Does Shape Matter? Spherical, Polyhedral, and Tubular Vesicles

Chapter 6

Downloaded by UNIV MASSACHUSETTS AMHERST on September 2, 2012 | http://pubs.acs.org Publication Date: May 5, 2004 | doi: 10.1021/bk-2004-0879.ch006

Does Shape Matter? Spherical, Polyhedral, and Tubular Vesicles 1,

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Alexander T. Florence *, Behrooz Nasseri , and Parinya Arunothyanun 1,2

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Centre for Drug Delivery Research, The School of Pharmacy, University of London, London WC1N 1AX, United Kingdom Current address: Government Pharmaceutical Organisation, Bangkok, Thailand *Corresponding author: email: [email protected] 2

Surfactant and lipid vesicles can be produced in a variety of geometries, discoidal, polyhedral, toroidal and tubular. Here some properties of spherical and polyhedral non-ionic surfac -tant vesicles have been compared flow being one parameter. Shape is less important than membrane characteristics in controlling release of entrapped drug, but the flow properties of vesicular suspensions are, of course, markedly dependent on shape. The elasticity and flow behavior of such vesicular systems have been little studied but we postulate by analogy with erythrocytes that these are pertinent to the behavior of these potential drug delivery systems in vivo, an attribute not discussed in the literature.

© 2004 American Chemical Society

In Carrier-Based Drug Delivery; Svenson, S.; ACS Symposium Series; American Chemical Society: Washington, DC, 2004.

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Introduction As most particulate drug delivery vectors are spherical, little attention has been paid to the influence of shape on the behavior of delivery systems. We have been interested for some time in non-ionic surfactant vesicle (niosome) design, and have as a result produced in aqueous media niosomes of a variety of shapes, namely discoidal, polyhedral, and tubular systems. (I) In non-aqueous solvents we have also observed spherical, tubular and toroidal inverse vesicles. (2) Other groups have found prolate vesicles and so-called non-axisymmetric "star-fish" vesicles. (3,4) As shape directly influences the flow properties of suspensions, the question of the effect of vesicle shape and properties on their rheological characteristics arises. Little work has been done on the rheology of liposomes or niosomes, yet one can postulate that the flow properties of spherical and aspherical vesicles in the capillary blood supply will be important. As Sackmann pointed out cell (and vesicle) membranes are "extremely soft with respect to bending and shearing but practically noncompressable with respect to lateral stretching", a construction which allows erythrocytes to travel "several hundred kilometers throughout our body" without loss of material. (5) The influence of deformability and hence shape on a range of blood cell behaviors has been discussed in detail. (6) Clear differences are seen in the rheology and properties of suspensions of spherical and polyhedral vesicles prepared from non-ionic surfactants and their behavior in capillaries in the laboratory. (7)

Results and Discussion There are two different, but related, effects of vesicle flow: the effect of vesicle shape on flow properties and the effect of flow on the shape of elastic vesicles (or cells), the latter being discussed theoretically. (8) There are several instances in the use of vesicles in drug delivery technology where capillary flow is of relevance (i) in the fate of elastic vesicles in their transport through the skin (9), (ii) in targeting, vectors arrival at target sites may be dictated not only by the diameter and surface characteristics of the carrier, but also by their elastic pro perties using the analogy of the fate of red cells (6), and (iii) in the production of delivery systems by extrusion technologies. (10-12) In this chapter we discuss issues surrounding the shape of niosomes and ask whether Theological characteristics matter in vivo. Based on in vitro data on the behavior of niosomes and liposomes under stress, we also consider the potential fate of vesicles in the capillary blood supply, with specific reference to vesicle damage or rupture and the consequent loss of entrapped solutes. Our proposition is that shape matters (i) because it affects flow and potential fate in vivo, through for example extravasation, and (ii) that as shape differences are determined by


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membrane composition there are secondary differences in the physical properties of vesicles of different shapes, including solute release and swelling, which must also be taken into account. In addition, the elasticity and visco-elasticity of vesicular systems may be important in their role as drug delivery vectors and may differentiate vesicles from inelastic solid nano- or microparticles.

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Solulan C24

Figure 1. The C G -cholesterol-Solulan C24 ternary phase-diagram. Regio 1, polyhedral vesicle (2-10 pm); region 2, spherical, helical and tubular v (0.5-10 pm); region 3, discomes (10-30 pm), large vesicles (40 pm) an spherical and helical vesicles (0.5-10 pm); region 4, discomes (12-60 pm possibly Solulan C24 micelles; region 5, cholesterol crystals; region spherical vesicles (0.5-10 pm); region 7, a clear liquid (Solulan C24 mi region 8, mixed micelles formed at elevated room temperature. (Repro with permission from reference 13. Copyright 1997.) i6

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Polyhedral systems are found in cholesterol-poor regions of the phase diagram, shown in Figure 1, for one of the typical systems we have studied, namely the three component mixture comprising cholesteryl-24-polyoxyethylene ether (Solulan C24), cholesterol and the non-ionic surfactant, C i G , a hexadecyl diglyceryl ether. (13) Typical spherical and asymmetric systems from such mixtures are shown in Figure 2. Shape is determined by the properties of the bilayer membranes at given temperatures. Polyhedral vesicles, for example, convert reversibly to spherical systems above a critical temperature. Differences in shape not only affect the capillary flow, but they translate into differences in osmotic behavior, membrane diffusion characteristics, and elastic properties, all 6

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of which are important in determining behavior as delivery vectors in vivo unless other unknown and over-riding factors impinge. (14) The release of LHRH from polyhedral niosomes is greater than the release from their spherical equivalents, the behavior of both systems being highly dependant on the medium in which release was measured. (15)

Figure 2. Photomicrographs showing (a) multi-lamellar polyhedral niosom an aqueous channel, (b) spherical niosomes madefromsorbitan monoste cholesterol and Solulan (45:45:10), (c) microtubules formedfrompolyhe niosomes when extrudedfromcapillaries smaller than their size, (d) a t microtubule also produced by extrusion, (e) discomes produced by incu surfactant I: Solulan C24 (50:50) (Cable, C Ph.D. thesis, University of St clyde, Glasgow, UK, 1990), (f) a hexadecane gel at room temperatur taining surfactant tubular aggregates dispersed in organic medium (Repro with permissionfromReference 2), and (g) toroidal vesicular structures isopropyl myristate formulation at the transition temperature between s gel phases. (Bars = 10 pm) (Reproduced with permissionfromreference Copyright 1999 Elsevier Science.) (See page 2 of color insert.)



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Figure 2. Continued. (See page 3 of color insert.)

Our studies on the flow properties of spherical and polyhedral vesicles in which estimates of surface hydration of non-ionic vesicle formulations were made, indicated clearly the higher viscosity of polyhedral systems (Figure 3). This work led us to consider the flow properties of vesicles in capillaries. Figure 4 shows the movement of an elastic spherical niosome and its deformation as the capillary thins. As shapes changes the contact area between the membrane and wall of the capillary increases, a phenomena which might well have some biological relevance. The behavior of a polyhedral vesicle is also shown. In narrow capillaries under pressure spherical vesicles, being visco-elastic, can survive intact. With polyhedral systems, as the capillary narrows and pressure is maintained, permanent shape changes occur. (11,12) Fusion of polyhedral nio somes under pressure in capillaries with diameters less than the diameter of the niosomes lead to the production of tubular systems up to 80 \im in length and approximately 1 jtim in diameter. (16) Can such fusion occur in vivo, for exam ple at a capillary bifurcation or during extravasation?



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T

Figure 3. Plot of reduced specific viscosity (rj - l)/C, versus concentratio lipid/surfactants at 25 °Cfor polyhedral niosome and spherical/tubidar nios suspensions, preparedfromC G /chotesterol/Solulan C24 in the ratios 91: and 45:45:10. (Reproducedfrom reference 7. Copyright 1999 American Chemical Society.) rel

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Figure 4. Flow of (a) a spherical vesicle in a capillary in which r =r m , (b) where r > r u and deformation of the elastic system occurs, and (c) polyhedral vesicles where flow patterns clearly differfromthose of spher systems. (Bar = 10pm) vesicle

vesicle

capi

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Much of the debate on whether the shape of vesicles matters is dependent on knowledge of the nature of the capillary blood supply and the forces exerted on and the damage done to vesicles as they move in capillaries. Earlier studies of doxorubicin remaining in and released from spherical niosomes after intravenous administration showed that around 60% of the drug remained within spherical niosomes eight hours after i.v. administration. (17) The extent to which this loss is due to normal diffusion or to the shear stress on the vesicles is not known. However, spherical vesicles can be stressed by micromanipulators and the outer bilayers removed, as shown in Figure 5, causing a rapid release of some of the encapsulated material (dye in this experiment). These might well be extreme forces but in experiments in which polyhedral vesicles were extruded through capillaries of decreasing diameter, from less than 1 fim to around 4 jam, the greater shear experienced by niosomes exiting through the narrower capillaries led to the greatest loss of entrapped carboxyfluorescein (Figure 6). The extent to which such assaults are commonplace in vivo is not known. The intrinsic release rates of spherical and polyhedral systems are ordinarily dependent on membrane properties. An additional complication in estimating what happens in vivo is that release from non-stressed systems is so different.


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Figure 5. Desquamation by micromanipulation of a Rhodamine B contai spherical liposome, leading to the release of the encapsulated dye. (See page 3 of color insert.)

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Micropipette tip size (pm)

Figure 6. Initial (T=0) levels of entrapped CF releasefrompolyhedra niosomes of size range 2-15 pm after extrusion via micropipette tips of re size.


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Conclusions This paper has simply posed the question "does the shape of vesicles matter?" and provides no concrete proof that in vivo it does. However we know that the closest relative of a lipid vesicle - a red blood cell - is influenced by its deformability, hence we maintain that shape matters. Shape differences are determined by membrane composition, hence there are differences in the physical properties of niosomes and other vesicles of various shapes, these some times leading to differences in the rate of release of contents and osmotic behavior. Elasticity is important in determining behavior in capillary beds and trafficking through cellular structures, but its true influence certainly requires more detailed investigation. Vesicle shapes are permanent only if the system has no elastic properties. Movement of intact vesicles across the skin and other semipermeable membranes is a function of their deformability. (9) Vesicles also show quite pronounced thermal fluctuations due to the "softness" of their membranes. (18) The presence of transition temperatures close to body tempera ture causes tubular structures to undergo shape transformation. It may be possible to take advantage of such shape changes to influence the movement of vesicles in vivo, for example after delivery to the eye or to intramuscular sites.

References 1. Uchegbu, I.F.; Florence, A.T. Non-ionic surfactant vesicles (Niosomes): Physical and Pharmaceutical Chemistry. Adv. Coll. Inter. Sci. 1995, 58, 155. 2. Murdan, S.; Gregoriadis, G.; Florence, A.T. Inverse toroidal vesicles: precursors of tubules in sorbitan monostearate organogels. Int. J. Pharm. 1999, 183, 47-49. 3. Döbereiner, H-G.; Evans, E.; Kraus, M.; Seifert, U.; Wortis, M . Mapping vesicle shapes into the phase diagram: A comparison of experiment and theory. Phys. Rev. E. 1997, 55, 4458-4474. 4. Wintz, W.; Dobereiner, H-G.; Seifert, U. Starfish vesicles. Europhys. Lett. 1996, 33, 403-408. 5. Sackmann, E. Membrane bending energy concept of vesicle- and cell -shapes and shape transitions. FEBS Lett. 1994, 346, 3-16. 6. Chien, S. Biophysical behavior of red cells in suspensions. In Surgenor, D.M. The Red Blood Cell. 1975, 11, New York: Academic Press. 7. Florence, A.T.; Arunothayanun, P.; Kiri, S.; Bernard, M-S.; Uchegbu, I.F. Some rheological properties of non-ionic surfactant vesicles and the determination of surface hydration. J. Phys. Chem. B. 1999, 103, 19952000.


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Bruinsma, R. Rheology and shape transitions of vesicles under capillary flow. Physica. A. 1996, 234, 249-270. Cevc, G,; Schätzlein, A,; Richardsen, H. Ultradeformable lipid vesicles can penetrate the skin and other semi-permeable barriers unfragmented. Evidence from double label CLSM experiments and direct size measurements. Biochim. Biophys. Acta. 2002, 1564, 21-20. Hope, M.J.; Nayer, R.; Cullis, P. In Liposome Technology; Gregoriadis G., 2 Ed.; CRC Press, Boca Raton. 1993. Florence, A.T.; Nasseri, B. Microfabrication of lipidic structures. Yakuzaigaku. J. Pharm. Sci. Tech. 2001, 61, 8-9. Arunothayanun, P.; Sooksawate, T.; Florence, A.T. Extrusion of niosomes from capillaries: approaches to a pulsed delivery device. J. Control. Rel. 1999, 60, 391-397. Uchegbu, I.F.; Schatzlein, A.; Vanlerberghe, G.; Morgatini, N.; Florence, A.T. Polyhedral non-ionic surfactant vesicles. J. Pharm. Pharmacol. 1997, 49, 606-610. Arunothayanun, P.; Uchegbu, I.F.; Florence, A.T. Osmotic behavior of polyhedral non-ionic surfactant vesicles (niosomes). J. Pharm. Pharmacol. 1999, 51, 651-657. Arunothayanun, P.; Turton, J.A.; Uchegbu, I.F.; Florence, A.T. Preparation and in vitro/in vivo evaluation of luteinizing hormone releasing hormone (LHRH)-loaded polyhedral and spherical/tubular niosomes. J. Pharm. Sci. 1999, 88, 34-38. Nasseri, B.; Florence, A.T. Microtubules formed by capillary extrusion and fusion of surfactant vesicles. Int. J. Pharm. 2003, in press. Uchegbu, I.F.; Double, J.A.; Turton, J.A.; Florence, A.T. Distribution, metabolism and tumoricidal activity of doxorubicin administered in sorbitan monostearate (Span 60) niosomes in the mouse. Pharm. Res. 1995, 12, 1019-1024. Sackmann, E.; Duwe, H.P.; Engelhardt, H. Membrane bending elasticity and its role for shape fluctuations and shape transformations of cells and vesicles. Faraday Discuss. Chem. Soc. 1986, 81, 281-290.


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Does Shape Matter? Spherical, Polyhedral, and Tubular Vesicles

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