Liposome extrusion process

Details Liposome extrusion process Liposome extrusion process

2001-11-20

2003-09-23

Lovering, Richard D. (Department: 1712)

Plastic and nonmetallic article shaping or treating: processes

Encapsulating normally liquid material

Liquid encapsulation utilizing an emulsion or dispersion to...

C424S450000, C428S402200

Reexamination Certificate

active

06623671

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a method of sizing liposomes, and more particularly to a sizing method which includes extruding liposomes through a branched-pore type aluminum oxide porous film.
BACKGROUND OF THE INVENTION
Liposomes are completely closed lipid bilayer membranes containing an entrapped aqueous volume. Liposomes may be unilamellar vesicles (possessing a single bilayer membrane) 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 (nonpolar) “tails” of the lipid monolayers orient toward the center of the bilayer while the hydrophilic “head” orient towards the aqueous phase.
The original liposome preparation of Bangham, et al. (J. Mol. Biol., 1965, 13:238-252) involves suspending phospholipids in an organic solvent which is then evaporated to dryness leaving a phospholipid film on the reaction vessel. Next, an appropriate amount of aqueous phase is added, the mixture is allowed to “swell”, and the resulting liposomes, which consist of multilamellar vesicles (MLVs), are dispersed by mechanical means. This technique provided the basis for the development of the small sonicated unilamellar vesicles (SUVS) described by Papahadjopoulos et al. (
Biochim. Biophys. Acta.,
1968, 135:624-638), as well as large unilamellar vesicles (LUVs). In addition, U.S. Pat. No. 4,235,871, issued Nov. 25, 1980 to Papahadjopoulos et al., describes a “reverse-phase evaporation process” for making oligolamellar lipid vesicles, also known as reverse-phase evaporation vesicles (REVs).
Alternative methods have been developed for forming improved classes of multilamellar vesicles which have been shown to have particularly improved properties such as, for example, higher active ingredient trapping efficiencies and loadability, better stability, less leakage, and greater ease of production. One such improved class of liposomes, denominated as stable plurilamellar vesicles (SPLVs), is described in U.S. Pat. No. 4,522,803, issued Jun. 11, 1985 to Lenk et al. Another such improved class, defined as monophasic vesicles (MPVs), is described in U.S. Pat. No. 4,558,578, issued May 13, 1986 to Fountain et al. Both of these classes of liposomes have also been characterized as having substantially equal interlamellar solute distributions. A general review of various methods for producing liposomes, including an extensive bibliography, is set forth in Deamer and Uster, “Liposome Preparation: Methods and Mechanisms”, in the
Liposomes,
edited by M. Ostro, pp. 27-51 (1983), incorporated herein by reference.
The administration of drugs encapsulated in or otherwise associated with liposomes has been proposed for use in a variety of drug delivery regimens in combination with or as an alternative to the administration of free drugs. In some applications, liposomes have been found to provide sustained release of drugs for extended periods, which can be of particular importance in the lengthy chemotherapy regimens often required for the treatment of various forms of cancer or AIDS-related illnesses. Another property of liposomes is their ability to be taken up by certain cells, such as phagocytes, such that they can deliver their active ingredient to the interior of the cells. This makes such liposome treatment particularly useful in treating intracellular infections, such as those associated with species of Mycobacteria, Brucella, Listeria, and Salmonella. Thus, drugs encapsulated in liposomes can be delivered for the treatment of such intracellular diseases without administering large amounts of free unencapsulated drug into the bloodstream. In addition, the mere association of certain drugs or other bio-active agents with liposomes has been found to potentiate or improve the activity of such drugs or bio-active agents, or to reduce their toxicity.
Liposomes behave like particles, and are commonly described in terms of average particle size and particle-size distributions. For certain uses of liposomes, particularly in the parenteral administration of drugs, it is important to size the liposomes to a desired average particle size, and to maintain a controlled particle-size distribution, particularly by sizing the liposomes so that substantially all of the liposomes are of a size below a predetermined maximum diameter. For liposomes intended for parenteral administration, one desirable size range is between about 100 and 1000 nm, preferably between about 100 and 500 nm. (As used herein, nm represents nanometer (10
−9
m) and um represents micrometer or micron (10
−6
m).) The maximum desired size range is often limited by the desire to sterilize the liposomes by filtering through conventional sterilization filters, which commonly have a particle-size discrimination of about 200 nm. However, overriding biological efficacy and/or safety factors may dictate the need for a particular particle size, either larger or smaller. Control of the size range of the liposomes may also improve the effectiveness of the liposomes in vivo, as well as the stability and leakage resistance of the liposomes.
The various methods for producing liposomes generally produce a suspension of liposomes of widely varying sizes, many of which exceed 1000 nm in average particle size. A number of methods have been proposed to reduce the size and size distribution of liposomes in such suspensions. In a simple homogenization method, a suspension of liposomes is repeatedly pumped under high pressure through a small orifice or reaction chamber until a desired average size of liposome particles is achieved. A limitation of this method is that the liposome size distribution is typically quite broad and variable, depending on the number of homogenization cycles, pressures, and internal temperature.
Small unilamellar vesicles (SUVs), generally characterized as having diameters below 100 nm, are composed of highly strained, curved bilayers. The SUVs are typically produced by disrupting larger liposomes via ultrasonication. It has been found that a narrow size distribution of such liposomes can only be achieved when the liposomes have been reduced to their smallest sizes, less than about 50 nm. Furthermore, this process may not be amenable to large-scale production, because it is generally conducted as a batch process with long-term sonication of relatively small volumes. In addition, heat build-up during sonication can lead to peroxidative damage to lipids, and sonication probes may shed titanium particles which are potentially quite toxic in vivo.
A method of sizing liposomes by filtration through a 200-nm Unipore™ polycarbonate filter is discussed in Szoka,
Proc. Natl. Acad. Sci. U.S.A.,
75:4194-8 (1978). A size-processing method based on liposome extrusion through a series of uniform straight-pore type polycarbonate membranes from about 1000 nm down to about 100 nm is described in Hunt et al., U.S. Pat. No. 4,529,561, issued Jul. 16, 1985. However, this method can be relatively slow, often requiring many passes through various size filters to obtain the desired particle-size distribution.
Vesicles may also be size-reduced using an extrusion process described in Cullis et al., U.S. Pat. No. 5,008,050, issued Apr. 16, 1991, incorporated herein by reference. Vesicles made by this technique are extruded under pressure through a filter with a pore size of 100 nm or less. This procedure avoids the problems of the above homogenization and sonication methods, and does not require multiple passes through decreasing size filters, as described in the above-cited U.S. Pat. No. 4,529,561. Such a process can provide size distributions of liposomes that are quite narrow, particularly by cycling the material through the selected size filter several times. In addition, it is believed that such extrusions may convert multilamellar vesicles into oligolamellar or even u

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