonated amine group) 
is added to the "alkalinized" carrier liquid, the system will tend to 
reequilibrate and a diffusion of said amphipatic compound into the core of 
the liposomes through the lipid membrane will occur. 
Techniques in which dehydrated and rehydrated liposomes are subjected to 
transmembrane loading also exist. For example, U.S. Pat. No. 4,673,567 
(SHIONOGI & Co.) discloses preparing "empty" MLV liposomes in an ion-free 
aqueous carrier liquid and dehydrating these liposomes by lyophilization; 
then the dried liposomes are rehydrated by suspending in a carrier liquid 
containing a drug like Fluorouracil, Cefalexin or the like, and incubation 
is carried out by heating for 5 min at 50.degree. C., whereby a 
significant portion of the drug dissolved in the carrier liquid becomes 
entrapped in the liposomes. The rationale behind this approach is that 
"freeze-drying liposomes produces structural defects in the bilayer 
membrane and heating above the transition temperature removes these 
defects" as acknowledged in an article by H. JIZOMOTO et al. in Chem. 
Pharm. Bull. 37 (1989), 3066-3069. However, as indicated in U.S. Pat. No. 
4,673,567, this method is hampered by a considerable reduction in the 
captured volume when the carrier liquid contains ionic solutes. For 
instance, from the data reported in Table 1, col.3 of this document, when 
using isotonic brine or 0.02 phosphate buffer as the carrier liquid, the 
transmembrane drug take-up was practically negligible, whereas when the 
drug was dissolved in pure Water a value of captured volume of 16.6 
.mu.l/mg of lipid was reported. Furthermore, it should be realized that in 
current practice, high values of captured volumes are not easily 
attainable. For instance, in a recent survey article: "The accumulation of 
drugs within large unilamellar vesicles exhibiting a proton gradient", by 
T. D. MADDEN & al., in Chemistry and Physics of Lipids 53 (1990), 37-46, 
the quoted captured value does not exceed about 1-2 .mu.l/mg of 
phosphatidylcholine. 
It can be seen from the foregoing brief summary that the techniques of the 
prior art for loading liposomes are complicated, expensive and not 
generally applicable to all types of drugs and media administrable via 
liposomes, namely ionic species are generally difficult to entrap. It was 
therefore the aim of the present inventors to increase the captured volume 
significantly, although avoiding tedious and expensive pretreatments of 
the film forming lipids (e.g. lyophilization as taught by H. JIZOMOTO in 
Chem. Pharm. Bull. 37 (1989), 1895-1898) and, simultaneously, efficiently 
condition the membrane forming lipids for enhancing the trans-membrane 
loading capacity toward substantially all kinds of solutes in aqueous 
media including ionic species. This has now been accomplished by embodying 
the method disclosed in the annexed claims which appears to be based on an 
osmotically controlled permeation process. 
In brief, in the present invention, one prepares liposomes by any available 
method, said liposomes, as made, being "empty". By "empty" liposomes, one 
wishes to say that the aqueous phase entrapped therein is only pure water 
or, otherwise, is made of very dilute solutions of non-ionic substances or 
electrolytes. Generally speaking, if solutes are present in the entrapped 
phase of the newly prepared "empty" liposomes, the osmolality thereof 
should not exceed about 0.2 Osm/kg; if the solutes are electrolytes, the 
ionic strength of the entrapped liquid should not exceed about 0.1. Then, 
once the so-called "empty" liposomes have been made, they are suspended in 
a carrier liquid containing one or more substances of interest to be 
encapsulated, and one proceeds to incubate the system at a temperature 
above the transition temperature T.sub.c for a time sufficient to ensure 
efficient vesicle loading by transmembrane permeation. 
It should be stressed at this stage that one of the chief factors in this 
invention relates to the absence, or the presence in only minute 
quantities, of solutes in the aqueous phase where the liposomes are 
initially prepared, particularly when the solutes are electrolytes. In 
this connection, it has been noted that the "quality" of the liposome 
vesicles, that is the extent of their ability to generate as high as 
possible a captured volume, strongly depends on the ionic strength of this 
aqueous liquid phase; naturally, this aqueous phase is also that one which 
is trapped within the core of the nascent "empty" liposomes at the time 
when they form. This situation contrasts strongly with the teaching of the 
prior art (e.g. U.S. Pat. No. 4,673,567) where the captured volume is 
affected only by ions present outside the liposome vesicles, i.e, the ions 
within the carrier liquid in which the liposomes are incubated after 
dehydration. Hence the fundamental unexpected feature now discovered by 
the present inventors is that the transient permeability of the membrane 
of the newly prepared liposomes depends mainly on the nature of the liquid 
in which the "empty" liposomes have been prepared initially, not on the 
liquid used for the post-incubation treatment. Actually, there is an 
inverse relationship between the concentration of electrolyte in the 
liquid used for making the empty liposomes and the captured volume of 
foreign substances encapsulated subsequently. The leaner this liquid, the 
higher the captured volume. 
For instance, in one embodiment of the invention, membrane-forming lipids 
are admixed with an aqueous liquid carrier, for instance water at pH of 1 
to 12, but preferably around neutrality, optionally containing diluted 
buffers and/or non-ionic stabilizers such as sugars or other polyols, and 
the carrier is maintained for a period of time at a temperature of a few 
degrees C. above the crystal/liquid transition temperature (T.sub.c) of 
the hydrated lipid, this being preferably within a narrow range of 
temperature values. The span of this range can be of about 20.degree. C., 
and the most preferred temperature is in the vicinity of the mid-point of 
this range, i.e. about 4.degree. to 10.degree. C. above T.sub.c. The time 
required to effect efficient hydration and conditioning of the lipids 
corresponds to the time required to obtain a homogeneous solution or 
dispersion thereof in the carrier phase, agitation being optional. 
Generally, a gentle swirl of the liquid is sufficient to ensure 
homogenization, but faster stirring is also possible if desired. It should 
be noted that the average size of the liposomes which form during 
hydration and conditioning of the lipids may depend on the rate and the 
mode of agitation. Generally, very slow agitation leads to liposomes of 
larger average size and internal capacity than when operating under more 
violent agitation. It should also be noted that all preliminary treatments 
of the lipids recommended in the prior art to increase the loading 
capacity of the liposomes, i.e. freeze-drying, thawing, evaporating from a 
solution into thin films on the walls of a laboratory flask, and other 
alike pretreatments, although harmless, are absolutely unnecessary in the 
method of the present invention; however, since these preliminary 
treatments are not harmful, they can be performed if desired. 
Irrespective of the ionically charged component, the lipids or mixture of 
lipids to be used in the present invention substantially include all 
compounds commonly used in the field of liposomes, i.e. 
glycerophospholipids, non-phosphorylated glycerides, glycolipids, sterols 
and other additives intended to impart modified properties to liposomic 
membranes. Preferably, they comprise at least a polarizable component 
(even in minor quantity), namely a cationic or anionic function carrying 
lipid or an ionizable tenside such as a fatty alcohol diphosphate ester, 
e.g. dicetyl phosphate (DCP) or a higher alkyl amine like stearylamine 
(SA). Charged phospholipids, i.e. fatty acid glycerides phosphatides like 
phosphatidic acid (PA), phosphatidylglycerol (PG), phosphatidyl-inositol 
(PI), phosphatidyl-serine (PS) from natural sources or synthetic (such as 
dipalmitoyl-phosphatidic acid (DPPA), dipalmitoyl-phosphatidyl glycerol 
(DPPG), etc.) are convenient polarizable lipid components. The 
glycerophospholipids may include for instance the following synthetic 
compounds: Dipalmitoyl-phosphatidyl-choline (DPPC), 
dipalmitoyl-phosphatidyl-ethanolamine (DPPE) and the corresponding 
distearoyl- and dimyristyl- phosphatidyl-choline and -ethanolamine (DSPC; 
DSPE; DMPC and DMPE). Phospholipids may also include natural phospholipids 
which have been subjected to more or less extensive hydrogenation, for 
instance egg and soy phosphatidylcholine. 
The glycolipids may include cerebrosides, galactocerebrosides, 
glucocerebrosides, sphingomyelins, sulfatides and sphingolipids 
derivatized with mono-, di- and trihexosides. The sterols, which should be 
used with parsimony, as too much may impede membrane permeation, encompass 
cholesterol, ergosterol, coprostanol, cholesterol esters such as 
hemisuccinate (CHS), tocopherol esters and the like. 
In order to perform the method of the invention, a proportion of lipid or 
mixture of lipids, with or without additives, is admixed into a volume of 
non-ionic aqueous liquid (or an aqueous liquid whose ionic strength does 
not exceed 0.1 and the osmolality of which does not exceed 0.2 Osm/kg), 
and hydration of the lipids is allowed to proceed until the mixture is 
homogeneous, by which time the desired liposome vesicles will have been 
formed. When the liquid phase is essentially water, the relative 
proportions of the lipids and the aqueous liquid are not critical but, 
evidently, a minimum of liquid is necessary to ensure correct a dispersion 
of the lipids therein. Normally, for 1 part by weight of lipids or 
mixtures of lipids and membrane forming additives, there is used at least 
20 parts of liquid phase. Excellent results are however observed with 
smaller lipid to liquid weight ratios, e.g. in the order of 0.1 to 1%. If 
the aqueous liquid used to make the empty liposomes is thereafter used as 
the carrier phase for incubation (i.e. the substance to be encapsulated is 
simply added to the liquid in which the liposomes have been made), it is 
obviously preferable that the amount of the liquid relative to the lipids 
be not too great, as this would lead to useless dilution of the substance 
to be encapsulated and lower entrapping yields. Notwithstanding, in case 
the liposome dispersion is too dilute, concentration of the vesicles can 
be brought about by centrifugation (in the range of 10.sup.3 to 10.sup.5 
g), or by partial evaporation of the liquid, or by ultrafiltration through 
suitably calibrated semi-permeable membranes. 
After the lipids have been added to the liquid phase, the system is allowed 
to homogenize upon standing with occasional shaking or under more constant 
agitation. The temperature at which this operation is brought about has 
been defined already before. The liquid may be raised to the desired 
temperature before or after adding the lipids. The preferred temperature 
will naturally depend on the kind of lipids or mixture of lipids used; 
however for the most commonly used lipids and lipid mixtures, the 
hydration and homogenization temperature will be selected in a range from 
about 40.degree. C. to 80.degree. C. 
The compositions of the liquid phases in which the liposome vesicles are 
generated to be embodied in this invention are very many. Besides pure 
water, solutions of diluted electrolytes like mineral salts, or of 
non-ionic species such as glycols or polyols stabilizers can be used. For 
example, the following non-ionic stabilizers can be mentioned: sugars like 
sucrose, glucose lactose and maltose, polyols like glycols, glycerol, 
sorbitol, mannitol, polyethylene-glycol, dextran, xanthan and the like. 
The time necessary to achieve hydration and conditioning of the lipids into 
liposomic vesicles of outstanding encapsulating properties may vary from a 
few minutes to several hours at the desired temperature, but heating times 
not exceeding about 30 to 60 min are generally preferred. 
Naturally, the method of the invention also applies to initial conditions 
for contacting the lipids and the aqueous carrier other than merely 
admixing the components together. For instance, as said before, other 
routes for making liposomes can be applied as well, e.g., first forming a 
lipid film on surfaces (like that of a round bottom flask, or of glass 
beads, or the interstitial surface of a bundle of wirelike materials) and 
then contacting or circulating an aqueous phase on said lipid film until 
hydration of the latter becomes effective. If desired, hydration with 
sonication can be effected. It has however been observed that the simplest 
preparation embodiments of the present invention lead to liposomes with 
the highest entrapping capacity. 
The liposomic vesicles obtained according to the invention are generally in 
a range of sizes of about 80 nm to about 5 .mu.m, sizes in the vicinity of 
300 to 2000 nm being preferred when therapeutic or diagnostic applications 
by injection are considered. These liposomes are preferably of the MLV 
type, but other kinds of liposomes can also be made depending on the 
choice of operational parameters. The size distribution of these liposomes 
is usually rather wide but, if narrower size distributions are desired, 
calibration techniques such as filtration under pressure or extrusion 
through microporous membrane can be successfully applied. It should be 
noted in this connection that the calibration of empty liposome versus 
that of loaded liposomes is advantageous because no substance being 
entrapped in the liposomes yet, no leakage thereof can occur during 
extrusion. Also extrusion of empty liposomes is easy because of their 
inherent low viscosity. Hence extrusion is preferably performed below Tc, 
e.g. at room temperature, or below, which provides optimal entrappment 
yields (high captured volumes) in the subsequent transmembrane loading 
steps. Furthermore, in the present invention, the type, size and 
concentration of the empty liposomes can be adapted to the needs before 
incubation, no loss of entrapped substance being involved in these 
operations. 
However, the fundamental advantage of the liposomes obtained in the present 
invention relates to their surprising loading capability toward most 
foreign substances to be encapsulated. This loading can be easily 
performed by simply incorporating the substance to be encapsulated in the 
liquid in which the liposomes have been formed or in another liquid 
carrier in which the empty liposomes are subsequently suspended, this 
liquid serving as the carrier phase for incubation with the substances to 
be encapsulated. The substances to be encapsulated are being brought 
either neat or in the form of solutions. Then, incubation of the system is 
carried out for a period of time, at a temperature above the lipid 
transition temperature T.sub.c. When the substance to be encapsulated is 
used neat, it will first dissolve in the liquid serving as carrier and 
from there it will permeate through the membrane and penetrate into the 
liposome core. A similar process will occur if the foreign substance is 
added in solution; here, the incubation carrier liquid will first become 
diluted by said solution and the dissolved substance will then penetrate 
into the liposomes as mentioned before. It is particularly interesting to 
note that, in contrast with the prior art, the vesicle transmembrane 
loading mechanism inherent to the present invention occurs satisfactorily 
even when ions are present in the carrier liquid used for incubation, said 
ions being either constituents of the incubation carrier itself (buffers 
or saline) or of the substances of interest to be encapsulated. It appears 
that when the water initially captured by the liposome vesicles is either 
pure or contains substances in low concentration, the inhibition to 
permeation noted in the prior art is overcome. What is particularly 
surprising in this invention is once a portion of an ionic substance has 
permeated the membrane during incubation, it does not inhibit the 
penetration of the remaining portion still in the carrier liquid. 
The time of incubation may vary in function to the rates of permeation into 
lipids typical of the substances to be encapsulated, the nature and 
concentrations of the liposomes in the carrier phase, and the temperature 
of incubation. The factor that will generally determine the end of the 
incubation time is the condition where the concentrations of the 
encapsulated substances are the same inside and outside the liposomes. At 
this moment, equilibrium has been reached and prolonging incubation has no 
further purpose. Of course, the higher the temperature, the faster 
equilibrium is established; however too high temperatures may be 
detrimental to the liposome properties, namely to the specific 
encapsulation capacity, i.e., the ratio of core volume to weight of 
lipids; hence the incubation temperatures may range from about T.sub.C to 
about 150.degree.-200.degree. C., the preferred range being from about 
40.degree. to 130.degree. C. It should be noted in this connection that if 
the incubation temperature is in the high portion of the given range, say, 
100.degree. to 150.degree. C., substantial sterilization of the liposomes 
will occur simultaneously with incubation. Alternatively, one may effect 
sterilization and incubation independently and subsequently. The heating 
means to bring the liposomes and the products to be encapsulated to 
incubation temperatures are conventional in the field and naturally 
include microwave heating means. It should however be remarked that the 
temperature of initially hydrating and conditioning the lipids for 
liposome formation is not to be confused with the incubation temperature, 
although both can be identical in some embodiments. Actually, the 
hydration temperature range is much narrower than the incubation 
temperature range; if hydrating and conditioning were carried out outside 
the given range, say at about 80.degree. C. with lipids or mixtures 
thereof having a T.sub.C around 45.degree.-55.degree. C., liposomes of 
inferior quality would be obtained, i.e., with lower entrapping capacity 
and lower volume to weight ratio. 
It should be noted that one further advantage of the present invention is 
that the concentration of lipids in the aqueous carrier used for 
incubation has no significant influence on the internalization capacity 
and efficiency of the liposomes toward foreign substances added to said 
aqueous carrier. Hence by concentrating the liposomes in the aqueous 
carrier, i.e. by increasing the lipid to carrier weight ratio, one may 
favorably influence the entrapment yield and reduce the amount of residual 
non entrapped substance to be recovered and reused afterwards. This can be 
illustrated by remarking that the ultimate concentration of the foreign 
substances in the liposome core only depends on the initial concentration 
thereof in the incubation carrier liquid, not on the total weight of the 
foreign substances used for encapsulation. Hence this total weight can be 
reduced for a given concentration by decreasing the amount of liquid used 
for incubation and, conversely, increasing the concentration of lipids in 
the carrier phase will lead to an increase of entrappment yield. 
The substances to be entrapped in the liposomes according to the invention 
include any imaginable therapeutically or diagnostically active compounds. 
As such, one may recite drugs like analgesics, narcotics, antibiotics, 
sulfamides, steroids, . . X-ray opacifiers, NMR contrast agents and the 
like. X-ray opacifiers include for instance organic iodinated compounds 
like 
N,N'-bis[2-hydroxy-l-(hydroxymethyl)-ethyl]-5-[(2-hydroxy-1-oxopropyl)-ami 
no]-2,4,6-triiodo-1,3-benzene-dicarboxyamide (iopamidol); metrizamide; 
diatrizoic acid; sodium diatrizoate; meglumine diatrizoate; acetrizoic 
acid and its soluble salts; diprotrizoic acid; iodamide; sodium 
iodipamide; meglumine diopamide; iodohippuric acid and the soluble salts 
thereof; iodomethamic acid; iodopyracetiodo-2-pyridone-N-acetic acid; 
3,5-diiodo-4-pyridone-N-acetic acid (Iodopyracet) and its diethyl ammonium 
salt; iothalmic acid; metrizoic acid and its salts; the ipanoic, iocetamic 
and iophenoxic acids and their salts; sodium tyropanoate; sodium opidate 
and other like iodised compounds. 
The following examples illustrate the invention:

EXAMPLE 1 
Thirty g of phospholipids (a 9/1 molar ratio of hydrogenated soy lecithin 
(Phospholipon 100H from NATTERMANNPHOSPHOLIPID GmbH, Koln, Germany) and 
dipalmitoylphosphatidic acid disodium salt (DPPA) with a trace amount of 
.sup.14 C-labeled tripalmitin (Amersham) in solution in chloroform (250 
ml) were introduced in a 10 1 reaction flask. After evaporation of the 
chloroform under reduced pressure, there were added 6 l of distilled water 
at 55.degree.-60.degree. C. (the transition temperature of the hydrated 
lipids used was 54.degree. C. as determined by differential scanning 
calorimetry) and the solid lipids were allowed to hydrate and distribute 
homogeneously through the liquid with occasional gentle shaking, whereby 
liposomes of the MLV type did form in high yield. After about 1 hour, the 
liposome suspension containing 5 mg/ml of lipids was extruded at 
60.degree. C. through a 2 .mu.m polycarbonate membrane (Nuclepore) and, 
after cooling to room temperature, it was concentrated to 30 mg/ml by 
microfiltration using a 0.22 .mu.m microfilter (Millipore). 
To the concentrated liposome solution, there was added 1 l of an aqueous 
solution containing 1040 g of (S)-N,N'-bis 
[2-hydroxy-1-(hydroxymethyl)-ethyl]-2,4,6-triiodo-5-lactamido-isophtalamid 
e (Iopamidol, an X-ray contrast agent produced by BRACCO INDUSTRIA CHIMICA, 
Milano) i.e. 520 g/l of covalent iodine at 60.degree. C. The resulting 
mixture (2 l) had an iodine concentration of 260 g/l and was incubated for 
about 30 min at 60.degree. C., after which time the iodine concentration 
outside and inside the liposome core had equalized. The resulting 
preparation was concentrated to 30 g lipids/l (Preparation A). 
Preparation A was analyzed for lipids and encapsulated iodine. For this, an 
aliquot (1 ml) was dialyzed against saline (NaCl 0.9% in water) until all 
iopamidol outside the liposomes vesicles had been removed (about 24 hours 
with 4 changes of the dialysis medium). The sample was then treated at 
50.degree. C. with 1/10th of its volume of a 10% sodium dodecyl sulfate 
solution in water and the liberated Iopamidol was measured 
spectrophotometrically at 260 nm. The corresponding amount of lipids was 
determined by counting with a scintillation counter using the residual 
radioactivity of the tripalmitin tracer. The results of the foregoing 
analysis showed that the encapsulation capacity measured as the 
iodine-to-lipid ratio (I/L) was consistently in the range of 3 to 5 mg (or 
more) of entrapped iodine per mg lipid, which means that the average 
internal captured volume of the liposome vesicles (calculated on the basis 
of an iodine concentration of 260 mg/ml) was about 12-19 .mu.l/mg of lipid 
(or more). 
Part of the preparation A of contrast agent-loaded liposomes of this 
example was diafiltered against buffered saline (0.9% NaCl, 10 mM 
Tris.HCl, pH 7.2) containing Na.sub.2 Ca EDTA (0.9 mM) using a 0.22 .mu.m 
membrane (Millipore). The resulting preparation (Prep B) as well as 
preparation A were usable directly for injection into the bloodstream of 
experimental animals, both of them providing x-ray opacification of blood 
vessels and organs (e.g. liver and spleen) with extremely favorable 
results. 
When, in the foregoing example, the Iopamidol was replaced by Iomeprol 
(N,N'-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-5-glycolamido-isophtalimide), 
another iodinated contrast agent from BRACCO INDUSTRIA CHIMICA (Milano), 
similar entrapping results were experienced. When in the foregoing 
example, the Iopamidol was replaced by B17500, an experimental non-ionic 
dimer made by BRACCO, I/L values in excess of 6, exceeding sometimes 7 
were obtained. Similar results were observed with Iotrolan, a non-ionic 
dimer produced by SCHERING AG. 
EXAMPLE 2 
REV liposomes were prepared according to the method of Szoka et al. (Proc. 
Natl. Acad. Sci. USA 75 (1978), 4194). Briefly, hydrogenated soy lecithin 
(Phospholipon 90H from NATTERMANN PHOSPHOLIPID GmbH, 912.2 mg) and DPPA 
(92.9 mg) were dissolved in 80 ml of a 1:1 mixture of chloroform and 
isopropylether. To this, were added 30 ml of distilled water and the 
mixture was emulsified by sonication (5.times.1 min.) using a Branson 
probe sonifier, while maintaining the temperature at 45.degree. C. Then 
the emulsion was evaporated at 45.degree. C. under reduced pressure in a 
rotary evaporator. After the evaporation of residual solvents was 
complete, and a small amount of distilled water had been added, a 
suspension of REV liposomes with 33 mg lipid/ml and an average size of 0.4 
.mu.m was obtained. Iopamidol (1.4 g) was dissolved in 2 ml of the 
suspension and the solution was incubated for 1 hour at 80.degree. C. I/L 
values (measured as described in Example 1) of 2.1-2.3 were obtained. 
Lower entrappment yields were obtained when there was used, for comparison, 
a Iopamidol solution (30 ml, 260 mg iodine per ml) instead of pure water 
for initially emulsifying with the organic solution of lipids. 
These experiments were repeated with REV liposomes prepared using 
dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidyl glycerol 
(DPPG) (molar ratio 9/1) with distilled water as the aqueous phase and 
then incubated with sodium diatrizoate (215 or 21.5 mg iodine/ml) for 20 
min. at 60.degree. C. For comparison, REV liposomes were also prepared 
with the same phospholipids using as the initial aqueous phase sodium 
diatrizoate solutions (215 and 21.5 mg iodine per ml, respectively) 
instead of distilled water. Although entrappment yields of the same order 
of magnitude were obtained with the two approaches i.e. I/L values of 
about 1, resp. 0.2 at 215, resp. 21.5 mg iodine/ml, the technique starting 
with empty liposomes gave still better results. 
EXAMPLE 3 
Liposomes of the SUV type were obtained by sonicating for 15 min. at 
60.degree. C., using a Branson probe sonifier, a suspension of MLV 
liposomes prepared in distilled water as described in Example 1. The 
supernatant obtained after centrifugation for 10 min. at 10'000 g was 
incubated with Iopamidol (final concentration 260 mg iodine/ml) for 20 
min. at 60.degree. C. An I/L value (measured as described in Example 1) of 
0.16-0.17 mg iodine per mg lipid was obtained, corresponding to a captured 
volume of 0.6 .mu.l/mg lipid. 
EXAMPLE 4 
MLV liposomes were prepared in distilled water as described in Example 1. 
Prior to extrusion, the liposome suspension was repeatedly frozen (at 
-75.degree. C.) and thawed (at 40.degree. C.) four times according to the 
method of Mayer et al. (Biochim. Biophys. Acta 817 (1985), 193). Both 
extruded (2 .mu.m) and non-extruded liposome suspensions were prepared and 
incubated at 60.degree. C. for 30 min. with a Iopamidol solution (260 mg 
iodine/ml). Extruded liposomes gave an I/L value of 5, whereas 
non-extruded liposomes gave an I/L value of 6.3. When, in a variant, 
extrusion was performed below the lipids transition temperature, e.g. at 
from room temperature to 50.degree. C., higher entrappment yields (I/L=8 
or more) were recorded. 
EXAMPLE 5 
Influence of temperature. 
MLV liposomes were prepared in distilled water as described in Example 1 at 
various temperatures, i.e. 55.degree., 60.degree., 65.degree., 70.degree., 
80.degree. C. They were incubated with Iopamidol (final concentration 260 
mg iodine/ml) at 60.degree. C. for 30 min. The following I/L values were 
obtained: 
______________________________________ 
Temperature of liposome 
I/L 
formation (.degree.C.) 
mg iodine/mg lipid 
______________________________________ 
55 4.2 
60 4.9 
65 4.5 
70 3.8 
80 3.1 
______________________________________ 
It can be seen that the optimal temperature for liposme formation is 
60.degree. C., i.e. 6.degree. above the transition temperature of the 
lipid mixture used (Phospholipon 100H and DPPA in a 9:1 molar ratio). 
The influence of the temperature of incubation was determined as follows: 
MLV liposomes were prepared in distilled water at 60.degree. C. as 
described in Example 1. Aliquots were then incubated at various 
temperatures with a Iopamidol solution (final concentration 260 mg 
iodine/ml). The following I/L values were obtained: 
______________________________________ 
Temperature of 
I/L 
incubation (.degree.C.) 
mg iodine/mg lipid 
______________________________________ 
40 3.0 
50 3.4 
55 4.7 
60 4.9 
65 4.1 
80 3.9 
______________________________________ 
The optimal temperature for incubation is in the range 
55.degree.-60.degree. C. i.e. 1.degree. to 6.degree. above the transition 
temperature of the mixture of lipids used. 
EXAMPLE 6 
MLV liposomes were prepared in distilled water at 60.degree. C. like in 
Example 1 with various lipid concentrations, then they were incubated for 
30 min. at 60.degree. C. with a iopamidol solution (260 mg iodine/ml) as 
described in Example 1. Aliquots were brought to 130.degree. C. for 
various time periods (see below), then rapidly cooled to room temperature. 
The following I/L values were measured: 
______________________________________ 
Duration of incubation 
I/L 
at 130.degree. C. (min.) 
mg iodine/mg lipid 
______________________________________ 
1 3.9 
2 3.8 
4 3.8 
6 3.7 
8 3.8 
10 3.6 
______________________________________ 
It can be concluded that the liposomes of the invention are not altered 
with regard to their loading capacity by exposure to sterilizing 
temperatures. 
EXAMPLE 7 
Influence of lipid concentration. 
MLV liposomes were prepared at 60.degree. C. in distilled water like in 
Example 1 using various lipid concentrations. Then they were incubated 
with a iopamidol solution (260 mg iodine/ml) as described in Example 1. 
The following I/L values were obtained: 
______________________________________ 
Lipid concentration 
I/L 
at formation (mg/ml) 
mg iodine/mg lipid 
______________________________________ 
2.5 3.8 
5.0 3.6 
10.0 3.5 
25.0 2.3 
50.0 1.9 
______________________________________ 
The best results are obtained at lipid concentrations of 2.5-10 mg/ml. 
MLV liposomes were prepared in distilled water at 60.degree. C. at a lipid 
concentration of 5 mg/ml. They were concentrated (between 5 and 35 mg 
lipid/ml) then incubated with a iopamidol solution (260 mg iodine/ml) as 
described in Example 1. The following I/L values were obtained: 
______________________________________ 
Lipid concentration 
I/L 
during incubation (mg/ml) 
mg iodine per mg lipid 
______________________________________ 
5 3.6 
9 3.8 
14 3.9 
18 4.0 
25 3.7 
35 3.7 
______________________________________ 
There is therefore no influence of the lipid concentration during 
incubation with Iopamidol on the trapping capacity. 
EXAMPLE 8 
MLV liposomes were prepared in distilled water, extruded through a 2 .mu.m 
membrane and concentrated to 35 mg lipid/ml as described in Example 1. To 
1 ml aliquots of the concentrated liposome suspension (but 3 ml in the 
case of Prep. A) were added 1 ml aliquots of the following solutions: 
Prep. A: Gd-DTPA (117 mM) labeled with a trace amount of .sup.153 Gd. 
Prep, B: a 4% lidocaine HCl solution in water adjusted to pH 7.2 with NaOH. 
Prep. C: a sodium diatrizoate solution (215 mg iodine/ml), 
Prep. D : a cis-platin solution in distilled water (10 mg/ml). 
Prep. E: an aqueous insulin solution (20 mg/ml) with pH adjusted to 7.5. 
Incubations were carried out at 80.degree. C. (Prep. A, Prep. B and Prep. 
C) or 60.degree. C. (Prep. D and Prep. E) during 30 min. The entrapped 
compounds were determined after dialysis by radioactive counting (Prep. 
A), HPLC (Prep. B), spectrophotometrically (Prep. C), atomic absorption 
(Prep. D). For Prep. E the non-entrapped insulin was removed by column 
chromatography on DEAE-A-50 Sephadex and the amount of entrapped material 
was measured by protein analysis (Method of Lowry). The following loadings 
and corresponding captured volumes were obtained: 
______________________________________ 
Sample Loading Captured volume 
______________________________________ 
Prep. A: 
0.25-0.35 .mu.mol Gd/mg lipid 
8.5-12 .mu.l/mg lipid 
Prep. B: 
0.35 .mu.mol lidocaine/mg lipid 
5 .mu.l/ml lipid 
Prep. C: 
0.8 mg iodine/mg lipid 
3.5 .mu.l/mg lipid 
Prep. D: 
5.9 .mu.g cis platin/mg lipid 
1 .mu.l/mg lipid 
Prep. E: 
0.18 mg insulin/mg lipid 
18 .mu.l/mg lipid 
______________________________________ 
High captured volumes were observed for all products tested. Prep. A was 
repeated replacing Gd-DTPA by Gd-BOPTA meglumine salt, a new contrast 
agent for MRI (code B-19030; formula: 
3-phenylmethoxy-2-N[2'-N'-{2"-N"-bis-(carboxymethyl)-aminoethyl}-N'-(carbo 
xymethyl)-aminoethyl]-N-(carboxymethyl)aminopropionic acid under 
developmetn at BRACCO and similar results were obtained. 
EXAMPLE 9 
Influence of the lipid composition. 
Various phospholipid mixtures were evaluated in a series of experiments 
carried out as described in Example 1. The following I/L values were 
obtained: 
______________________________________ 
Lipid composition (molar ratio) 
I/L 
______________________________________ 
Phospholipon 100H/DPPA.Na.sub.2 (9.9/0.1) 
3.0 
Phospholipon 100H/DPPA.Na.sub.2 (9.5/0.5) 
3.7 
Phospholipon 100H/DPPA.Na.sub.2 (9.25/0.75) 
4.2 
Phospholipon 100H/DPPA.Na.sub.2 (9/1) 
4.9 
Phospholipon 100H/DPPG (9/1) 4.8 
Phospholipon 100H/Cholesterol/DPPA.Na.sub.2 (4.5/4.5/1).sup.a 
1.3 
Phospholipon 100H/Cholesterol/DPPA.Na.sub.2 (6.75/2.23/1).sup.b 
2.2 
Phospholipon 100H 1.4 
Phospholipon 100H/Stearylamine (9/1) 
1.7 
DPPC/DPPA.Na.sub.2 (9/1).sup.c 
4.0 
DPPC/DMPC/DPPA.Na.sub.2 (4.5/4.5/1).sup.d 
3.6 
Phospholipon 100H/DCP.Na (9/1) 
3.0 
Phospholipon 90H/DSPA.Na2 (9/1) 
______________________________________ 
Legend: 
DPPG: dipalmitoyl phosphatidylglycerol sodium salt 
DPPC: dipalmitoyl phosphatidyl choline 
DMPC: dimyristoyl phosphatidyl choline 
DCP.Na: dicetylphosphate sodium salt 
The liposomes were prepared at the temperatures: 
.sup.a 40.degree. C. 
.sup.b 50.degree. C. 
.sup.c 50.degree. C. (i.e. 6.degree. C. above the transition temperature 
of the mixture of phospholipids). 
.sup.d 40.degree. C. (i.e. 4.degree. C. above the transition temperature 
of the mixture). 
EXAMPLE 10 
MLV liposomes were preared in distilled water as described in Example 1. 
After extrusion and concentration they were incubated with various 
concentrations of Iopamidol, in the absence (Series A) or presence (Series 
B) of NaCl. In the experiments of Series C, MLV liposomes were prepared 
directly in the iopamidol solution in the presence of various 
concentrations of NaCl. The following I/L values were obtained: 
______________________________________ 
Iopamidol conc. 
NaCl I/L capt. vol 
(mg I.sub.2 per ml) 
(mM) (mg I.sub.2 per mg lipid) 
(.mu.l/mg) 
______________________________________ 
Series A 
100 0 1.7 17 
200 0 2.8 14 
260 0 3.5 14 
300 0 4.0 13 
370 0 4.4 12 
Series B 
215 56 2.1 10 
215 565 0.7 3 
Series C 
215 56 1.8 8 
215 565 0.15 0.7 
______________________________________ 
Thus increasing iopamidol concentrations resulted into increased loading, 
with no major impact on the captured volume (Series A). The presence of 
salt reduces the loading as well as the captured volume (Series B). 
Nevertheless higher loadings are achieved with the technique of the 
invention compared to the classical MLV technique (Series C). 
EXAMPLE 11 
MLV liposomes were prepared in various aqueous solutions (instead of 
distilled water) at 60.degree. C., then after extrusion and concentration, 
they were incubated for 30 min. at 60.degree. C. with a iopamidol solution 
(260 mg iodine/ml) (see Example 1). The following I/L values were 
obtained: 
______________________________________ 
Medium used for the formation 
I/L 
of MLV's (mg I.sub.2 /mg lipids) 
______________________________________ 
Distilled water (as reference) 
4.2-4.4 
10 mM Tris/HCl pH 7.2, 0.9 mM EDTA 
2.9-3.1 
6 mM NaCl 3.0-3.3 
56 mM NaCl 2.2-2.4 
560 mM NaCl 0.7-0.8 
146 mM trehalose 2.1-2.3 
274 mM mannitol 1.5-1.8 
iomeprol solution (260 mg iodine/ml) 
0.7 
(calc. as iopamidol) 
______________________________________ 
As seen in these experiments, a decrease in the entrapment of Iopamidol is 
observed in all cases when the vesicles are formed in a medium containing 
already a solute. The presence of ionic species such as NaCl at ionic 
strengths above 0.1 or of non electrolytes at Osmolalities of more than 
200 mOsm/kg are particularly detrimental.