Liposomes structure

Cationic quaternary ammonium compounds such as distearyldimethylammonium-chloride (DSDMAC) used as a softener and as an antistatic, form hydrated particles in a dispersed phase having a similar structure to that of the multilayered liposomes or vesicles of phospholipids 77,79). This liposome-like structure could be made visible by electron microscopy using the freeze-fracture replica technique as shown by Okumura et al. 79). The concentric circles observed should be bimolecular lamellar layers with the sandwiched parts being the entrapped water. In addition, the longest spacings of the small angle X-ray diffraction pattern can be attributed to the inter-lamellar distances. These liposome structures are formed by the hydrated detergent not only in the gel state but also at relatively low concentrations. 

Figure 10.11 Liposome structures, including multilamellar vesicles (MLV) and large unilamellar...

Mixtures of phospholipids in aqueous solution will spontaneously associate to form liposomal structures. To prepare liposomes having morphologies useful for bioconjugate or delivery techniques, it is necessary to control this assemblage to create vesicles of the proper size and shape. Many methods are available to accomplish this goal, however all of them have at least several steps in common (1) dissolving the lipid mixture in organic solvent, (2) dispersion in an aqueous phase, and (3) fractionation to isolate the correct liposomal population. 

Activation of PE residues with these crosslinkers can proceed by one of two routes the purified PE phospholipid may be modified in organic solvent prior to incorporation into a liposome, or an intact liposome containing PE may be activated while suspended in aqueous solution. Most often, the PE derivative is prepared before the liposome is constructed. In this way, a stable, stock preparation of modified PE may be made and used in a number of different liposomal recipes to determine the best formulation for the intended application. However, it may be desirable to modify PE after formation of the liposomal structures to ensure that only the outer half of the lipid bilayer is altered. This may be particularly important if substances to be entrapped within the liposome are sensitive or react with the PE derivatives. 

The use of artificial cells for biotechnology applications is simplified if the replication and reproduction characteristics are removed so that an artificial cell does one particular job. A liposome structure would continue to uptake molecules from the environment and synthesise proteins or cofactors, but the molecular inventory within the liposome is comparatively small. 

Figure 13.15 Generalized liposome structure. Refer to text for details...

Another concern with freeze-drying LEH is the instability of liposome structure upon lyophilization. Vesicle formation occurs in the presence of bulk water and when water is removed, loss of structural integrity is inevitable. Fusion, crystal formation, and phase transition are observed, resulting... 

Poulain FR, Allen L, Williams MC, et al. Effects of surfactant apolipoproteins on liposome structure implications for tubular myelin formation. Am J Physiol 1992 262(6 Pt 1) L730-L739. 

Less frequently used at present is electron spin resonance spectroscopy, which is based on the use of spin probes as model componnds or covalent spin labeling of drugs. Microviscosity and micropolarity of the molecnlar environment of the probe can be derived from electron spin resonance spectra. Moreover, the spectra allow us to differentiate isotropic and anisotropic movements, which result from the incorporation of the probe into liposomal structures. Quantitative distribution of the spin probes between the internal lipid layer, the snrfactant, and the external water phase is to be determined noninvasively. On the basis of the chemical degradation of drugs released from the lipid compartment, agents with reductive features (e.g., ascorbic acid) allow us to measure the exchange rate of the drugs between lipophilic compartments and the water phase [27,28]. 

Measurements of the quantities of glycolipids inserted into the membrane have also been reported by a technique based on the use of C-labeled lipid anchors. In this method, the carbohydrate (a-o-Man) was covalently coupled to the anchor at the surface of a pre-formed vesicle. Indeed, the liposome structure was shown to remain intact in the treatment. Nevertheless, the measurement of the incorporated mannose was performed after separation of bound and unbound material by centrifugation. The yields of coupling were shown to increase with the increase of the initial mannose/ C-anchor ratio, but non covalent insertions were displayed at high initial mannose concentrations. Therefore, the aforementioned method was not as accurate as could have been expected for the use of radioactive materials [142]. Radiolabeled phospholipids were also used for such determinations thus the amounts of glycosphingolipids incorporated into liposomes were quantified by the use of H-phospholipids whereas the amounts of glycolipids were determined by a sphingosine assay [143]. 

Another complication is the fact that the phospholipids are not soluble in water, forming in many cases liposomal structures, but they are soluble in electrolyte solutions. Thus, the mere addition of an electrolyte to a phospholipids-stabilized emulsion may physically affect the structure of the mesophase and, hence, the stability of the whole system. 

It should be noted that liposomes in effect mimic cell walls and proteins are to be found in nature associated with cell walls. However, the association may differ physical according to the conformation for the protein under consideration. For example some proteins fold so that they are exposing hydrophobic regions which lit into the hydrophobic regions of the liposomal structure (or cell wall). The net effect is that the transbilayer protein exposes its hydrophilic regions both inside and outside of the liposomal structure to the water surrounding the structure (Figure 9.6). 

El Maghraby, G.M., A.C. Williams, and B.W. Barry. 2000. Skin delivery of oestradiol from lipid vesicles Importance of liposome structure. Int J Pharm 204 159. 

The fact that no apparent fusion of the vesicles is revealed by the atomic force microscopy (AFM) does not prove the liposome structural integrity (Fig. 4c, d). Analysis of the profiles of the embedded vesicles show that they are immersed in the film, suggesting the immersion by two different modes of the capping film layers (1) exponential between the vesicles, and (2) linear on the vesicle top [82], Evidence of vesicle stability is proved by a direct release study of the vesicle-encapsulated CF marker, as shown in Fig. 4f [82]. Similar results were found for DPPC vesicles filled with ferrocyanide ions [77], No considerable release of the markers, at least during the first few hours after embedding, points to vesicle integrity. 

In this chapter we will provide information about the basic characteristics of liposomes staring from their building blocks, that is, phospholipids. After this, liposome structure, physicochemical properties, and stability, which are most important for their in vivo performance, will be discussed as well as methods used for liposome preparation, characterization, and stabilization. Following this first part which is more technological, we will move into the biological part and talk about the fate of conventional liposomes and sterically stabilized liposomes, as well as liposomal drugs, after in vivo administration by different routes [mainly intravenous (i.v.), intraperitoneal (i.p.), or subcutaneous (s.c.)] and also give some information about other possible routes for in vivo administration of liposomes. Finally, specific applications of liposomes in therapeutics will be presented, some in more detail, mainly for the therapy of different types of cancer. 

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