Phospholipids and Liposomes

Lecithin (Section 12.17) has traditional use as an emulsifying, wetting, dispersing and stabilising agent in pharmaceutical preparations. It is also a recognised source of phosphatidyl choline. 

Stealth liposomes, containing therapeutic drugs and coated with polyethylene glycol, may assist evasion of immune systems and enable targets to be reached in the body. 

Liposomes were introduced into commercial cosmetics and face creams about 1987, and have potential value in medicinal creams and ointments as tissue promoters. Because of their high water content, moisturising properties are claimed. Liposomes containing dyestuffs in their aqueous core are used for diagnostic tests in immunology. 

Phosphoglycerides have been prescribed to treat AIDS-related infections and compounds of this type have also been described as artificial lung surfactants [45,46]. 

Co-polymers utilising phospholipids have other medical uses (Section 12.15). 

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Alving, C. R. (1984). Natural antibodies against phospholipids and liposomes in humans, Biochem. Soc. Trans.. 12. 342-344. 

Some steroid molecules (estrone, estradiol, and estriol) have phenolic hydroxyl in the ring A (Figure 29.12) and therefore, are able to react as free radical scavengers. In 1987, Japanese authors [264,265] showed that all these compounds inhibited iron adriamycin- or iron ADP-ascorbate-dependent phospholipid and liposomal lipid peroxidation. Later on, most attention was drawn to the study of antioxidative properties of estradiol-17(3 (estrogen E2) it has been proposed that E2 antioxidant activity may contribute to cardioprotection observed after estrogen therapy in postmenopausal women. The necessity for the phenolic hydroxyl has been shown by studying the effects of several estrogens on LDL oxidation. It was found [266]... 

Preparation of Phospholipids and Liposomes, sn-2 DHA-inserted phosphatidylcholine (2-DHA-PC) was enzymatically synthesized from soy lysophosphatidylcholine via phospholipase (PL)A2-mediated esterification as previously reported (5). Then, sn-2 DHA-inserted phosphatidylserine (2-DHA-PS) was prepared from 2-DHA-PC through PLD-mediated transphosphatidylation (6). Liposomes with 70%... 

That vesicles and liposomes form at all is a consequence of the amphi-pathic nature of the phospholipid molecule. Ionic interactions between the... 

Barenholz, Y., Amselem, S., and Lichtenberg, D. (1979). A new method for preparation of phospholipid vesicles (liposomes). French press, FEES Lett.. 99, 210-214. 

Senior, J., and Gregoriadis, G. (1982). Stability of small unilamellar liposomes in serum and clearance from the circulation The effect of the phospholipid and cholesterol components. Life Sci., 30. 2123-2136. 

Figure 14-22. Formation of lipid membranes, micelles, emulsions, and liposomes from am-phipathic lipids, eg, phospholipids.

A further partihon system based on the use of liposomes, and commercialized under the name Transil [110, 111], has shown its utiUty as a UpophiUcity measure in PBPK modeling [112]. Fluorescent-labeled liposomes, called fluorosomes, are another means of measuring the rate of penetration of small molecules into membrane bilayers [113, 120]. Similarly, a colorimetric assay amenable to HTS for evaluating membrane interactions and penetrahon has been presented [116]. The platform comprises vesicles of phospholipids and the chromahc Upid-mimehc polydiacetylene. The polymer undergoes visible concentrahon-dependent red-blue transformahons induced through interactions of the vesicles with the studied molecules. 

To use liposomes as delivery systems, drug is added during the formation process. Flydrophilic compounds usually reside in the aqueous portion of the vesicle, whereas hydrophobic species tend to remain in the lipid proteins. The physical characteristics and stability of lipsomal preparations depend on pH, ionic strength, the presence of divalent cations, and the nature of the phospholipids and additives used [45 47],... 

Osterherg, T. Svensson, M. Lundahl, P, Chromatographic retention of drug molecules on immohilized liposomes prepared from egg phospholipids and from chemically pure phospholipids, Eur. J. Pharm. Sci. 12, 427 139 (2001). 

Perhaps the simplest solvent dispersion method is that developed by Batzri and Korn (1973). Phospholipids and other lipids to be a part of the liposomal membrane are first dissolved in ethanol. This ethanolic solution then is rapidly injected into an aqueous solution of 0.16M KC1 using a syringe, resulting in a maximum concentration of no more than 7.5 percent ethanol. Using this method, single bilayer liposomes of about 25 nm diameter can be created that are... 

Phospholipid concentration was determined using our modification of Bartlett s procedure (49,53). Cholesterol concentration and purity were determined by HPLC or enzymatically by cholesterol oxidase (49,53). Purity of phospholipids as raw materials, and the extent of their hydrolysis during various steps of liposome preparation and liposome storage, were assessed by TLC and enzymatic determination of the increase in level of nonesterified fatty acids (10,38,49-51,53). 

Numerous methods have been developed for attaching ligands to the surface of liposomes for reviews, see Refs. (3,6 ). For peptides, they fall into two major categories (i) covalent coupling of the peptides to preformed liposomes that contain functionalized hydrophobic anchors such as, e.g., derivatives of phos-phatidylethanolamine (PE) or (ii) incorporation of lipopeptides, obtained by conjugation of peptides to hydrophobic anchors (fatty acids, phospholipids), into liposomes either during the preparation of the vesicles or by postinsertion into preformed vesicles. In this section, we will briefly discuss these techniques and focus on the ones we have been using in our own work. 

Finally, besides conventional liposomes that are made from natural (e.g., egg yolk and soybean) or synthetic phospholipids, novel liposomes called archaeosomes that are prepared from the polar ether lipids extracted from various archaeobacteria proved also interesting for the design of vaccines as peptide antigen carriers (71) and as powerful self-adjuvanting vaccine delivery vesicles that promote both humoral and cell-mediated immunity (72). Related to this, one can mention that pseudopeptides, which are less prone to proteolysis when conjugated to liposomes, were also competent in triggering a humoral immune response (73). 

The two examples from our work we are going to describe below are the design and study of liposomal diepitope constructs combining either (i) B and T-helper (Th) peptide epitopes, which induced particularly powerful humoral responses (21) (Fig. 3) or (ii) CTL and Th epitopes, which provided a powerful antitumor vaccine (74) (Fig. 4). For the production of these constructs we have conjugated peptides that contain a cysteine residue either at the N- or C-terminus, to the surface of preformed liposomes by reaction with thiol reactive functionalized phospholipids and/or PamaCys lipopeptide anchors (Fig. 2). To that end, we have developed strategies that give, in aqueous media, high... 

In principle, liposomes can enter (target) cells through different pathways by direct fusion of liposomes and the plasma membrane (1) or by an endo-cytic uptake mechanism. Other liposome-cell interactions that have been described in the literature are absorption, phospholipid and protein exchange, and cell-induced leakage of liposome contents (2,3). 

Figure 1 Schematic structures of micelle and liposome, their formation and loading with a contrast agent, (a) A micelle is formed spontaneously in aqueous media from an amphiphilic compound (1) that consists of distinct hydrophilic (2) and hydrophobic (3) moieties. Hydrophobic moieties form the micelle core (4). Contrast agent (asterisk gamma- or MR-active metal-loaded chelating group, or heavy element, such as iodine or bromine) can be directly coupled to the hydrophobic moiety within the micelle core (5), or incorporated into the micelle as an individual monomeric (6) or polymeric (7) amphiphilic unit, (b) A liposome can be prepared from individual phospholipid molecules (1) that consists of a bilayered membrane (2) and internal aqueous compartment (3). Contrast agent (asterisk) can be entrapped in the inner water space of the liposome as a soluble entity (4) or incorporated into the liposome membrane as a part of monomeric (5) or polymeric (6) amphiphilic unit (similar to that in case of micelle). Additionally, liposomes can be sterically protected by amphiphilic derivatization with PEG or PEG-like polymer (7) [1].

Liposome-encapsulated tretinoin has been tested in hairless mice as well as in man. The animal experiment has demonstrated the favorable uptake of the retinoid, whereas the liposomal lipids appear to be more retained in the homy layer [53], Moreover, with phospholipid-based liposomes belonging to the gel-state type, tretinoin penetration in murine skin appears to be confined to the epidermis [54] and, thus, is close to prednicarbate penetration described above. In patients with acne vulgaris, we could demonstrate a better tolerability of liposomal tretinoin as compared to a commercial gel while efficiency remains the same [55],... 

A liposome is a spherical vesicle with a membrane composed of a phospholipid and cholesterol (less than 50%) bilayer. Liposomes can be composed of naturally derived phospholipids with mixed lipid chains (such as egg phosphatidylethanolamine) or... 

Regarding other pseudostationary phases for measurement of lipophilicity or lipophilicity-related properties (e.g., intestinal absorption, brain penetration), there are several reports on the use of vesicles such as phospholipid bilayer liposome (56-58), lysophospholipid micelle (59), DTAB/SDS vesicle (60), and double-chain synthetic surfactant vesicle (61), which are described in other chapters. 

Fig. 1 Schematic illustration of a liposome encapsulating a hydrophilic drug. The membrane contains phospholipid and cholesterol molecules. (Reprinted with permission from Ref. 3 with slight modification. Copyright 1998 Adis International.)... 

Fig. 5 Relationship between the log Ks values obtained by chromatography on entrapped egg yolk phospholipid (EPL) liposomes and the log Poct values from oc-tanol/water partitioning analyses, for neutral drugs (upper line) and for positively (center line) and negatively (lower line) charged drugs. (Reprinted with permission from Ref. 27. Copyright 2001 Elsevier Science.)...

Vesicles for use as materials can be divided into two categories naturally occurring vesicles, or liposomes, which are composed of natural amphiphiles, usually phospholipids and polymer vesicles, which are generally composed of block copolymers. 

Glycolipid incorporated liposomes have found extensive use as sensors for the detection of Escherichia coli bacteria. Liposomes prepared using a diacetylene and a glucosyl lipid underwent a chromatic transition upon the addition of E. coli (Ma et al. 1998). The chromatic transition is sensitive to the diyne and glycolipid stmc-ture (Ma et al. 2000). An optimized vesicle assembly, consisting of a maltotriosyl lipid, phospholipid, and diyne, detected E. coli at a concentration of 2x10 cells/mL... 

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