PROCESOS Y PROCEDIMIENTOS

Water dispersible, poorly soluble amphiphiles, including non-ionie surfactants are known to self-assemble into spherical uni- or multi-lamellar vesicles (Azmin et al,

1985; Uchegbu & Florence, 1995). However, various tubular (Furhop & Helfrich, 1993; Chiruvolu et a l, 1994; Uchegbu & Florence, 1995), disk-like (Walter et al,

& Bensimon, 1995) vesicle structures also form as a result of amphiphile self­ association. Highly structured ‘geodesic’ multivesicular structures arising from the association of many small non-ionic surfactant vesicles have also been described (Sternberg et a l, 1995). The specific shapes obtained are governed by the type and amount of surfactant used, which in turn is governed by the geometric shape that the surfactants possess. External factors such as temperature, which affect the properties of the components of the lamellar membrane also play a part in the form and dimensions of the vesicles as shown in Figure 1.6.

Membrane activation (eg. Heat above T„, sonication)

or Adding of Cholesterol Restabilised membrane (eg. Cooling) Symboh ^ C16EO5 O Choieivterol ^r^^olulan C24

Figure 1.6: The schematic represents the typical orientations o f amphiphiles giving rise to specific shapes o f polyhedral and spherical vesicles and also the factors affecting shape and molecular conformation o f polyhedral niosomes. For the polyhedral vesicles, the bilayer membranes exist in a “gel” state, where the fatty acyl chains o f the lipid or surfactant membranes are closely packed and relatively ordered, whereas for the spherical vesicles, the side chains are capable o f rotational motion existing in a “fluid” state. (Modified from Arunothayanun, PhD thesis. University o f London 1998.)

Vesicles may be harvested after controlled swelling which gives rise to a broad spectrum of different sizes and shapes. Spherical shapes are most commonly encountered as these shapes have minimal bending energies (Deuling & Helfrich, 1976) and the richness of all the shapes clearly indicate that lipid swelling and liposome detachment is a dynamic process. Changes in vesicle shape may be due to temperature or pressure changes, the addition of various amphiphiles or adsorbents, mechanical, electrical or magnetic treatments, or to adhesion (Deuling & Helfrich, 1976; Kas & Sackmann, 1991; Lasic, 1993; Lipowsky & Sackmann, 1995). Since the discovery of these structures in the mid 1960s (Bangham & Home, 1964) studies on vesicle shape and changes in shape have constituted a considerable part of experimental and theoretical vesicle research. In the early days of lipid vesicle discovery, it was recognised that lipid vesicle shape behaviour reflects to a large extent the bilayer nature of lipid membranes (Helfrich, 1974). Further analysis of vesicle shape revealed some general features of lipid vesicle shape behaviour that depended on the layered membrane structure, and not on the structural and compositional details of the membrane monolayers (Svetina & Zeks, 1996; Seifert,

1997).

The shape changes of a vesicle can be altered when certain conditions are applied. One of the factors which can affect the shape of a vesicle is the volume of solute enclosed. For example for a given area of the vesicle membrane (A), the vesicle volume (V), being practically equal to the volume of the internal solution, can have any value between zero and the volume of the sphere with radius Rg = (A/4ji)^^^.

osmotic state of the internal and external solutions. For any vesicle volume smaller than the volume of the sphere, the vesicle is flaccid and can assume an infinite number of shapes (Svetina & Zeks, 1996), as seen in Figure 1.7.

Many cellular processes, notably endocytosis and exocytosis involve changes of membrane conformations and thus depend on membrane mechanical properties. The role of membrane mechanics is also indicated by characteristic cellular shapes, such as the disc shape of the red blood cell. It seems, therefore, natural to extend the concepts to develop in studies of lipid vesicles to analysis of the shape behaviour of cells and subcellular vesicles, as well as multicellular layered systems involving closed surfaces (Svetina & Zeks, 2002). However, cellular processes are in general highly controlled, and their successful performance depends on the intactness of many proteins and their complexes. The connection between the shape behaviour of simple vesicular structures, such as phospholipid vesicles and cellular systems may therefore be by chance; hence more complex studies could shed light on such matter while providing us with insight knowledge into cellular shape transformations.

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Figure 1.7: Reproduced from Svetina and Zeks who describe the figure as follow, ‘ y4

series o f vesicle shapes as observed by phase contrast microscopy. This microscopy senses the p a rts o f vesicles in which the path o f the optical beam through the membrane is the longest; therefore, the equatorial contours o f vesicles are seen representing the equatorial cross-sections o f vesicles. In the fir s t row are three shapes belonging to the cup-shape class (1-3) an d a shape belonging to the disc-shape class (4). In the second row are shapes belonging to the cigar-shape (5) and pear-shape (6-8) classes. In the third row are some examples o f shapes with a relatively sm all vesicle volume/membrane area ratio. Shape 9 is term ed a codocyte, shape 10 is a torocyte, shape I I is a starfish, and shape 12 is a worm shape. The fourth row shows shapes characterized by narrow necks connecting nearly spherical vesicle parts. Shape 13 has two invaginated spheres within a large sphere. Shape 14 is com posed o f a large sphere an d two sm all evagi nated spheres. Shape 15 has a sm all sphere in between two large spheres, whereas shape 16 has (in addition to a large mother sphere) fiv e sm all spheres arranged in a row and a single sm all sphere connected to it a t another position. D ata are from : shapes 1-4, 13, and 14 (Kas & Sackmann, 1991), 5-8 (Kas et a l , 1993), 9 and 16 (Svetina et a i, 2001), 10-12 (J. Majhenc, unpublished data), and 14 (Farge & Devaux, 1 9 9 2 f\

Research on soft matter can be very fruitful, mainly due to the fact that such materials have the capability to take on new shapes when exposed to external stimuli. The possibilities of simple experiments in inducing such changes make this type of research attractive. Once formed, vesicles can remain stable for a number of days if not weeks. Vesicles are also characterised by good mechanical stability; one can puncture a giant vesicle with a micro-needle, make microinjections and manipulate them, without destroying or transforming them (Fischer et a l, 2002).

The formation of a vesicle can be tailored in such way that the size falls within the micrometer range, hence, manipulation of large vesicles can be carried out while the object is viewed live under a light microscope. Thus, this type of research does not depend on statistics and the average behaviour of a population but an individual vesicle. Nevertheless, each individual vesicle may behave differently, thus, one research aim is to produce equal individuals that behave reproducibly. The information obtained from visualisation of large vesicles would not necessarily apply to smaller vesicles in every detail, but visualisation is paramount and hence larger systems have to be employed. In the future new modes of microscopy might allow the study of sub-micrometer vesicles.