Drug – bio-affecting and body treating compositions – Preparations characterized by special physical form – Liposomes
Reexamination Certificate
1999-12-14
2004-12-28
Kishore, Gollamudi S. (Department: 1615)
Drug, bio-affecting and body treating compositions
Preparations characterized by special physical form
Liposomes
C424S001210, C424S009321, C424S009510, C424S417000, C424S489000, C424S502000, C428S402200, C264S004100, C264S004300
Reexamination Certificate
active
06835394
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the preparation and use of vesicles and related encapsulating membranes made in aqueous solution from amphiphilic polymers and related molecules.
BACKGROUND OF THE INVENTION
Membranes that are stable in aqueous media are heavily relied upon for compartmentalization by biological cells. For instance, the outermost plasma membrane of a cell separates the inside of a cell from the outside and, like most cell membranes, it is a self-assembled, complex fluid of biological molecules, primarily lipids and proteins. Only a few molecules, such as water and small, uncharged organic molecules, significantly permeate the membrane. A biomembrane also possesses stability and other thermo-mechanical properties that are not unrelated to passive permeability and are certainly central to cell function (see, e.g. Lipowsky and Sackmann, Eds.,
Structure and Dynamics of Membranes from Cells to Vesicles, Handbook of Biological Physics
. vol 1 (Elsevier Science, Amsterdam, 1995); Bloom et al.,
Q. Rev. Biophys
. 24:293 (1991)).
The same characteristics of permeability and thermo-mechanical stability in addition to biocompatibility—also affect how lipid vesicles that are assembled in vitro and that are also known as liposomes can effectively encapsulate and deliver a long list of bioactive agents (Needham et al., in
Vesicles
, M. Rosoff, Ed. (Dekker, N.Y., 1996), chap. 9; Cevc & Lasic in
Handbook of Biological Physics
, chaps. 9-10, 1995; Koltover et al.,
Science
281:78 (1998); Harasym et al.,
Cancer Chemother. Pharmacol
. 40:309 (1997)). The typical liposome is comprised of one or more bilayer membranes, each approximately 5 nm thick and composed of amphiphiles such as phospholipids. Each bilayer exists as a temperature- and solvent-dependent lamellar phase that is, in its surface, in a liquid, gel, or liquid-gel coexisting state. Because of a certain intrinsic biocompatibility of phospholipid vesicles, many groups have developed them for use as encapsulators and delivery vehicles. Vesicles surrounded by a lipid bilayer can range in diameter from as small as tens of nanometers to giants of 0.5-40 microns.
Phospholipid vesicles are materially weak and environmentally sensitive. Transit through the digestive tract, for example, can expose liposomes to a host of solubilizing agents. Repeated transit through the microcirculation can also tear apart giant phospholipid vesicles which cannot withstand high fluid shear. Smaller phospholipid vesicles may not fragment, but they tend to adhere, and are thus cleared from circulation. Circulating cells suppress their own adhesion partly through a brushy biopolymer layer, known as the glycocalyx, which faces the environment. The glycocalyx has, to some extent, been mimicked in liposome systems by the covalent addition to lipids of hydrophilic polyethyleneglycol (PEG) polymer chains. To maximally extend a vesicle's circulation lifetime (about ten hours), a suitable PEG weight ranges between about two and five kilograms/mole.
To further counteract mechanical forces imposed on their membranes, cells often also possess a sub-membranous network of cross-linked proteins (Alberts et al., in
Molecular Biology of the Cell
, 3
rd
ed., pp489-493 and 800-1, Garland Publ., Inc., New York, 1999). The red blood cell, as an example, survives repeated deformation through the microcirculation without fragmentation, but only because it has a cross-linked network of peripheral membrane proteins. Without such a network, the cells cannot withstand such circulation for more than a few hours even with a glycocalyx (Schmid-Schoenbein et al.,
Blut
52(3):131 (1986)). With a normal membrane network, red blood cells circulate in humans for more than 100 days. In terms of measurable properties, the network imparts a shear elasticity that is only achievable with a cross-linked structure.
Past efforts to enhance the stability of lipid lamellae against shear and other factors, resulted in the synthesis of many different modified lipid molecules with polymerizable double bonds. Such bonds were located either at the surfactant head group, or more, commonly, at different locations on the hydrophobic tails (Fendler et al.,
Science
223:888 (1984); Liu et al, Macromolecules 32:5519 (1996)). This approach clearly had the ability to generate covalently inter-connected poly-amphiphiles when reacted after self-assembly into membranes per ordinary lipids. However, a fully, covalently interconnected network of lipids requires complete cross-linking of the membrane of a vesicle, and the full extent of cross-linking achievable with cross-linkable lipids appears to be difficult to ascertain. O'Brien's group (Sisson et al.,
Macromolecules
29:8321 (1996)) has used solubility in hexafluoropropanol to estimate a degree of polymerization up to at least 1000. This corresponds to a vesicle diameter of about 10 nanometers, if one assumes complete cross-linking within and between layers of the bilayer, and a typical lipid area of about 0.5 square nanometers per lipid. Detergent induced leakage of entrapped solutes was strongly inhibited by cross-linking. It is clear, however, that no fully cross-linked lipid vesicle larger than several hundred nanometers has been reported.
A cross-liinkable amphiphile related to cross-linkable phospholipids has been made by Komatsu et al.,
J. Am. Chem. Soc
. 119:11660 (1997)). Tetrakis(aminophenyl)porphyrin contains four hydrophobic bixin side chains that each terminate in a small hydrophilic carboxylate group and harbor approximately ten (photo)reactive double bonds along the backbone of each bixin chain. When dissolved in the organic solvent, tetrahydrofuran (THF), and rapidly injected into a one-eighth volume of water and sonicated, the synthetic molecules reportedly formed vesicles. However, the resulting membranes are porous. Irradiation led to what was claimed to be the first spherical membrane structure of molecular thickness, which was considered a single, dehydratable, balloon-shaped polymer molecule insoluble in a predominantly organic solvent, such as 95% ethanol. Electron micrographs showed spherical particles of less than 100 nm, while collapsed particles studied by atomic force microscopy were reported to have a height of about 7 nm. Whether the cross-linked shells were truly semi-permeable vesicles or were highly porous macromolecular shells, as Komatsu et al. suggested, leaves open the question of whether, to date, a wholly cross-linked vesicle of any size has actually been produced. Certainly, no cross-linked vesicle larger than several hundred nanometers has been reported.
Small amphiphiles of natural origin, such as phospholipids have inspired the engineering of high molecular weight analogs, which also self-assemble into complex phases in aqueous media similar to those observed for phospholipids. For example, vesicles have been assembled in aqueous solution by Uchegbu et al.,
J. Pharm
. &
Pharmacol
. 50:453 (1998)) using the naturally occurring macromolecule chitosan modified by the covalent attachment of many fatty acid pendant groups. The resulting self-assembled vesicles were 300-600 nanometers in diameter, and were shown to be bio- and haemocompatible. Although such modified natural products have disadvantages of variability in the natural polymer and a lack of precise control in covalent modification, the assembly of membranes from amphiphiles of high molecular weight has the potential to improve vesicle stability. The overall approach has similarities to lipid cross-linking, but a primary distinction lies in the fact that, with cross-linking, self-assembly of the membrane must occur first.
Many semi- or partially-synthetic, amphiphilic molecules are also significantly larger (in molecular weight, volume, and linear dimension) than phospholipid amphiphiles, and have therefore been called “super-amphiphiles” (Comelissen et al.,
Science
280:1427 (1998)). Cornelissen et al. used polystyrene (PS) as a hydrophobic fraction in their series of non-synthetic, natural block copolym
Bates Frank S.
Discher Bohdana M.
Discher Dennis E.
Hammer Daniel A.
Lee James C-M.
Dilworth Paxson LLP
Kishore Gollamudi S.
McConathy Evelyn H.
The Trustees of the University of Pennsylvania
LandOfFree
Polymersomes and related encapsulating membranes does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Polymersomes and related encapsulating membranes, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Polymersomes and related encapsulating membranes will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3291878