Method of loading preformed liposomes using ethanol

Details Method of loading preformed liposomes using ethanol Method of loading preformed liposomes using ethanol

1996-01-18

2002-09-10

Kishore, Collamudi S. (Department: 1615)

Drug, bio-affecting and body treating compositions

Preparations characterized by special physical form

Liposomes

C424S001210, C424S009321, C424S009510, C424S417000, C264S004100, C264S004300, C264S004600

Reexamination Certificate

active

06447800

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of liposomes. More particularly this invention relates to the field of loading liposomes or releasing solutes from liposomes by transmembrane permeation.
BACKGROUND OF THE INVENTION
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single membrane bilayer) or multilamellar vesicles (onion-like structures characterized by multiple membrane bilayers, each separated from the next by an aqueous layer). The bilayer is composed of two lipid monolayers having a hydrophobic “tail” region and a hydrophilic “head” region. The structure of the membrane bilayer is such that the hydrophobic (non-polar) “tails” of the lipid monolayers orient toward the center of the bilayer while the hydrophilic (polar) “heads” orient towards the aqueous phase.
A variety of liposome types are known and include multilamellar vesicles (MLV's), single unilamellar vesicles (SUV's), large unilamellar vesicles (LUV's), stable plurilamellar vesicles (SPLV's), frozen and thawed multilamellar vesicles (FATMLV's), reversed phase evaporation vesicles (REV's) as described in U.S. Pat. Nos. 5,049,392, 5,204,112 and 5,262,168.
One of the primary uses for liposomes is as carriers for a variety of materials such as drugs, cosmetics, diagnostic reagents, biological materials such as proteins, hormones, antibodies, nucleic acids and polypeptides, and the like.
So far, several methods have been developed for liposome loading. The simplest method of loading is a passive entrapment of a water soluble material in the dry lipid film by hydration of lipid components. The loading efficiency of this method is generally low because it depends on the entrapping volume of the liposomes and on the amount of lipids used to prepare them. Loading efficiency can be increased by the dehydration-rehydration method in which preformed liposomes are dehydrated in the presence of solute and subsequently reconstituted. Disadvantages include heterogenous size, difficult standardization and low reproducibility.
Recently ethanol has been employed to generate interdigitated fusion vesicles (IFV) composed of saturated phospholipids. This method produces large vesicular structures which exhibit large trap volumes (10-20 L/mole) and therefore high trapping efficiencies (P. L. Ahl et al. (1994) “Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume”
Biochimica Et Biophysica Acta
1195:237-244). It is known that acyl chain interdigitation can be induced by small, amphipathic molecules such as ethanol (F. Zhang et al (1992) “Titration calorimetric and differential scanning calorimetric studies of the interactions of n-butanol with several phases of dipalmitoylphosphatidylcholine”
Biochemistry
31:2005-2011; E. S. Rowe and T. A. Cutrera (1990) “Differential scanning calorimetric studies of ethanol interactions with distearoylphosphatidylcholine: transition to the interdigitated phase”
Biochemistry
29: 10398-10404; J. A. Veiro et al. (1988) “Effect of alcohols on the phase transitions of dihexadecylphosphatidylcholine”
Biochimica Et Biophysica Acta
943:108-111; E. S. Rowe (1987) “Induction of lateral phase separations in binary lipid mixtures by alcohol”
Biochemistry
26:46-51; S. A. Simon (1984) “Interdigitated hydrocarbon chain packing causes the biphasic transition behavior in lipid/alcohol suspensions”
Biochimica Et Biophysica Acta
773:169-172), but only for saturated lipids and in the absence of cholesterol. The formation of IFV occurs when small vesicles (<200 nm) are induced to form sheets of interdigitated phase lipid by the addition of 5 M ethanol at temperatures below the gel to liquid crystalline phase transition (T
c
) of the phospholipid. When the temperature is raised above T
c
, the sheets spontaneously form large bilayer vesicles which are now stable above or below T
c
once ethanol has been removed. It is well known that ethanol can induce an interdigitated organization of phospholipids when it is added to hydrated bilayers composed of saturated phospholipids. However, interdigitation does not occur for unsaturated phospholipids. (P. L. Ahl et al. (1994) “Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume”
Biochimica Et Biophysica Acta
1195:237-244; H. Komatsu et al. (1993) “Effect of unilamellar vesicle size on ethanol-induced interdigitation in dipalmitoylphosphatidylcholine”
Chemistry & Physics of Lipids
65:11-21; J. W. Zeng and P. L. Chong (1991) “Interactions between pressure and ethanol on the formation of interdigitated DPPC liposomes: a study with Prodan fluorescence”
Biochemistry
30:9485-9491; L. L. Herold (1987) “13C-NMR and spectrophotometric studies of alcohol-lipid interactions”
Chemistry & Physics of Lipids
43:215-225). DPPC has been studied the most in this regard and it has been shown that small DPPC vesicles will collapse in the presence of ethanol to form extended sheets of lipid in an interdigitated state (P. L. Ahl et al. (
1994
) “Interdigitation-fusion: a new method for producing lipid vesicles of high internal volume”
Biochimica Et Biophysica Acta
1195:237-244).
More recently another method for liposome loading has involved adding solutes to pre-formed intact liposomes. Typically, higher loading efficiencies are obtained. In this method, conditions are provided under which the 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. A transmembrane chemical potential is employed to drive the substance to be loaded into the liposome. Commonly, the transmembrane potential is created by a concentration gradient which is formed by having differing concentrations of a particular species on either side of the liposomal membrane. Neutralization of the concentration gradient is coupled to flow of the substance being loaded into the liposome. pH gradients (U.S. Pat. Nos. 4,946,683; 5,192,549; 5,204,112; 5,262, 168; 5,380,531), Na+/K+ gradients (U.S. Pat. Nos. 5,171,578; 5,077,056) and NH
4
+ gradients (U.S. Pat. No. 5,316,771) have been used to load a variety of drugs into liposomes. One limitation of using ion gradients is that the substance being loaded must be an ionizable or protonatable substance. Therefore, the substances loaded by these methods are typically ionizable compounds, often weakly acidic or basic or amphipathic molecules. Other chemical potential driven methods for liposome loading after liposome formation have used a concentration gradient of the solute itself to drive the loading process by employing precursor liposomes with low ionic strength interiors and raising the temperature above the crystal/liquid transition temperature T
c
or temporarily disrupting the liposome membrane with shear stresses (U.S. Pat. Nos. 5,393,350; 5,104,661 and 5,284,588). Despite the availability of these methods for liposome loading, it is still desirable to have alternative methods which do not have the limitations of the methods described above. This invention fulfills this and other needs.
SUMMARY OF THE RELATED ART
1. H. Komatsu et al. (1993) “Effect of unilamellar vesicle size on ethanol-induced interdigitation in dipalmitoylphosphatidylcholine”
Chemistry and Physics of Lipids
65:11-21; discloses that DPPC unilamellar vesicles are capable of becoming interdigitated in the presence of ethanol and that this tendency increases with increasing vesicle size.
2. E. S. Rowe and T. A. Cutrera (1990) “Differential scanning calorimetric studies of ethanol interactions with distearoylphosphatidylcholine: transition to the interdigitated phase”
Biochemistry
29: 10398-10404; discloses effect of dilution on the ethanol-induced interdigitated state of saturated phosphatidylcholine multilamellar liposomes.
3. Komatsu et al. (1991) “Effect of cholesterol on the ethanol-i

Affiliated with

Also associated with

Rating

Method of loading preformed liposomes using ethanol does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Method of loading preformed liposomes using ethanol, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Method of loading preformed liposomes using ethanol will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-2824863