Bioehimica et Biophysiea Acta, 457 (1976) 259-302 © Elsevier Scientific Publishing C o m p a n y , A m s t e r d a m - Printed in T h e N e t h e r l a n d s BBA 85163

NEW

ASPECTS

OF

LIPOSOMES

D. A. T Y R R E L L , T. D. H E A T H , C. M. C O L L E Y a n d B R E N D A E. R Y M A N

University of London, Department of Biochemistry, Chafing Cross Hospital Medical School, Fulham Palace Road, London W6 8RF (U.K.) (Received April 23rd, 1976)

CONTENTS I.

I1.

General considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

260

A. I n t r o d u c t i o n a n d historical b a c k g r o u n d . . . . . . . . . . . . . . . . . . . . .

260

B. L i p o s o m e structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

260

C. Physical properties o f liposomes . . . . . . . . . . . . . . . . . . . . . . . .

262

D. N e w applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263

Methodology

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

263

A. Preparation of liposomes . . . . . . . . . . . . . . . . . . . . . . . . . . .

263

B. Preparation o f h o m o g e n e o u s s u s p e n s i o n s o f liposomes . . . . . . . . . . . . . .

265

C. R e m o v a l o f non-associated solutes from liposomes . . . . . . . . . . . . . . . .

266

D. L i p o s o m e s a n d proteins . . . . . . . . . . . . . . . . . . . . . . . . . . .

268

1. L i p o s o m e - p r o t e i n interactions . . . . . . . . . . . . . . . . . . . . . . . 2. Protein e n t r a p m e n t in liposomes . . . . . . . . . . . . . . . . . . . . . .

268 271

E. L i p o s o m e s a n d e n t r a p m e n t o f small molecules . . . . . . . . . . . . . . . . .

272

F. Quantitative expression o f e n t r a p m e n t . . . . . . . . . . . . . . . . . . . . .

273

G. S u m m a r y

274

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

III. U p t a k e o f liposomes in vivo . . . . . . . . . . . . . . . . . . . . . . . . . . .

274

A. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

274

B. I n vivo fate o f intravenously injected liposomes . . . . . . . . . . . . . . . . . C. Experimental models to test the efficacy o f liposomes as carriers o f therapeutic agents

274 276

D. Effect o f liposome modification (size, charge, lipid c o m p o s i t i o n etc.) o n uptake by tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

277

E. Alternative routes o f a d m i n i s t r a t i o n o f liposomes . . . . . . . . . . . . . . . . F. S u m m a r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

279 280

IV. Interactions o f liposomes with cells in culture . . . . . . . . . . . . . . . . . . .

280

A. I n t r o d u c t i o n . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

280

B. M e t h o d o l o g y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

281

C. Earlier observations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. U p t a k e o f liposomally e n t r a p p e d materials into cultured cells . . . . . . . . . . .

282 282

E. M e c h a n i s m o f liposome uptake into cultured cells . . . . . . . . . . . . . . . .

283

F. T h e role o f cholesterol in cell m e m b r a n e s . . . . . . . . . . . . . . . . . . . . G. Direction o f liposomes to target ceils . . . . . . . . . . . . . . . . . . . . . . H. S u m m a r y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

285 285 286

260 V. Immunological aspects ofliposomes . . . . . . . . . . . . . . . . . . . . . . . . A. Antigenic lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Complement mediated lysis of liposomes . . . . . . . . . . . . . . . . . . . . C. The adjuvant properties of liposomes . . . . . . . . . . . . . . . . . . . . . D. Hypersensitivity reactions to antigens in liposomes . . . . . . . . . . . . . . . . E. Other aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Summary and conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . .

286 286 288 290 291 291 294 294

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

297

I, GENERAL CONSIDERATIONS

IA. Introduction and historical background For m a n y years work has been in progress to find methods of directing drugs and other therapeutic molecules to specific sites in the b o d y in order to achieve tissuespecific treatment of various conditions. Chang [1,2] entrapped enzymes in nylon or collodion microvescicles and administered these intraperitoneally to animals, but as these vesicles are composed of non-natural materials they accumulate in the body. Until 1971, liposomes (phospholipid bilayer vesicles) had been extensively studied as models for biological membranes; one aspect of these studies involved the entrapment within liposomes of small solute molecules in order to measure the permeability of the model membranes to these molecules [3]. It was proposed [4,222] that liposomes could be used to entrap enzymes which could then be administered intravenously to patients for the treatment of inherited storage diseases. Liposomes have an advantage as carriers in vivo in that they m a y be formed from natural molecules which can be metabolised in the body. F r o m this work [4] the use o f liposomes as carrier vesicles has been extended to solutes other than enzymes, including chelating agents [5], antibiotics [6], drugs, with particular emphasis on anti-tumour drugs [7], cell-modifying c o m p o u n d s [8] and hormones [9]. In this review we intend to consider recent developments in the use of liposomes in living systems, including the factors governing the encapsulation of therapeutic agents in the liposomes, the fate o f the liposomes after administration in vivo and the factors governing tissue uptake. Immunological aspects o f liposomes will also be discussed. The review is intended to be representative, not exhaustive, and work published up to Spring 1976 is covered. Although some discussion o f earlier uses o f liposomes is given, the reader is referred to a number of informative reviews for further details [10-17,18*].

lB. Liposome structure When phospholipids are suspended in an excess o f aqueous solution they * After this review had been prepared, a preprint of an article by Poste et al. was received, which further reviews this field.

26,1 Lipid BiIayer LarnelIae

~.~

~l,um

~

25 nm

Aqueous (a)

Spaces

(b)

(c)

Fig. 1. Schematicrepresentationsofliposomes. (a) Multilamellarliposome. (b) Enlarged viewof(a). (c) Small, unilamellar liposome.

spontaneously form multilamellar concentric bilayer vesicles, with the lipid layers separated by layers of aqueous medium (Fig. 1). Electron micrographs of such liposomes are found in many publications, such as those of Hauser et al. [19], Gregoriadis et al. [4] and Bangham [223] and are therefore not shown here. These "liquid crystal vesicles" (liposomes) can be formed from many different phospholipids, and the composition most used has been egg phosphatidylcholine, with or without cholesterol and with or without anionic lipids, e.g. phosphatidic acid, or cationic lipids, e.g. stearylamine; they can also be formed from whole lipid extracts of cell membranes, e.g. erythrocyte ghosts [20]. Under normal conditions cholesterol can be incorporated into liposomes up to a maximum of one cholesterol molecule per phospholipid molecule [21], although some workers have reported up to 2 : 1 cholesterol:phospholipid in highly sonicated liposome preparations [22]. Where higher proportions of cholesterol than this are reported there is considerable doubt as to the presence of all the cholesterol in liposomal form, as much of it may be present as crystals of cholesterol. Phospholipids may form other structures in the presence of water [23], dependent on the relative proportions of water and lipid, though in the presence of excess water the liposome is the preferred configuration for most phospholipids [24]. Phosphatidylethanolamine does not form liposomes but forms non-closed structures and, therefore, does not sequester any volume of water within the lipid structure [24]. In addition, phospholipids only form liposomes at temperatures at which their fatty acyl chains are fluid, therefore dipalmitoylphosphatidylcholine will not form liposomes below its transition temperature of 41 °C [25]. The separation between the bilayers is determined by the balance between the repulsive forces between the layers, probably mainly electrostatic interactions between headgroups and hydration forces of the head-groups, and attractive forces which are consistent with the expected van der Waal's forces between the layers [26]. The equilibrium distance between bilayers of pure egg phosphatidylcholine in water is 2.75 nm [26]. This separation, and thus the volume of aqueous phase within the liposome, can be

262 increased by the addition of up to 10 mol ~ of charged lipid (either anionic or cationic) into the phospholipid [27]. The entrapped volume can be estimated by measuring the degree of entrapment of a non-ionic, non-permeable solute, e.g. glucose, within the liposomes. The entrapped volume is then the fraction of solute entrapped × the total volume of the preparation. Despite the comments of Rahman et al. that only pure lipids will form liposomes [28], in our experience liposomes can be readily formed from commercially available impure phospholipid preparations. Reeves and Dowben even state that in their system pure lipids would not form vesicles at room temperature but that it was necessary for the phospholipids to be highly oxidised [29]. Nevertheless, in order to be able to compare results in experimental systems, it is advisable to use pure lipids. Phospholipids of suitable purity can be prepared using the methods of Papahadjopoulos and Miller [24] or Bangham et al. [10].

IC. Physicalproperties of liposomes This ability of liposomes to entrap water and therefore solutes within a closed lipid bilayer structure led to their use as models of biological membranes for permeability studies; phosphatidylcholine vesicles are permeable to water, ions and non-electrolytes [25,30,31], although the permeabilities depend on the chemical composition of the liposomes. Positively charged liposomes (e.g. phosphatidylcholine plus a positively charged lipid such as stearylamine) are impermeable to cations, while negatively charged liposomes (e.g. containing phosphatidic acid) are permeable to cations; however the permeability of all liposomes to protons is low [3]. Anions diffuse rapidly through negatively and positively charged lipid membranes and also through uncharged lipid membranes [30,224]. In general i t has been found that increasing the degree of saturation or the length of the phospholipid fatty acyl chains produces a decrease in the permeability of the bilayers to all solutes [3]. This is due to the increase in the transition temperatures resulting from these modifications, which leads to a decrease in fluidity of the chains [32]. A decrease in permeability can also be produced by the addition of cholesterol to naturally occurring phospholipids [3], again by decreasing the fluidity of the bilayer structure [33]. The permeability of liposomes to ions may be selectively increased by the addition of ionophores such as valinomycin [34]. The addition of some proteins such as lysozyme [35], myelin basic protein [36] and immunoglobulins [37] to a liposome suspension increases the leakage of anions and glucose from the liposomes, while general anaesthetics, such as ether and chloroform, produce an increase in cation permeability of the bilayers, but no increase in glucose permeability [38]. Liposomes, therefore, are capable of sequestering aqueous solutions, and the rate at which the solutes leak out of the liposomes is dependent on both the nature of the solute and the composition of the liposome. By modification of the composition of the lipid bilayers it is possible to reduce the leakage of particular molecules, although any small molecule not having too many hydrogen bonds will leak rapidly

263 from the liposome irrespective of lipid composition, while large molecules such as proteins will not leak out unless the structure of the liposome is disrupted [39]. Liposomes have also been used as models of biological membranes to study many other aspects of membranes, including molecular motions within membranes (studied by nuclear magnetic resonance [33] and electron spin resonance [40]),the binding of local anaesthetic molecules to membranes [41,42] and the actions of the anaesthetics on these membranes [43], the freezing of living cells [44], mechanisms of inflammation in gout [214], the reconstitution of membrane-bound ion-transport systems [45], and interactions of antibiotics with membranes [46,47].

ID. New applications In earlier work the liposome was investigated mainly as a model membrane. More recently, however, a rather different approach has emerged in which liposomes have been considered as carrier vesicles of possible biological and therapeutic use. The methods of preparation of such liposomes are at present extremely variable and this variation, together with the many factors to be considered when using standard methods of liposome preparation in the entrapment of solute molecules, will be discussed in Section II, as will the nature of the interactions of proteins, drugs and other solutes with liposomes. The problems of determining whether a molecule is truly entrapped within the aqueous spaces of the liposome, or whether it may be associated with the lipid structure, are also considered in Section 11. The potential use of liposomes in man necessitates the production of sterile, pyrogen-free preparations of liposomes which require special conditions for their preparation (Section II). The sites of uptake of liposomes after administration in vivo, the factors governing rate and site of uptake and the possibilities for tissue specific direction of liposomes will be discussed in Sections II1 and IV, including work done on the uptake of liposomes by cells in culture. Another growing field is the immunological properties of liposomes, as models for complement-mediated lysis, as immunologically active structures when antigenic lipids are used in their preparation, and when virus subunits are attached to the liposomes, and as adjuvants for possible use in man; these aspects of the field are covered in Section V. In conclusion, we shall discuss the present applicability of liposomes in various therapeutic fields, some of the problems which require further investigation, and possible future developments.

1I METHODOLOGY

1114. Preparation of liposomes The original and still most commonly used method of making liposomes is that in which the lipid is first dried down under vacuum from organic solvent as a very thin film on the walls of a rotary evaporator flask. The lipid is then suspended by adding an aqueous solution and gently shaking the flask. The lipid swells spontaneously and then gradually falls from the walls of the flask into aqueous suspension

264 as very large multilamellar liposomes (hand-shaken liposomes), about 1 /~m in diameter. This method has been described in detail by Bangham et al. [10]. In addition to this system, a number of more recent methods for making liposomes have been documented and are considered here because they will clearly be of use in the future in biological studies involving liposomes. Batzri and Korn [48] have described a method in which a solution of lipid in ethanol is injected into an aqueous solution through a fine hypodermic needle. This produces a suspension of very small liposomes about 25 nm in diameter. Redwood et al. [49] have employed this method to incorporate erythrocyte sialoprotein (glycophorin) into liposornes, and have demonstrated the presence of the protein within the membrane by agglutination of the liposomes with lectins. Methods in which aqueous solutions of protein and lipid in detergent are mixed and then dialysed to remove the detergent have also been employed by a number of workers for membrane reconstitution studies. Slack et al. [50] have incorporated an (Na+,K+)-dependent ATPase into liposomes, Hinkle et al. [51 ] have incorporated mitochondrial cytochrome oxidase, and Warren et al. [52] have incorporated a (Ca2+,Mg2+)-dependent ATPase. The method involves the mixing of lipid in an aqueous detergent solution with the protein, followed by dialysis of the mixture against successive batches of detergent-free buffer. Because the critical micelle concentration of the detergent is very high compared to that of the phospholipid, dialysis removes the detergent and leaves behind the protein and lipid, which together form liposomes. If Ca z+ or Mg 2+ is incorporated into the suspension and dialysis medium, and provided that a portion of the phospholipid is negatively charged, multilamellar liposomes are formed. The method developed by Eytan et al. [53] for the preparation of liposome-membrane protein complexes involves lysophosphatidylcholine incorporations by liposomes to induce lipid-protein interactions. They claim there is a biological precedent for this, since phospholipase A2 is present in mitochondria and may produce high local concentrations of lysophesphatidylcholine to facilitate insertion of other proteins into the membrane. A further method of liposome preparation that seems worthy of note here is that developed by Deamer et al. [54] for the study of ion diffusion across lipid bilayers. Ethereal lipid solution is injected slowly into the aqueous phase through a fine needle. The aqueous phase is held at 60 °C by a water jacket, which causes the ether to spontaneously evaporate. As a result the lipid forms very large unilamellar vesicles about 1 # m in diameter. The entrapment of aqueous phase per/~mol of phospholipid is about ten times that of hand-shaken liposomes and about 40 times that of very small liposomes. Although in this method relatively small quantities of lipid can be handled, i.e. about 4-8 /zmol of lipid per ml of aqueous suspension, the percentage entrapment of solute is comparable to that achieved when as much as 160 #mol of phospholipid per ml is used for the preparation of hand-shaken liposomes. The method seems very attractive from this viewpoint, and it will be interesting to see if these liposomes show differences from other liposome preparations in their behaviour when administered to animals. However, the temperature of 60 °C may impose limitations when using labile molecules. Large unilamellar liposomes may also be prepared by the method described

265 by Papahadiopoulos et al. [55]. Small unilamellar liposomes are fused into large sheets or cochleate cylinders by the addition of calcium ions. When further treated with ethylenediaminotetraacetic acid (EDTA) the sheets form large unilamellar vesicles. The only limitation of this method is that it has only been shown to work when phosphatidylserine is used in the preparation of the liposomes.

lIB. Preparation of homogeneous suspensions of liposomes The heterogeneity of liposomes with respect to size can be a limitation on their use in certain investigations. In the study of the interaction of liposomes with cells in culture, many workers have aimed for homogeneous suspensions of small liposomes. Moreover, Juliano and Stamp [56] have recently shown that the rate and site of uptake in vivo of small and large liposomes may be quite different. The preparation of liposome suspensions of uniform size may well prove to be of increasing importance in the future, and it therefore seems pertinent at this point to give some consideration to the methods currently available. Sonication (ultrasonic irradiation) has long been used as a means of reducing the size of very large, multilamellar liposomes. Sonic cleaning baths or probe type sonicators may be used. The latter are, in general, faster, although Bangham et al. [10] express reservations concerning the use of probe sonicators. In addition to their objections it should be mentioned that it is more difficult to maintain sterility when using a probe sonicator in the preparation of sterile, pyrogen-free liposomes for therapeutic purposes. Many workers have used brief sonication as a means of rendering liposome suspensions more amenable to chromatographic separation of liposomes from nonentrapped solute [4,9,57-62], since gel chromatography for removal of solutes may be very difficult without prior sonication. Huang [63,64] was the first to attempt to prepare a homogeneous suspension of small liposomes, which he called "smallest possible liposomes", and which have a diameter of 25 nm. After prolonged sonication the lipid suspension was chromatographed on Sepharose 4B which produced two peaks: a void volume peak containing all larger liposomes and a single included peak containing the very small liposomes. Judicious cutting of fractions yielded a suspension of very small liposomes which were shown to be homogeneous by rechromatographing on Sepharose 4B and by analytical ultracentrifugation. In our laboratory we have also used Sepharose 2B for this purpose. The main disadvantage of this method is that these Sepharoses adsorb lipid and must be pre-equilibrated with lipid sonicates to avoid large losses [63]. Johnson [65] avoided this problem by using a method of exhaustive sonication in a sonic bath. Suspensions so produced were, however, contaminated with slightly larger vesicles. Most recently, Roseman et al. [66] have prepared homogeneous suspensions by prolonged sonication followed by centrifugation at 150000 × g for 4 h, which sediments all but the small unilamellar vesicles. Liposomes made by methods described so far for the preparation of homogeneous suspensions have been used mainlyfor physical studies. Although similar methods have been used for biological studies [8] it is clear that there are

266 limitations if labile compounds, such as enzymes, are being incorporated into the liposomes. Prolonged sonication could destroy their biological activity. For such compounds the method of Batzri and Korn [48] seems perhaps most promising, although this would not be suitable if the compound to be entrapped were labile in 7~o ethanol. The method described by Slack et al. [50] might possibly be used for non-diffusable solutes, although the compound would have to be stable in detergent. The preparation of homogeneous suspensions of large liposomes is, unfortunately, not possible since, unlike small liposomes, every one is different. It is, however, possible to isolate multilamellar liposomes free from very small liposomes, enabling studies of two distinct populations [56]. Perhaps the nearest thing to a homogeneous suspension of large liposomes is that produced by the method of Deamer et al. [54].

IIC. Removal of non-associated solutes from liposomes When liposomes are prepared in the preser~ce of a solute a proportion of the solute is entrapped, but most remains in free solution; it is usually necessary to remove this. The three commonly used methods for the removal of solutes are gel filtration, centrifugation and dialysis. Ultrafiltration has not been used extensively, although it would appear a feasible procedure for removal of small solutes. In our hands it has proved a useful method for concentrating liposomes after chromatography, and Batzri and Korn [48] have used it for a similar purpose. Dialysis has been used for the removal of small solutes [14]. It is also a convenient method for studying the leakage of small molecules from liposomes [30]. Gel-filtration has a possible disadvantage in that lipid may be adsorbed by the gel bed, as mentioned above. This phenomenon is documented for Sepharose 4B [63], and in our laboratory has been found to occur with Sepharose 2B. It has not, however, been reported for any of the other gels available. Gel filtration is a rapid and simple method, since even very small liposomes are excluded by all but the highest fractionation range gels. Thus, by selection of the correct gel, it is possible to perform a separation in which the liposomes are excluded and the solute totally included. Centrifugation has proved useful in our laboratory in two cases when otherwise we might have used gel chromatography, where a large number of different lipid composition liposomes were required at the same time [68], and also where sterile, pyrogen-free liposomes were required for administration to a patient [69]. Sterility of the final preparation is easily achieved by passage through a Millipore filter, but in order to pass rigorous pyrogen testing sterility is needed throughout the entire preparation. Centrifugation using sterile centrifuge tubes was found to be the best method for maintaining such aseptic conditions. In general, the wide range of centrifugation conditions used by various workers in the field to spin down liposomes seems rather perplexing, and Table I illustrates the point. It seems reasonable to suggest that the disparity here reflects the variation in the size of the liposomes. According to Johnson [73] it is possible to spin down multilamellar liposomes at 700 x g for 10 rain. Roseman et al. [66] have stated that it is possible to purify the very small vesicles by centrifugation at 150000 >~ g for

M + U

M

M

M + U

M

Mg2+/CaZ+-dependent ATPase N A D H / c y t o c h r o m e b5 reductase Bovine s e r u m a l b u m i n

M M ~- U M ÷ U

M

M M ÷ U

Original solution

Cytochrome c Poly(I):poly(C) Various

Actinomycin D

E D T A or A c t i n o m y c i n D 22Na+

7 : 2 : 1 Phosphatidylcholine: cholesterol :dicetyl p h o s p h a t e 7 : 2 : 1 Phosphatidylcholine: A m y l o g l u c o s i d a s e cholesterol :dicetyl p h o s p h a t e Various None

Various Phosphatidylcholine: cholesterol : dicetyl p h o s p h a t e 7 : 2 : 1 Phosphatidylcholine: cholesterol :dicetyl p h o s p h a t e Phosphatidylserine Various Various Commercial phospholipid preparation (Asolectin) Phosphatidylcholine

Additional substances present

M = multilamellar liposomes, U = unilamellar liposomes. Lipid c o m p o s i t i o n s expressed as m o l a r ratios.

36 000

6000

6 000

6 000

450 780 1 500 5 400-10 800

30

4 7

C o m p o s i t i o n o f liposomes

M

M

M

M

M M÷U M M

M

M M

Pellet

L i p o s o m e s present in

C O N D I T I O N S U S E D B Y V A R I O U S I N V E S T I G A T O R S IN L I P O S O M E P R E P A R A T I O N S

D u r a t i o n of centrifugation ( g min) × 10 -3

CENTRIFUGATION

TABLE I

U

--

--

U÷M

--U --

--

-U

Supernatant

66

4

79

78

75 76 56 77

74

5,28,70-72 73

Reference

I'J

268 4 h, since such treatment removes all but the very small vesicles from suspension. It therefore seems likely that between these two extremes there is a variety of liposome sizes each requiring a different number of g rain for sedimentation. Consequently, centrifugation may be of limited use when working with sonicated suspensions, since many small liposomes may be lost in the supernatant fractions.

IID. Liposomes and proteins 1. Liposome-protein interactions. The feature of liposomes that has made them such a widely used model is their ability to entrap solutes. In the case of proteins, however, there are a number of reports of protein interaction with liposomes as well as the many dealing with protein entrapment, and it seems pertinent to consider this, since such interactions of liposomes and proteins are of biological importance. Moreover, any liposome-protein interactions should be examined closely for their possible implications when liposome entrapment of protein is under consideration. Recently a number of purified proteins have been shown to associate with liposome bilayers and these are listed in Tables II A and B. A number of aspects of protein-lipid interactions have been examined, including the effect of proteins on the permeability of liposomes to small solutes [36,37,75,79,89] and the morphology of the protein-lipid complex produced [82,83] All these models provide useful information on the nature of lipid-protein interactions and will, no doubt, contribute to the elucidation of membrane structure. Investigations of the interactions of non-membrane proteins with liposomes have, so far, shown that, with the possible exception of immunoglobulins, positively charged protein is associating with negatively charged liposomes and vice versa, and the interaction is, at least initially, electrostatic. Kimelberg et al. [75] have some evidence to suggest that the electrostatic interaction with phosphatidylserine liposomes of the basic proteins they have studied is followed by hydrophobic interaction, since high salt concentrations inhibit the binding of these proteins, but do not reverse the binding once it has occurred. In contrast, the X-ray data of Sogor and Zull [91] show no effect of bovine serum albumin on the packing of the phosphatidylcholine hydrophobic side chains, and their diffusion studies show that the liposome-bovine serum albumin complex is not markedly different in permeability to K + and some sugars, from similar liposomes prepared in the absence of protein. Hence, hydrophobic interactions of bovine serum albumin with lipid do not appear to occur. This is interesting since the earlier work of Sweet and Zull [79] suggested that bovine serum albumin did affect glucose release from liposomes. Sogor and Zull [91] state that the earlier observation is probably due to transient disruption of the liposomes, since all early experiments were performed by adding bovine serum albumin to preformed liposomes. This might explain why bovine serum albumin interaction with negatively charged liposomes at pH 3.5 is proportional to the dicetyl phosphate concentration, and is the same whether the liposomes are sonicated or unsonicated [79]. The binding of immunoglobulins to liposomes is, perhaps, a rather special phenomenon and, as indicated by Weissmann et al. [37], seems related to observed

Phosphatidylcholine dicetyl, phosphate Phosphatidylcholine Phosphatidylcholine, cholesterol, phosphatidylserine Phosphatidylserine 9:1 Phosphatidylcholine: dicetyl phosphate Phosphatidylcholine, soyabean or microsomal lipids Phosphatidylcholine Phosphatidylcholine Phosphatidylcholine

50 49

81

82 83

77

84

78,85

86

87 88

Myelin hydrophobic protein Iafluenza virus subanits

MgZ+/CaZ+-dependentATPase

D-fl hydroxybutyrate dehydrogenase Cytochrome b5 Cytochrome bs-NADH reductase Cytochrome b5

T(is) peptide from glycophorin Cytochrome b5 Phosphatidylcholine Various

Phosphatidylcholine Beef heart mitochondria phospholipids Acidic phospholipids

80 51 36

Rhodopsin Cytochrome oxidase Myelin basic proteins P~, P2 and AI (Na +, K+)-dependent ATPase Erythrocyte sialoprotein (Glycophorin) Spectrin

Lipid composition

Reference

Proteins studied

MEMBRANE PROTEIN-LIPOSOME COMPLEXES EXAMINED

TABLE II

Effect on permeability, Stokes radius, and sedimentation coefficient of liposomes Permeability of liposomes, circular dichroism of peptides Interaction studied using a fluorescence probe

Enhancement of enzyme activity by interaction with unilamellar vesicles Interaction of cytochrome b5 and the reductase

Morphology (visualised by freeze fracture) Morphology (visualised by electron microscopy using negative staining) Effects on permeability + ultrastructure

Conditions required for interaction

Activity of enzyme in reconstituted state Morphology, + lectin agglutinability of complex

Effect on spin labelled phosphatidylcholine Activity of enzyme in reconstituted state Effect of protein on liposome permeability

Aspects examined

90 79,91

37

92 93 94

Methaemoglobin Bovine serum albumin

Immunoglobulirls

Glutamate dehydrogenase Malate dehydrogenase Streptolysin O

i

Release of solutes from liposomes

3:1 phosphatidylserine: phosphatidylcholine Phosphatidylcholine 7 : 2: 1 phospbatidylcholine: cholesterol :dicetyl phosphate 7 : 2 : 1 phosphatidylcholine , cholesterol :stearylamine 7: 2: 1 phosphatidylcholine: cholesterol :stearylamine 7: 2 : 1 phosphatidylcholine: cholesterol :dicetyl phosphate 1 : 1 Cardiolipin:phosphatidylcholine Tissue lipid extracts 6:6:1 phosphatidylcholine: cholesterol: dicetyl phosphate

89

Effect on enzyme activity Effect on enzyme activity Morphology (C shapes and rings observed under electron microscope)

~ Permeability of liposomes to sugars -v K ÷, X-ray analysis of structure Effect on permeability of liposomes

Release of Rb ÷ from liposomes i Stoicheiometry of complex, pH at which complex forms

Effect of protein on diffusion of Na ÷ through bilayer

Phosphatidylserine

75

1

Aspects examined

Cytochrome c Lysozyme Ribonuclease Poly-L-lysine Haemoglobin

Lipid composition

Reference

Proteins studied

SOLUBLE PROTEIN-LIPOSOME COMPLEXES STUDIED

TABLE 111

I,O "---...1

271 interactions of immunoglobulins and heat aggregated immunoglobulins with cells. There are perhaps a few guidelines here for those attempting to entrap proteins. It would seem advisable to work with protein and liposomes of the same charge. Failure to do so could result in interactions that are initially electrostatic, but subsequently hydrophobic, and such interactions may be irreversible. Moreover, pH should be controlled. This may seem an obvious statement, but it is rarely pointed out that many of the charged lipids used are strongly acidic or basic. It is, therefore, important to use either a salt of the lipid or to buffer adequately for the concentration of charged lipid being used. Subject to these provisions, entrapment is feasible, and likely under such conditions to be the sole reason for lipid-protein interactions. However, the work of Dodd [92] on the inhibition of glutamate dehydrogenase by cardiolipin/phosphatidylcholine sonicates contains perhaps a further caution. In this work it has been shown that a negatively charged protein binds to negative liposomes, and it emphasises the point that even if a protein is net negatively charged it does not necessarily follow that it will not bind to negatively charged liposomes. In each case the evidence for entrapment must be examined closely, and in the next section the means of proving that a protein is truly entrapped within a liposome will be examined. 2. Protein entrapment in liposomes. To date, numerous proteins have been entrapped in liposomes and these are summarised in Table IV. For many investigations it may be sufficient to generate a stable lipid-protein complex. In other cases, however, it may be necessary to entrap proteins within liposomes with no proteinlipid interaction, and for such cases one must be able to prove that entrapment is the sole reason for the association of the protein with the liposomes. In the original paper on lysozyme entrapment in liposomes, Sessa and Weissmann [57] give a number of criteria for proof of entrapment: (i) That the entrapment of an enzyme should demonstrably be a function of the internal aqueous volume of the liposomes. Their proof of this was that the entrapment of the enzyme paralleled the entrapment of glucose and that any increase in the proportion of charged lipid over 10~ did not increase entrapment. The effect of

TABLE IV PROTEINS REPORTED TO HAVE BEEN ENTRAPPED IN LIPOSOMES Protein

Reference

Protein

Reference

Lysozyme Amyloglucosidase fl-D-fructofuranosidase Human serum albumin Bovine serum albumin

57 4 58 59 68

Dextranase a-Mannosidase insulin Hexokinase I Glucose-6-phosphate dehydrogenase

60 61 9

Peroxidase Obelin Asparaginase

95-97 98 99

fl-Galactosidase Glucocerebrosidase

62

100

272 varying the lipid composition and the ionic strength of the suspension medium on the trapped volume of liposomes has been studied by Bangham et al. [27]. They found that for a given suspension medium any increase in charged lipid over 10~o did not increase trapped volume. (ii) The enzyme activity, when associated with liposomes, should not be apparent until detergent or other membrane disrupting agents are added (latency). Moreover, the unmasking of the enzyme should be simultaneously matched by the release of glucose or other small molecules from the liposomes. (iii) The enzyme should not interact with preformed liposomes if the two are mixed together. It should be emphasised that none of these points, on its own, is rigorous proof of entrapment. Enzyme latency could simply be due to adsorption of the enzyme to the lipid bilayer, with a consequent masking of activity. Increasing salt concentrations could be reducing "apparent entrapment" by inhibiting electrostatic binding rather than reducing real entrapment by reduction of the internal aqueous volume. There are, in addition, some special problems associated with the proof of protein entrapment. One cannot be sure what effect sonication may have on lipid-protein interaction, nor can one easily predict the conditions which may exist whilst the lipid is swelling in the solution. Clearly, additional methods for proving that no protein is present on the outside of the liposome, without evoking enzyme activity measurements, would be an advantage. Numerous compounds that react with proteins have been used to study the location of proteins in membranes [10t]. One of the more commonly used methods involves [12Sl]lactoperoxidase [102]. However, doubt has recently been cast on the validily of this method since it seems to label extensively both internal and external components in cell membranes including lipids [103]. liE. Liposomes and entrapment of small molecules The ability of liposomes to sequester solutes within their internal aqueous volume was first demonstrated using small molecules, and much work has centred on the permeability of liposomes to small molecules. Of particular relevance in this review is the entrapment of low molecular weight compounds of therapeutic interest within liposomes, in which instance efforts are directed towards decreasing leakage. Before discussing which drugs have been entrapped, consideration will be given to the lipid composition of liposomes, since this is of particular importance when entrapping and retaining small molecules in liposomes. Lipids capable of forming the typical liposome structure on their own include phosphatidylcholine, sphingomyelin, cardiolipin, phosphatidylserine, phosphatidylinositol, and phosphatidic acid. A number of other natural and synthetic lipids can form liposomes when mixed with the "structural" lipids mentioned above. These include phosphatidylethanolamine, dicetyl phosphate, stearylamine and cholesterol. If neutral lipids such as phosphatidylcholine are used to make liposomes, a very small interlamellar spacing is produced, and the level of aqueous phase entrapment is low. If, however, charged lipid is introduced, the bilayers repel one another, and entrap-

273 ment of aqueous phase is thus increased. Addition of charged lipid in excess of 10 mol ~ of the total, however, does not increase entrapment [27]. Increasing the ionic strength of the aqueous phase causes charge quenching and a consequent reduction in the level of entrapment. Thus, when preparing liposomes it is often an advantage to use 10 mol ~ charged lipid, and to keep buffer molarity to a minimum. Cholesterol decreases the permeability of liposomes in which the phospholipids are above their transition temperature. This reduces the level of leakage of small molecules and is thus a useful addition when entrapping small solutes. An alternative means of reducing such leakage is the use of lipids such as dipalmitoylphosphatidylcholine. This is a synthetic phospholipid with a transition temperature of 41 °C (cf. egg phosphatidylcholine which has a transition temperature o f - - 1 5 °C). Liposomes can be formed from this lipid at 50 °C and then cooled below the transition temperature. Liposomes prepared from dipalmitoylphosphatidylcholine exhibit a reduced movement of the hydrocarbon side-chains when below 41 °C, and, as a consequence, there is a reduction of the diffusion of small molecules across the bilayer. This lipid has been used with limited success in our laboratory for the entrapment of 5-fluorouracil [104], and in the entrapment of actinomycin D and benzyl penicillin [6]. It is also possible to minimise leakage when working with ionic compounds by using lipids of the same charge as the ion to be entrapped. Electrostatic repulsion reduces the rate of diffusion of the compound across the bilayer. Drugs which are lipid-soluble may be incorporated into the lipid phase of liposomes by dissolving them in a non polar solvent together with the lipid prior to the formation ofliposomes [6,105]. Whilst leakage of small molecules from liposomes can be reduced by the means described above, there are some special problems concerning leakage of drugs from liposomes. Certain drugs, such as 5-ftuorouracil, are amphiphilic and thus soluble in both polar and non-polar environments and this leads to rapid leakage from liposomes. Moreover, the interaction of proteins with liposomes can cause an increase in the rate of diffusion of small molecules across the liposome bilayers. Consequently interaction of liposomes containing small molecules with serum components will, in some cases, cause an increased leakage of drug from the liposomes, Further discussion on work related to entrapment of materials of therapeutic interest occurs in this review in Sections III and IV.

IIF. Quantitative expression of entrapment The expression of the amount of material (protein, small molecule or ion) entrapped in liposomes is often expressed as the percentage of starting material which becomes liposomally associated. This has become very misleading in comparing results from different workers for the following reasons. When the proportion of lipid used to make liposomes is increased compared with the volume of aqueous phase, the fraction of the aqueous phase enclosed between the bilayers, and hence the entrapment, will increase. Careful consideration will make it apparent that this will not cause greater entrapment per mg liposomal lipid, whereas when expressed as a

274 percentage of starting material entrapment is considerably increased. It has already been stressed that true entrapment is a function of the trapped aqueous volume and should parallel that of a marker for this such as glucose. If the entrapment of a particular molecule exceeds the value expected by this criterion, then factors such as electrostatic or hydrophobic interaction with the lipid bilayers are contributing to the amount of material which becomes associated with the liposomes. We thus propose that the entrapment of material in liposomes should be expressed as a function of liposomal lipid and, if possible, be related to the volume of the internal aqueous space.

IlG. Summary Liposomes may be prepared by a number of methods, some of which produce homogeneous suspensions and seem feasible methods for the entrapment of labile biological molecules. Proteins may be entrapped in liposomes, provided due consideration is given to the net charge of the protein and lipid, and provided that the evidence for entrapment is established for every protein. However, protein-liposome association may be sufficient for many investigations. Small molecules of therapeutic interest may be entrapped in liposomes; although there are problems involving the leakage of these compounds from the liposome, leakage can be minimised by careful selection of the appropriate lipid composition for the liposomes.

IIl. UPTAKE OF LIPOSOMES IN VIVO

IliA. Introduction Liposomes were first considered as possible therapeutic carriers of a glucamylase from Aspergillus niger in the treatment of glycogen storage diseases by Leathwood and Ryman, and successful entrapment of the enzyme was achieved, together with separation of the liposomes from free enzyme [4]. The idea has now been widely extended and a variety of other clinical applications have been proposed, such as the entrapment of drugs for cancer chemotherapy [6,7,106,107] and of chelating agents for alleviating heavy metal poisoning [5,28,70-72,108]. In this section we will discuss in vivo experiments which have been performed to demonstrate the feasibility of such applications. IIIB. In vivo fate of intravenously injected liposomes Early experiments in rats showed that protein-containing liposomes were rapidly removed from the circulation following intravenous injection and were localised mainly in the liver and spleen; only small amounts were found in other tissues [59]. This has been confirmed using entrapped EDTA [28] and various drugs [6,7,109]. Furthermore, subfractionation of the livers of animals receiving liposomes containing entrapped radioactive proteins or enzymes showed a preferential localisation of the radioactivity or enzyme activity in the mitochondrial-lysosomal fraction. Using density gradient centrifugation, the liposomes were found to be associated

275 with the lysosomal fraction [58]. Further evidence for the lysosomal localisation of liposomally entrapped protein has come from a study of the fate of entrapped and non-entrapped neuraminidase injected into rats [110]. The uptake of liposomes into the liver poses the question as to which cell type is involved in the uptake. The liver is composed predominantly of parenchymal cells, with a smaller proportion of Kupffer cells, which form part of the reticuloendothelial system and are known to be highly phagocytic. The actual distribution of liposomes between the two cell types is still not entirely clear. Early work showed by autoradiography that liposomes appeared to penetrate both types of liver cell [59]. These experiments used radioactively labelled cholesterol in the liposomal membrane, and it is possible that the phenomenon of cholesterol exchange between cell membranes and liposomes may have affected the result. Recent work has suggested that cholesterol does, in fact, exchange with blood constituents [111,112], and it is certainly known that phosphatidylcholine exchanges between liposomes in the presence of a protein purified from beef liver [113]. Thus, such exchange processes may well occur in vivo. Later work, using electron microscopy with Nitroblue Tetrazolium as the entrapped liposome marker, showed only Kupffer cell involvement in liposome uptake [114]. The same group of workers have published observations of electron micrographs of liver tissue of cancer patients given liposomes in which no liposomes could be seen in Kupffer cells but were present in about onethird of the parenchymal cells [115]. More recently, the same group has proposed that liposomes appear in Kupffer cells very soon after injection, but after a few hours they are also found extensively in parenchymal cells [97]. This work involved the use of electron microscopy to follow peroxidase entrapped in the injected liposomes. Other workers using electron microscopy have shown liposomes associated with both Kupffer and parenchymal cells, although the relative numbers seen in each cell type were not determined [71]. de Barsy et al. [116] have studied the cellular distribution of antibody-containing liposomes in the livers of new-born rats (in an attempt to interfere with normal lysosomal enzyme activity), and observed heavily swollen Kupffer cells and normal parenchymal cells, although the latter cells contained multilamellar concentric structures. Finally, using selective destruction of parenchymal cells by pronase digestion, Tanaka et al. [117] showed 7 0 ~ of liposomally associated radioactivity with the Kupffer cell fraction 15 minutes after injection. Thus, it is apparent that knowledge in this particular area is still incomplete and much of the previous work is confusing and open to criticism. An alternative approach to the problem may be the separation of homogeneous populations of Kupffer and parenchymal cells, using modifications of the Berry and Friend technique for preparing isolated liver cells [118], such as those described by Crisp and Pogson [119] or Munthe-Kaas and Seglen [120]. However, preliminary work in our laboratory has shown that neither method gives a pure population of a single cell type, and work is in progress to attempt to modify these methods for liposome uptake studies. An elegant method of demonstrating the in vivo fate of liposomes has recently

276 been produced. McDougall et al [112] have studied the fate of pertechnetate (99mTcO4-) entrapped in liposomes in mice by gamma-counting of the tissues. The technique has been extended using a scanning gamma-camera to obtain a visual image of the tissue distribution of liposomes [122]. In our laboratory [123] we have been attempting to improve the efficiency of this technique by directly binding the pertechnetate to liposomes in the presence of reducing agents such as stannous chloride and ascorbate as catalysts. We are, at present, applying this technique to a variety of problems. Preliminary scans on turnout-bearing rats have shown no uptake of liposomes by the tumours. Similarly, in a study of the uptake of liposomally-entrappeal cations into tumour-bearing mice, Kimelberg et al. [124] have shown little radioactivity in the turnouts. We are, at present, initiating a programme of scanning of human tumour-bearing patients to determine whether any particular tumour has an affinity for liposomes. It may be necessary to attach turnout-specific antibodies to liposomes to promote uptake into tumours, and this scanning technique will prove invaluable in such studies. II1C. Experimental models to test the efficacy of liposomes as carriers of therapeutic agents The ability of liposomes to deliver therapeutic agents in vivo has been investigated using animal models. If dextran is administered intraperitoneally to rats, a proportion of the dose is taken up and stored in the liver. When dextranase entrapped in liposomes is administered intravenously to such dextran-loaded rats, the stored dextran is broken down (as detected by the loss of 3H label after administration of [3H]dextran), thus giving an experimental model of the treatment of a storage disease [60,67]. In another study the therapy of mannosidosis (a disorder associated with loss of a zinc dependent a-mannosidase activity and accumulation of mannose rich material) has been simulated by administering a-mannosidase entrapped in liposomes to zinc-deficient rats [61]. Recently, amyloglycosidase-containing liposomes have been administered to a glycogen storage disease patient, with an apparent reduction of liver glycogen and a marked decrease in liver size [69]. In the therapy of storage diseases it would obviously be an advantage if the half-life of proteins delivered to lysosomes by liposomes could be increased. Attempts have been made to do this by cross-linking proteins using glutaraldehyde [125] or by concurrent treatment with cathepsin inhibitors, such as pepstatin and chloroquine, in liposomes [126], the latter method with little success. Recently, Dean [127] has used liposomes containing pepstatin in a perfused liver system to demonstrate the importance of lysosomes in the degradation of intracellular proteins. He has also [128] investigated the binding of cytoplasmic proteins to liposomes in a model study of a possible mechanism for the uptake of such proteins into lysosomes. Investigating the possibilities of liposomes as carriers of chelating agents in the therapy of heavy metal poisoning, Rahman et al. [106,108] have shown the disappearance of plutonium from livers of mice treated with diethylenetriaminepentaacetic acid (DPTA) entrapped in liposomes. Finally, one application which has proved

277 unsuccessful is the use of liposomally entrapped glutathione. Glutathione in the free state protects against paracetamol-induced liver necrosis in rats; entrapped glutathione is less effective [129].

IIID. Effect of liposome modification on uptake by tissues It is appreciated that only a few diseases affect solely the liver (the predominant site of uptake of intravenously injected liposomes) and attention has, therefore, turned to finding a means of directing liposomes to other tissues. This has led to a study of the distribution of injected liposomes following modifications in charge, size lipid composition and attachment of macromolecular probes to the surface. The route of administration has also been varied. These factors will now be considered in relation to the types of application that such a modification might have. The basic lipid composition of liposomes used in early in vivo experiments was a mixture of phosphatidylcholine, cholesterol and dicetyl phosphate (or phosphatidic acid), frequently in a molar ratio of 7 : 2 : 1. The first step was to investigate the effect of changes in composition on liposome uptake and subsequent tissue distribution. However, for reasons which will become apparent, the effect of varying the size of the liposomes will be discussed before the effects of varying composition and charge, although the chronological order of experiments to investigate these factors has been somewhat the reverse. It is also important to make the distinction between the rate of uptake and the amount of uptake into various tissues, since this can be confusing when comparing different sets of results. The methods available for obtaining populations of liposomes homogeneous with respect to size have been discussed in Section lIB. Until recently, studies on factors other than size were carried out using a mixed population of liposomes of a wide range of sizes. In vivo the size effect is best demonstrated by the work of Juliano and Stamp [56]. They showed a longer half-life in the circulation for unilamellar as compared with multilamellar structures, using neutral or positively charged liposomes. They could discern no differences using negatively charged liposomes containing phosphatidylserine, although in our laboratory we have also found differences in half-lives for large and small anionic liposomes containing dicetyl phosphate [126]. Juliano and Stamp showed a two phase clearance of liposomes from the circulation, as had previously been observed by Gregoriadis and Neerunjun [125]. This involved a rapid phase of clearance followed by a slow phase. Using a preparation of liposomes homogeneous with respect to size, Juliano and Stamp showed a more linear clearance of liposomes from the plasma, suggesting that the two phase clearance might be due to size heterogeneity. They also found, in agreement with Gregoriadis and Neerunjun [125], that negatively charged liposomes were cleared most rapidly although, in contrast, they found no difference in the clearance of positively charged and neutral liposomes. Turning attention to the tissue distribution of liposomes, the only significant difference with respect to size is that the spleen appears to have a marked preference for large liposomes [126]. Other published experiments to determine tissue

278 distribution following modifications in lipid composition and charge have no defined rigorous criterion of size homogeneity and hence care has to be exercised in comparing results. Jonah et al. [72] have published extensive data showing the variation in tissue distribution using liposomes of a wide variety of lipid compositions. In another study Rahman et al. [105] claim to have shown a difference in tissue distribution when actinomycin D was entrapped either in the lipid or aqueous phase of liposomes. However, when entrapped in the aqueous phase the drug leaks rapidly. In vivo tissue distribution differences could be explained by the in vivo leakage of the drug which then behaves as if administered free. It should also be noted that the lipid compositions of liposomes used when the drug was entrapped in the lipid or aqueous phase were different. It is difficult to imagine why a drug leaks from the aqueous phase of liposomes and not from the lipid phase, since in the former case the drug must pass through the lipid layers to escape, i.e. it must pass through the environment in which it remains in the latter case. These authors also report [72,105] a relatively high concentration of liposomally entrapped material in the lungs shortly after injection. This may well be a result of the large unsonicated liposomes used, as there have been no such reports from other workers using sonicated liposomes. Colley and Ryman [7] have shown changes in the tissue distribution of methotrexate-containing liposomes of different compositions. However, these are relatively minor variations, as most of the injected liposomes are taken up by the liver. In our laboratory [126] we have extracted lipids from a variety of rat tissues. On injecting liposomes made from these into rats we found no differences in tissue distribution which cannot be explained by charge and size effects. It can be inferred from these experiments that the only tissues having a great affinity for liposomes are the liver and spleen, and that the relative rates at which these tissues take up liposomes and the amounts which they take up appear to be governed by the charge and size of the liposomes only. These results were foreshadowed by the work of van Deenen [130] who injected lipid extracts of rat thyroid gland into other rats and found that these, which were almost certainly liposomes, were taken up predominantly by the liver and spleen. It has thus become apparent that more subtle modifications of the liposome membrane will be necessary to achieve any altered tissue distribution and specific direction of liposomes to target cells or tissues. The method which has been proposed for the specific direction of liposomes is still very much in its infancy, but remains promising. This involves the attachment of target specific molecules, such as antibodies, to the outside of liposomes. There is no reported experimental work using tissue specific antibodies in vivo, although preliminary work has been done in vitro [131,132]. The use of desialated glycoproteins incorporated into the liposomal membrane has led to a slight increase in liver uptake of liposomes [132]. Clearly, much remains to be discovered concerning the precise mechanism of liposomal uptake before surface alterations can be made to alter the process. The main problem at present appears to be that of trying to avoid liposome uptake by the liver whilst, at

279 the same time, attempting to make the liposome attractive to another tissue. Tanaka et al. [117] have reported a decrease in the hepatic uptake of liposomes when rats are preinjected with methyl palmitate. However, there may exist some doubt concerning the validity of these observations, since the lipid composition used (phosphatidylcholine :cholesterol :dicetyl phosphate, in a molar ratio 3:9:1) contains more cholesterol than can be incorporated into this amount of phosphatidylcholine in the form of liposomes [21,22]. Present investigations are concerned with attempting to determine interactions between liposomes and serum components in order to gain insight into processes which might occur before liposomes can enter cells. Black and Gregoriadis [111 ] have shown the association of an az-macroglobulin with liposomes, irrespective of the charge on the liposome, and other work from our own laboratory has shown that serum components may indeed have an effect on liposome uptake in vitro and in the perfused liver [133]. This is further discussed in Section IVB. IIIE Alternative routes of administration of liposomes Several studies have been carried out using routes of administration other than the intravenous route used in the experiments so far described. Several routes have been studied by McDougall et al. [122] using the scanning technique which has been considered earlier (Section IIIB). Rahman et al. [106,134] have used the intraperitoneal route to study the effectiveness of liposomally entrapped actinomycin D against ascitic tumours in mice. A similar study has been reported more recently [135]. The fate of intravenously administered liposomes containing actinomycin D has been documented [109,136]. In experiments using the intraperitoneal route the drug was found to have reduced toxicity when given in liposomes, and it also increased the survival times for tumour-bearing mice [106]. However, in contrast, the intraperitoneal administration of methotrexate-containing liposomes to tumour-bearing mice increased the toxicity of the drug at least one hundred-fold compared with the free drug [137]. In other work, liposomally entrapped asparaginase has been found to be effective in completely curing tumour-bearing mice, but doses needed were in excess of those required when the enzyme was non-entrapped. However, liposomal entrapment protected against adverse immunological reactions [99]. The oral route of administration has recently become of great interest with the findings of Patel and Ryman [9] that insulin administered orally in liposomes (phosphatidylcholine:cholesterol:dicetyl phosphate, molar ratio 10:2: 1) can cause a reduction in blood glucose in diabetic rats, whereas free insulin given orally causes no change. More recently, Gregoriadis et al. [138], using normal rats, found a reduction in blood glucose only when the insulin-containing liposomes were made from phosphatidylinositol. In order to determine the suitability of liposomes as vehicles for the local release of drugs, Segal et al. [74] injected liposomes (containing various radiolabelled compounds) directly into the rat testicle and found that large multilamellar liposomes delayed the release of entrapped substances. Similar results were found by Gregoriadis et al. [138] using the intraperitoneal and intramuscular routes for administration of

280 polyvinylpyrrollidone-containing liposomes. Furthermore, Arakawa et al. [139] examined the release of various liposome-associated compounds (including insulin) from intramuscular sites and showed that some delay in absorption occurs when compared to the free compound. As in the work of Tanaka et al. [117] they use a proportion of cholesterol above that which is generally thought to be the maximum incorporated into liposomes. Finally, some recent work has involved the direct administration of liposomes into the brains of experimental mice in order to determine the suitability of such vesicles as carriers of drugs in the treatment of tumours and viral infections of the central nervous system [140], and the reported toxicity of dicetyl phosphate when used in the preparation of the liposomes is of considerable interest. The pharmacological effects of intravenously injected sonicated phosphatidylserine liposomes have been investigated by Bruni et al. [141]. They found an increase of glucose levels in the blood and brain and a decrease in adrenal catecholamine content in mice injected with such liposomes. These observations need to be further extended to establish whether these effects taking place in the central nervous system reflect liposome penetration into brain fluids. IIIF. Summary Considerable advances in the knowledge of the in vivo fate of liposomes have been made in the past few years and novel methods, such as the scanning technique using 99roTe-labelled liposomes, have been evolved to follow their uptake. However there still remain areas where considerable work is necessary to elucidate precisely the processes involved in this uptake. In particular, the relative importance of the Kupffer and parenchymal cells of the liver in removing liposomes from the circulation is still uncertain and, as yet, there is no clear knowledge of particular interactions of liposomes with serum components. If, in the future, it proves possible to direct liposomes to specific tissues, the therapeutic applications will be manifold although, at present, the use of liposomes as carriers of therapeutic agents is limited by their rapid uptake by the liver and spleen.

IV. INTERACTIONS OF LIPOSOMES WITH CELLS IN CULTURE IVA. Introduction The rapid expansion of tissue culture in recent years has afforded another system in which to study the interactions of liposomes with living cells. In the past, liposomes have yielded considerable information about the mechanisms of membrane permeability and transport. Present ideas on membrane structure propose that bilayer regions of membranes are involved in intermembrane phenomena such as fusion. Thus, liposome-cell systems have become very important in a study of such processes.

281

IVB. Methodology Experiments on the interactions between liposomes and cultured cells have to date followed the same general pattern. It is thus pertinent to begin with a brief discussion of the way in which such experiments have been carried out. Many different cell lines have been used in these studies, mainly monolayers of fibroblasts, with occasional use of other cell types in suspension culture. As with in vivo work (Section III) there has been the usual diversity in the methods used to prepare the liposomes themselves. This has been discussed in Section II and will only be mentioned here if considered of relevance to the conclusions drawn from a particular piece of work. It should be noted that the term "vesicles", as used in this section, is synonymous with unilamellar liposomes. This has been adopted in accordance with the terminology found in the literature on the interaction of liposomes with cells in culture. In general, fibroblasts are grown to confluent monolayers on glass or plastic and incubated with liposomes either in complete growth medium (containing serum) or in a simple balanced salt solution. At the end of the incubation period the ceils are washed thoroughly with several changes of medium and then harvested by traditional tissue culture methods such as trypsinisation or simply scraping the cells from the surface. Cells in suspension culture are incubated with liposomes in growth medium and separated from the liposome-containing medium by a series of washings and low-speed centrifugations. Ceils can then be analysed for markers initially present in the lipid layers or the aqueous spaces of the liposomes. These markers are usually radioactive tracers or possibly enzymes. Tissue culture lends itself to the techniques of both light and electron microscopy and these have been extensively used in following the fate of liposomes in these experiments. Variations in the uptake and the type of process involved can thus be investigated by variations in the composition, charge, size and fluidity of the liposomal membrane. One of the aspects which should be considered in the interactions of liposomes with cells in vitro, and indeed which has already been mentioned in relation to in vivo work in Section Ill of this review, is the importance of serum components. As has been indicated, culture experiments on liposome uptake into cells have been carried out in the presence and absence of serum. In the presence of serum, liposomes are exposed to a variety of proteins, and it is probable that interactions occur with these proteins, some of which may lead to liposomal attachment and recognition by cells. Indeed, our preliminary work has shown that a number of these factors have an effect when liposomes are incubated with cultured cells. Supporting the work of Black and Gregoriadis [111], the a globulin fraction reduces the attachment of positively charged liposomes to cells, suggesting the charge on the liposome is altered. In addition, serum albumin seems to promote cholesterol exchange with ceils when radiolabelled cholesterol is used as a liposomal marker [133]. Some of these interactions are summarised in Table V. In addition, studies have previously been carried out concerning the interaction of serum and tissue phospholipases with liposomes as models for protein/phospholipid

282 TABLE V SOME EFFECTS OF SERUM COMPONENTS ON THE INTERACTIONS OF LIPOSOMES WITH CELLS Serum component

Effect

Reference

Albumin ~-Globulins

Promotes transfer of cholesterol from liposomes to cells Give negative charge to positive and neutral liposomes Reduce attachment of positive liposomes to cells Reduce attachment of positive liposomes to cells

133 111 133 133

/~-Globulins Immunoglobulins 1 Heat-aggregated H immunoglobulins ~ Heat-aggregated immunoglobulins

Interact with liposomes and increase permeability Promote phagocytosis when attached to liposomes

37 96, 152

interaction [225]. Any possible attack by such enzymes on liposomes should ~dso be considered both in vivo and in vitro.

IVC. Earlier observations The first observations of the interactions between liposomes and cells in culture were made by Magee and Miller [142] who attempted to define the conditions for liposomal attachment to cells. Using multilamellar liposomes and a monolayer nf M L cells they found that cationic liposomes (stearylamine supplying the positive charge) attached almost instantaneously to cells, but little or no association of anionic (dicetyl phosphate-containing) liposomes was found. They also demonstrated that the cells were protected from virus infection by cationic liposomes containing antiviral antibody to a degree of up to 10000 times that afforded by the free antibody under the same conditions. An extension of this work was a combined in vivo and in vitro study of the induction of interferon by poly (I):poly(C) associated with cationic liposomes [143]. In vivo in mice, liposomes containing poly(I):poly(C) were much more effective than free poly(I):poly(C) in inducing serum interferon. In vitro using L-999 mouse cells, the liposome associated material was not, however, very effective in stimulating anti-viral resistance. IVD. Uptake of liposomally entrapped materials into cultured cells As a result of early work in experimental animals, the advocates of [iposomal entrapment of enzymes as a potential method of enzyme replacement therapy investigated systems to assess the feasibility of such a carrier; cells in culture have provided such models. Gregoriadis and Buckland [144] showed that fl-fructofuranosidase (invertase) entrapped in anionic liposomes could cause the disappearance of vacuoles of stored sucrose (induced by exposure of the cells to the disaccharide) in mouse peritoneal macrophages and human embryo lung fibroblasts. More recently [145,146] a report has been made of the use of amyloglucosidase entrapped in anionic liposomes to reduce glycogen levels in skin fibroblasts from a patient with Type II

283 glycogen storage disease (Pompe's disease). The fate of horseradish peroxidase, liposomally administered to cells in culture, has also been followed [147]. This enzyme is a useful marker as its activity can be followed by electron microscopy [148] as well as by conventional enzyme assay. The authors report that initial interaction between cationic liposomes and monolayers of Hela cells appears to be electrostatic, but they were unable to tell whether liposomes entering cells did so by phagocytosis or fusion. Much of the entrapped enzyme did not find its way into cells and a considerable degree of liposomal aggregation on the outside of cells is reported. Pinocytosis of free horseradish peroxidase by fibroblasts has since been reported [149]. Recently the uptake of actinomycin D-containing liposomes into tumour cells which are resistant to this drug has been studied [150]. The resistant cells have reduced permeability to the drug, and its introduction by means of liposomes restores their sensitivity. This provides a novel approach to cancer chemotherapy in vitro.

IVE. Mechanism of liposome uptake into cultured cells The relative importance of the two types of process, i.e. phagocytosis and fusion, by which liposomes may enter cells has formed a major area of interest in tissue culture experiments. A series of papers has been published on the subject using a wide variety of cell systems but there seems, as yet, no absolute definition of the conditions required for one process or the other. It is perhaps of interest to note here some early experiments on the activation and release of lysosomal enzymes from isolated leucocytic granules by liposomes [ 151 ]. Although not strictly in tissue culture, the experiments were designed to study factors responsible for the fusion of lysosomal and vacuolar membranes during phagocytosis. Liposomes were inactive in producing degranulation unless the charged lipid dicetyl phosphate was present in the liposomat membrane [151]. The phagocytosis of liposomes by cells has been studied by Weissmann et al. [96], who demonstrated that peroxidase-containing liposomes were incorporated into the phagocytes of the smooth dogfish (Mustelis canis) by phagocytosis to a much greater extent when coated with heat aggregated isologous IgM. The enzyme was followed into the lysosomal apparatus of the cells by ultrastructural histochemistry. More recently [152] this group has shown that heat aggregated IgG promotes the uptake of hexosaminidase A-containing liposomes into the lysosomes of Tay-Sachs leucocytes. The fusion of mammalian cells in vitro has become an area attracting much attention in an attempt to investigate cellular control mechanisms and the interactions between viruses and cells. Martin and MacDonald [153] have used lipid vesicles as model viruses and studied the attachment to the "host cell". Haywood [154] has studied the "phagocytosis" of Sendai viruses by liposomal model membranes. The virus particles were only enveloped by liposomes containing gangliosides which serve as Sendai virus receptors. Lysophosphatidylcholine and many other agents have become well known tools for the induction of cell fusion in vitro [155]. Papahadjopoulos et al. [156] showed

284 how mammalian cells could be fused in vitro by unilamellar lipid vesicles, and investigated the effect of charge, fluidity and cholesterol content. Anionic vesicles containing phosphatidylserine were necessary for fusion, which was increased when lysophosphatidylcholine was present in the vesicles, although this was more toxic to ceils. Fluid vesicles (in which the lipids are above their transition temperature) gave more fusion than solid vesicles (below the transition temperature). These studies have been extended to fusion between vesicles [157]. Calcium ions were found to be necessary for vesicle-vesicle fusion and the results obtained closely paralleled those found for vesicle-cell fusion. The kinetics of Ca 2÷ induced vesicle fusion have also been studied [158]. More recently concanavalin A has been used as an agent to induce vesicle-vesicle fusion [159]. There has been a series of papers attempting to distinguish between endocytosis and fusion of vesicles by cells. Grant and McConnell [160] showed that solid phase dipalmitoylphosphatidylcholine vesicles fused with the mycoplasma Acholeplasma laidlawii. The foreign lipid was incorporated into the mycoplasma membrane. However, only a small portion of vesicle contents (radiolabelled deoxyglucose) were transferred to the organism. Papahadjopoulos et al. [8,161,162] have studied the uptake of vesicles into 3T3 and L929 cells, and erythrocyte ghosts. They report that the charge of the vesicles or the presence of azide or iodoacetate (inhibitors of energy coupled processes) have little effect on uptake. Cyclic [3H]AMP incorporated into the vesicles inhibited the growth of cells when administered in fluid vesicles but not in solid vesicles (although in the latter case considerable numbers of vesicles were taken up by cells). This suggested that endocytosis may be a predominant process in the latter case, vesicles and vesicle contents being taken into the lysosomal apparatus. These results have been criticised by Batzri and Korn [48] who postulate that the cyclic AMP was associated with, rather than entrapped in, the positively charged vesicles and may have interfered with cell growth by processes not involving fusion. Further work by Poste and Papahadjopoulos [163] has followed the uptake of liposomes into cells using radiolabelled dipalmitoylphosphatidylcholine incorporated into the vesicles with radiolabelled sucrose entrapped. They report similar results to those when following cyclic [3H]AMP uptake in that it is the physical state of the lipids in the vesicles which determines the predominant pathway of uptake. The work of Batzri and Korn [48,164] has been a study of the interaction of vesicles with the plasma membrane of Acanthamoeba castellonii. Using inhibitors of pinocytosis, and visualizing the process by electron microscopy, they propose that at the optimum temperature of uptake unilamellar egg phosphatidylcholine vesicles are endocytosed, whereas multilamellar egg phosphatidylcholine and unilarnellar dipalmitoylphosphatidylcholine vesicles fuse with the cell membrane. A third group has also studied these processes using another system. Huang and Pagano [165,167] investigated the interaction of vesicles with Chinese hamster fibroblasts and proposed a rather complex two-step mechanism for fusion. Because of the insensitivity of vesicle uptake to inhibitors of energy metabolism, they restrict themselves to a discussion of fusion, lipid exchange and simple adsorption to

285 cell surfaces as being factors responsible for uptake. By comparison of uptake of radiolabelled lipids and markers for the aqueous space they suggest that fusion is the major process at 37 °C (90~) and the remainder is probably accounted for by phospholipid exchange. These experiments were carried out using uncharged vesicles. The process of lipid exchange is one which should be carefully considered in any study of vesicle-cell interactions. There have been reports that some tissues such as heart [168], liver [169-171] and brain [172] contain specific exchange proteins which catalyse the transfer of phospholipids and cholesterol between membranes and liposomes [113,173-180]. The possibility that such exchanges might lead to difficulty in interpretation of data obtained from liposome-cell interactions when radiolabelled lipids are used to follow liposome fate is obviously a very real one, both in vitro and in vivo (see Sections IIIB and IVB). As mentioned in Section IA, much of the work concerning the interaction of liposomes with cells has recently been summarised by Poste et al. [18]. IVF. The role of cholesterol in cell membranes The plasma membranes of red blood cells, bovine lymphocytes and Ehrlich ascites tumour cells can be enriched with cholesterol by incubating with liposomes containing a high proportion of cholesterol [181-183]. These in vitro observations can be explained by processes such as exchange/transfer, endocytosis and fusion, depending upon the cell type and the liposomes used [182]. lnbar and Shinitzky [184,185] have studied the role of cholesterol in an ascites form of virally transformed lymphoma ceils. The increase in cholesterol in the membranes of these cells, caused by incubation with 1 : 1 phosphatidylcholine :cholesterol liposomes, inhibited development of the tumour in vivo. This leads to the suggestion that cholesterol may have a special role in determining the fluidity of cell membranes, especially those of malignant ceils. Indeed, it has been proposed [186] that nuclear magnetic resonance studies can differentiate between normal and malignant tissue; this is presumably due to a difference in membrane fluidity. Liposomes have also been used [187] to enrich the cholesterol content of platelets in an attempt to assess the role of cholesterol content in platelet aggregation (induced by adrenalin or ADP, or in serotonin release). Another interesting piece of work concerning cholesterol in membranes has been carried out by Johnson [73]. The uptake of liposomes into macrophages in culture has already been mentioned [144]. Johnson [73] found that macrophages were unable to digest liposomes containing a high proportion of cholesterol. This result may have immunological significance and will be referred to again in Section V of this review. IVG. Direction of liposomes to target cells One of the most interesting and potentially most useful applications of liposomes is the possible directing of them to target cells. This has recently been achieved in a tissue culture system [131,132]. Liposomes containing an anti-tumour drug and associated with antisera to various cell lines were found to have a greater affinity for

286 those cells to which the particular antiserum had been specifically raised. This is the first demonstration of the specific direction of liposomes using macromolecular probes.

1VH. Summary In summary, it is apparent that cells in culture provide a very useful model system in which to study the potential use of liposomes as tools for modification of cell behaviour. It may be possible to introduce new molecules and biologically active materials into different compartments of cells and different cells by modification of the liposomal membrane. Experiments in culture have so far provided a substantial basis for such studies. The present picture, with regard to the mechanism of vesicle uptake into cells, is still not entirely clear, but with a growing pool of experimental data the relative importance of the various processes, e.g. endocytosis, fusion, etc., should become defined.

V. IMMUNOLOGICAL ASPECT OF LIPOSOMES

VA. Antigenic lipids It has been known for a long time that many naturally occurring lipids display antigenic properties, and may thereby give rise to the production of, and interact with, antibodies. A comprehensive review of this subject has been published by Rapport and Graf [188]. Possibly the best known example of an antigenic lipid is cardiolipin, antibodies to which are present in patients with syphilis [188]. It is only recently, however, that the importance of the bilayer structure in producing antisera to lipids has been realised. Moreover, it is possibly true to say that, in studies of antigenic lipids, the liposome structure was playing an important role before the investigators concerned realised that they were, in fact, using liposomes. Rapport [189] demonstrated an absolute requirement for phosphatidylcholine when producing antibodies to cytolipin H. lnoue and Nojima [190] found that a mixture of cardiolipin, phosphatidylcholine, cholesterol and methylated bovine serum albumin was effective in producing specific a ntisera to cardiolipin. Elimination of cholesterol produced little effect but, just as for cardiolipin H, the absence of phosphatidylcholine produced a non-immunogenic preparation. Kataoka and Nojima [191] showed that a similar mixture of phosphatidylinositol with phosphatidylcholine, cholesterol, and methylated bovine serum albumin was effective in producing antisera to phosphatidylinositol. In addition, they demonstrated that removal of phosphorylcholine from the phosphatidylcholine with phospholipase C after formation of the antigen preparation did not affect immunogenicity, although pretreatment of the phosphatidylcholine with phospholipase C rendered it ineffective as an auxiliary lipid. It seems likely that in all these studies a membrane-like structure is being formed which thereby presents the lipid in an antigenically active form.

287 The importance of the bilayer structure for the formation of antisera to lipids has been amply demonstrated by the recent work of Kinsky and his co-workers [192]. They have synthesised a series of lipid antigens which they have used to immunise animals. Initially Uemura et al. [192] raised antisera to dinitrophenylcaproylphosphatidylethanolamine (Dnp-cap-phosphatidylethanolamine) and dinitrophenylcaproyllysophosphatidylethanolamine (Dnp-cap-lysophosphatidylethanolamine) by incorporating the antigen into liposomes (sphingomyelin :cholesterol :dicetylphosphate 7:2: 1) and mixing the liposomes with complete Freund's adjuvant prior to injection. The antigen with complete Freund's adjuvant but without liposomes was ineffective. A non-immunogenic preparation was also produced if the antigen was mixed with preformed liposomes and then mixed with complete Freund's adjuvant. An adjuvant was necessary to produce an immune response. Although the best adjuvant for this purpose was complete Freund's adjuvant, incomplete Freund's adjuvant, and also lipopolysaccharide from Salmonella minnesota, did cause the production of low-titre antisera to the lipid antigen incorporated into liposomes. Liposomes made from sphingomyelin:cholesterol:dicetylphosphate (molar ratio 7:2:1) and sphingomyelin :cholesterol :stearylamine (molar ratio 7:2:1) were equally effective in causing an immune response to incorporated antigen, and proved to be more effective than liposomes made from phosphatidylcholine:cholesterol:dicetylphosphate (7:2:1). The authors [192] suggest that this may be due to the greater stability of sphingomyelin liposomes. In a subsequent paper, Uemura et al. [193] have shown, in contrast to earlier findings, that liposomes containing Dnp-cap-phosphatidylethanolamine without adjuvant can produce an immune response, although this is very much lower than that obtained when complete Freund's adjuvant is used. The synthesis and study of the lipid antigen azobenzenearsonyltyrosylphosphatidylethanolamine (ABzAs-Tyr-phosphatidylethanolamine) by Nicolotti et al. [194] has proved a most interesting addition to the field. The soluble hapten, azobenzenearsonyltyrosine (ABzAs-Tyr), produces only a cell mediated response and no humoral response; animals immunised with this compound produce a delayed hypersensitivity reaction when given an intradermal injection of azobenzenearscnyl bovine serum albumin, but have no antibodies to ABzAs-Tyr in tbeir blood. If, however, an animal is immunised with azobenzenearsonyl bovine serum albumin it prcduces antibodies but no cell mediated response. ABzAs-Tyr-phosphatidylethanolamine in liposomes induces both humoral and cell mediated immunity when used as an immunogen. Furthermore, it is possible to elicit delayed hypersensitivity in primed animals using both azobenzenearsonyl bovine serum albumin and ABzAs-Tyr-phosphatidylethanolaminein liposomes. Thus, it may well prove possible to use ABzAs-Tyr-phosphatidylethanolamine in liposomes to study the cell mediated immune response in vitro. The mixing of ABzAs-Tyrphosphatidylethanolamine and Dnp-cap-phosphatidylethanolamine in the same liposomes, and the use of these "hybrid" liposomes to immunise guinea pigs, produces two interesting results described by Kochibe et al. [195]. The presence of ABzAsTyr-phosphatidylethanolamine stimulates the production of antibodies to Dnp-

288 cap-phosphatidylethanolamine, whilst increasing amounts of Dnp-cap-phosphatidylethanolamine inhibit the humoral, but not the cell mediated, response to ABzAsTyr-phosphatidylethanolamine.

VB. Complement-mediated lysis of liposomes A most interesting area of research which has grown up alongside recent studies on the immunogenicity of antigenic lipids in liposomes, discussed above, is the complement-mediated lysis of liposomes. The system was first developed by Kinsky and his collaborators, and a review of the early work has been documented by Kinsky [[4]. It has been found that liposomes containing glucose, and having a lipid antigen incorporated into the lipid bilayers, release the glucose in the presence of antiserum to the lipid antigen and complement, whilst if either antiserum or complement was omitted no glucose was released. Moreover, no glucose was released by antiserum and complement from liposomes not containing the specific antigen concerned. Clearly this lysis of liposomes by complement is an excellent model which parallels the normal action of complement upon cells, in which antibody binds to the cell surface antigen, followed by the "cascade" of complement components [196], and culminates in cell lysis. Early experiments were performed using liposomes made from whole sheep erythrocyte lipid and antisera raised in rabbits to sheep erythrocytes. Complementmediated lysis of liposomes has now been shown to occur with a range of pure lipid antigens including galactocerebroside [197], globoside [197], lipid A and lipopolysaccharide from S. minnesota [198,199], galactolipids [200], and sialosphingolipids from sea urchins [201]. In addition, Kinsky et al. have used a range of synthetic phospholipid antigens including dinitrophenyl-phosphatidylethanolamine (Dnpphosphatidylethanolamine), dinitrophenyllysophosphatidylethanolamine (Dnp-lysophosphatidylethanolamine) [202], fluoresceinisothiocyanylphosphatidylethanolamine [203], dinitrophenylcaproyllysophosophatidylethanolamine[203] and, finally, azobenzenearsonyltyrosylphosphatidylethanolamine[194]. Significantly, the N-substituted glycerophosphorylethanolamines are incapable of inducing complement-mediated lysis since they do not form a part of the lipid bilayer and, therefore, cannot cause binding of antibody and subsequent complement damage [192]. The antigen :phospholipid ratio found to give maximal release of glucose was established as 1:250 [197] for Forssmann hapten, a lipid antigen. In contrast, for Dnp-phosphatidylethanolamine and Dnp-lysophosphatidylethanolamine an antigen:phospholipid ratio of 1:25 was required [202] for maximal release. The reason this high level of antigen was required proved to be the dissimilarity between the antigenic determinant of the lipid antigen (dinitrophenol) and the antigenic determinant of the dinitrophenyl bovine serum albumin used to raise antisera (dinitrophenyllysine). This was established when Dnp-capphosphatidylethanolamine was synthesised and found to be required in similar quantities to the naturally occurring lipid antigens in order to cause maximal glucose release from liposomes with lipid antigen incorporated [203]. This was expected, since dinitrophenylcaproate is very similar in its structure to dinitrophenyllysine. Sphingomyelin liposomes were shown to release less of their entrapped glucose under

289 maximal release conditions that phosphatidylcholine liposomes [14]. Confirmation has come from Alving et al. [200] who suggest that this may he due to a partial burying of the antigenic determinant within the hydrophobic region of the bilayer, which may occur since the average fatty acyl chain length of naturally occurring sphingomyelins is greater than that of naturally occurring phosphatidylcholines. According to Kinsky [14], most lipid antigens must be incorporated "actively". That is, they must be introduced by dissolving them with the liposome-forming lipid in organic solvent, prior to drying down and resuspension in aqueous solution. This clearly indicated that the antigen must be a part of the lipid bilayer in order to cause complement lysis. The only lipid antigens that can be incorporated "passively", that is, introduced after the liposomes have been formed, are substances which may be expected to associate with the liposomes in aqueous solution, such as the N-substituted derivatives of lysophosphatidylethanolamine [192.202]. The complement-mediated lysis of liposomes has proved an important tool in a number of ways. Since complement and antibody have been shown to act on a model lipid membrane in much the same way as they act on cells, this provides strong incidental evidence that cell membranes contain regions of exposed lipid bilayer. Moreover, since in the complement-mediated lysis of liposomes pure components are used, it has been possible to show that the action of complement does not involve the degradation of lipids, since thin-layer chromatography of liposome phospholipids after complement lysis of phosphatidylcholine liposomes revealed no degradation products [204] and, indeed, phosphatidylcholine analogues not susceptible to phospholipase attack but capable of forming liposomes when used did not in any way inhibit complement-mediated lysis [205]. Several workers have used this system to detect antibodies to antigenic lipids [194,198,199,202,203], and Knudsen et al. [206] have used it to assay complement. Alving and his co-workers [207,208] have used the complement-mediated lysis of liposomes to study the inhibition of complement action by retinal. In addition to glucose, a number of other water-soluble compounds have been shown to be released from liposomes by complement. Kataoka et al. [62] have demonstrated the release of enzymes from liposomes containing lipid antigen by complement lysis. The largest of the enzymes studied was fl-galactosidase, which is of similar dimensions to the complement "pits" observed in cell membranes with the electron microscope. To attempt to clarify the nature of the lesions in the complement attack on lipid membranes, Wobschall and McKeon [209] have studied step conductance increases in bilayers induced by antibody-antigen-complement action. Six et al. [210] have developed a more sensitive system involving the release of umbelliferyl phosphate (UmP) from liposomes. This is then cleaved by alkaline phosphatase to give the fluor umbelliferone which may be detected fluorimetrically and gives a very sensitive monitoring of solute release from liposomes. Prior to the development of this method, only multilamellar, unsonicated liposomes had been used to study complement lysis, since it was not possible to detect a release of glucose from highly sonicated unilamellar vesicles [206]. Using UmP, however, release of solute from

290 unilamellar liposomes could be detected, and under optimal conditions the level of release was found to be 9 0 ~ of the entrapped UmP, a much higher level than found for multilamellar liposomes, probably explained by the fact that for the latter the innermost bilayers are not damaged by complement. The situation is complicated, however, by the findings of Humphries and McConnell [211], who do not observe any release of a spin label from small liposomes, with lipid antigen incorporated, by the action of appropriate antibody and complement. Since a method involving a spin label should be even more sensititive than a fluorescent method [210], this leaves the situation requiring further clarification.

VC. The adjuvant properties of liposomes In addition to the importance of [iposomes in the production of antibodies to antigenic lipids, liposomes also have an adjuvant effect upon protein antigens. This was first demonstrated by Allison and Gregoriadis [212] using diphtheria toxoid as an antigen, and has subsequently been confirmed and expanded in our own laboratory using bovine serum albumin [68]. It is fairly clear that the adjuvant property of liposomes is a physical rather than a chemical effect. Data reviewed in Section Ill shows that when injected intravenously, liposomes are taken up mainly by the liver and spleen. Interestingly, intravenous administration is a very poor method of immunising mice with material in liposomes; intraperitoneal and subcutaneous administration being routes which lead to a greater immune response [68]. It seems likely that liposomes injected by these two routes will remain at the site of injection for a long period. Thus, liposomes may well exert their adjuvant effect by the same method as many other particulate adjuvants, that is, by retaining a "depot" of antigen at the site of injection. The phagocytosis of liposomes by cells such as macrophages may also be an important factor. This is suggested by the fact that the incorporation of more than 30 tool ~,, cholesterol into liposomes markedly reduces the immune response to the antigen entrapped within them [68]. This level of cholesterol has also been shown to reduce the rate of digestion of liposomes by macrophages [73]. In addition to [iposomes containing more than 30 tool ~ cholesterol, a number of other lipid compositions have been shown to have little or no adjuvant effect on the antigen concerned. Allison and Gregoriadis concluded that positively charged liposomes (7:2:1 phosphatidylcholine:cholesterol:stearylamine) do not exert an adjuvant effect on the antigen diphtheria toxoid [212], although in our laboratory this has not proven to be the case when bovine serum albumin was the antigen studied [68]. Using bovine serum albumin, low anti-bovine serum albumin titres were observed when the liposomes were made from 9:l phosphatidylcholine:phosphatidylserine, 9:1 phosphatidylcholine:phosphatidylinositol; and 9 : I sphingomyelin:dicetyl phosphate [68]. There seems no obvious explanation for this observation regarding the low immune response when phosphatidylserine and phosphatidylinositol are used. However, the result with sphingomyelin may be due to the high transition temperature of this lipid (42 °C). Liposomes made from lipids

291 with transition temperatures above the ambient are known to behave differently from liposomes made from tipids whose transition temperatures are below the ambient, when interacting with cells in tissue culture (see Section IV). The use of sphingomyelin may affect the uptake of the bovine serum albumin-containing liposomes by cells and thus affect the immunogenicity of the preparation. The history of adjuvants is as old as that of immunology, and it has long been known that certain amphiphilic substances are adjuvants. In 1956 Weiss and Dubos [213] reported that sphingomyelin had an adjuvant effect upon methanol extracts of tubercle bacilli. No details of their preparation were ~iven, but it seems Quite likely that their preparations consisted of liposomes intimately associated with an antigen. It is only recently, however, that immunology and biophysics have come together, and thereby brought the realisation that certain adjuvants exist in the form of liposomes and that this may be profoundly involved in their adjuvenating ability. VD. Hypersensitivity reactions to antigens in liposomes

Allison and Gregoriadis [215] have claimed that whilst liposomes enhance the immune response they also prevent any adverse hypersensitivity reactions to the antigen by shielding it from antibodies. They have so far demonstrated for diphtheria toxoid, that liposomal entrapment of antigen prevents Arthus type hypersensitivity reactions when the material is injected into the footpad of a primed mouse, and also prevents the death of mice with high circulating antibody when injected intravenously [99,215]. In contrast to this finding however, Nicolotti and Kinsky [194], as mentioned earlier, have presented evidence showing that azobenzenearsonyltyrosylphosphatidylethanolamine incorporated into the lipid bilayers of liposomes is effective in eliciting a delayed type hypersensitivity reaction in guinea pigs immunised with the antigen in liposomes, together with Freund's complete adjuvant. Since the lipid presented on the outside of the liposome produces a delayed hypersensitivity reaction, it seems likely that a protein similarly disposed would also elicit delayed hypersensitivity reaction. If a protein or lipid is presented in a liposome in a form which can undergo interactions leading to a delayed hypersensitivity reaction, it seems likely that such a liposome-antigen complex, if injected into an animal with a high titre of circulating antibodies to the antigen, would bring about an immediate hypersensitivity reaction. VE. Other aspects

A number of studies have been reported in which purified cell membrane components bearing specific receptor sites have been shown to orient themselves in liposomes in a manner in which the receptor site is available for interaction with receptor specific compounds. Haywood [216] has studied the interaction of Sendai virus with liposomes. Virus particles were found to bind to positively charged liposomes and to liposomes containing gangliosides. The interaction of the virus with positive liposomes was shown to be an electrostatic association, by the observation that positive liposomes

292 did not inhibit agglutination of erythrocytes by Sendai virus. Hence, the positive liposomes were binding to the viruses but not competing with erythrocytes for the specific haemagglutinin glycoprotein on the virus surface. The binding of liposomes containing gangliosides to Sendai virus, however, was due to a specific ganglioside-haemagglutinin glycoprotein interaction, since liposomes containing gangliosides did inhibit agglutination of erythrocytes by the virus. Moreover, the interaction was in no way due to non-specific electrostatic attraction, since both the Sendai virus and the liposomes containing gangliosides are net negatively charged. The liposome structure is necessary for the interaction of Sendai virus with gangliosides, since pure gangliosides in aqueous suspension did not inhibit haemagglutination. Pure gangliosides form a micellar structure rather than the bilayered liposome structure. In the same study, the binding of Sendai virus to liposomes containing gangliosides was visualised under the electron microscope using negative staining. Interaction of virus with liposomes was usually found to involve several of the surface "spikes" of the virus and not simply one spike. Not surprisingly, such close association of virus and liposome leads to fusion of the two [217] and also, under some conditions, an envelopment of the virus by the bilayer which resembles phagocytosis [154]. Mooney et al. [218] have performed similar studies using erythrocyte lipid liposomes and Sindbis virus. Surolia et al. [219] have studied agglutination of liposomes containing gangliosides by the lectin from Ricinus cornmunis. They observed an increase in turbidity of suspensions of liposomes containing gangliosides when lectin was added, and they have calculated rate constants for the reaction. They report that this reaction was inhibited by lactose and that ganglioside micelles could not inhibit the precipitin reaction between guargum and lectin [219]. Thus, gangliosides must be present in liposomes to compete for lectin specific sites. Lectin interactions with liposomes have been studied in a slightly different context by Redwood et al. [49], who have incorporated erythrocyte sialoprotein (glycophorin) into liposomes and shown that such liposomes may be agglutinated by wheat germ agglutinin. Thus the sialoprotein is oriented in a way which enables its carbohydrate receptor site to interact with the agglutinin. Specific antibody interactions with liposomes with antigen incorporated have been used as a means of purifying anti-glycolipid antibodies [220]. Influenza virus surface proteins have also been shown by Almeida et al. [83] to orientate themselves in liposomes in a manner which, under the electron microscope, appears similar to their disposition in the intact influenza virus (Fig. 2). Moreover, the "virosomes" so formed can be agglutinated by anti-influenza antisera (unpublished observations). The production of the virosome was prompted by the knowledge that liposomes were adjuvants, and the need for a non-pyrogenic influenza vaccine. Whole influenza virus is a good immunogen, but is also pyrogenic. Influenza surface proteins are not pyrogenic, but also are not very good immunogens. It is hoped that the combination of non-pyrogenic lipid with the subunits should produce a nonpyrogenic, but immunogenic, preparation.

293

C q ~ D Fig. 2. The incorporation of influenza virus subunits into liposomes to form virosomes. A. Whole influenza virus. B. Suhunits have been extracted from virus with detergent and then dialysed to remove detergent, i n the absence of detergent they form star-shaped aggregates, as seen here, C. Highly sonicated liposomes (9:1 phosphatidylcholine:dicetyl phosphate). D. Virosomes - subunits mixed with sonicated liposomes and briefly sonicated to induce formation of the virosome. This field also shows non-liposomally associated suhunits. Magnification x 270 000. Micrographs provided by courtesy of Dr. J. D. Almeida.

294

VF. Summary Liposomes have proved to be important tools in immunology in a number of ways. Studies on naturally occurring antigenic lipids have shown that antisera are raised most successfully when the antigen is mixed with lipids that are known to form liposomes. Recent studies on synthetic antigenic lipids have shown that the lipid must be incorporated into liposomes and injected with complete Freund's adjuvant to produce high-titre antisera. Liposomes with lipid antigens incorporated have also proved useful as a model for the study of complement-mediated lysis. Liposomes have been found to exert an adjuvant effect on protein antigens if the antigen is associated with the liposome. The antigens, of influenza virus tsurface subunits), have been incorporated into liposomes. The resultant structure appears, under the electron microscope, to be very similar to the influenza virus from which the protein is extracted. A number of lipids and proteins known to have receptors for viruses or lectins have been incorporated into liposomes, and such liposomes have been observed to interact specifically with the virus or lectin concerned.

Vl. SUMMARY AND CONCLUSIONS Until about six years ago liposomes were mainly used as a research tool in the membrane field to attempt to understand the properties of the lipid bilayers believed to form part of the structure of biological membranes. While this aspect of liposome research continues to produce important information, interests in the liposome field have now diversified and include direction towards the possible uses of lipid vesicles as carriers of molecules of therapeutic interest into cells. Although early workers were quick to point out the many potential uses of liposomes in the therapy of storage diseases and in the direction of entrapped drugs to specific tissues [4,58,221], so far these possibilities have not been fully developed, though many significant advances have been made. Despite many modifications to the composition of liposomes they are still taken up after intravenous injection in vivo predominantly by liver and spleen, and it seems likely that, before work can progress in the tissue specific direction of liposomes, some way must be found to prevent their rapid sequestration by these tissues; attempts to achieve this by saturation of the liver with carbon particles have been unsuccessful [125], although a recent paper claims that liver uptake of liposomes can be prevented by prior injection of a highly sonicated suspension of methyl palmitate in Tween 20 [117]. Also, in order to compare more meaningfully the results of different groups of workers, unifying criteria will need to be defined relating to the size distribution (and associated therewith the sonication conditions) as well as to the lipid composition of the liposomes. The multiplicity of compositions and sonication and centrifugation times used serves only to confuse what is already a complex field. Although even crude phospholipids will readily make liposomes, and may well be perfectly satisfactory when a therapeutic

295 system is eventually developed, at the present time pure lipids must be used for correct assessment of the effects of variation in the lipids. The preparation of "smallest-possible liposomes" necessitates prolonged sonication, which may lead to breakdown of any protein materials being entrapped and also, if conditions are not strictly controlled, to peroxidation of the lipids. If probe sonication is used, titanium particles are shed by the probe and must be removed by low-speed centrifugation prior to further purification of the liposomes, since without this precaution the titanium co-chromatographs with the liposomes on gel-filtration. In the preparation of liposomes for human use, strict sterile conditions must be maintained. This can be achieved by minor modifications to standard techniques. Many authors have automatically assumed entrapment of the molecules of solute within the aqueous spaces of the liposomes. However, it now seems probable that in many cases association of the molecules with the lipid bilayers may be occurring, and most authors fail to provide sufficient evidence to prove that true entrapment has taken place. In some cases this will be unimportant when the result desired is solely to prepare drugs etc. in a directable, particulate form. It will only be of significance if the exposure of the protein or drug on the surface of the liposome leads to the production of antibodies to the solute molecule, or to interaction of the molecule with pre-existing antibody (administration in liposomes originally being intended to prevent this), or to an enzyme protein being active and affecting blood components. As the liver appears to sequester the majority of liposomes given intravenously, it was hoped that the liposomal carrier system might be of use in the treatment of hepatomas, but preliminary work (using pertechnetate scanning) indicates that hepatomas may not have the same ability to take up liposomes as do normal liver cells [123]. The attachment of tissue-specific antibodies to the surface of liposomes in an attempt to achieve their uptake by tissues other than liver and spleen has not yet been reported in vivo but does, however, lead to an increase in their uptake by cultured cells of the type for which the antibody is specific [131,132]. Although it is recognised that the properties of cultured cells may be markedly different from those of the original tissue, further investigation is likely to produce methods capable of use in vivo for the direction of liposomes. The effects of liposomal membrane fluidity on the intracellular distribution of the liposomes have been the object of much study, but no definite conclusions can be drawn since the results of different experimenters are contradictory. Liposomally entrapped enzymes can certainly have effects within cultured cells [144], and this, with the results of in vivo studies [60], provides encouragement for further work. The recent interest in the incorporation of cytochromes into liposomes [78,85] provides a non-medical new aspect of liposomes, and one which may lead to a better understanding of mitochondrial and other membrane-bound enzymes. One therapeutic use of liposomes which appears promising is the entrapment and administration of chelating agents in liposomes. The use of such liposomes has been shown to decrease the level of plutonium in the livers of mice and may, therefore, be useful in the treatment of patients who have been accidentally exposed to radio-

296 active materials [5]. By the choice of suitable chelating agents it may be possible to remove deposits of other metals from the tissues, such as the iron which accumulates in the liver in haemochromatosis and haemosiderosis. Liposomes have found much use in the study of immunological processes, and it appears that the liposome structure itself is important in the production of an immune response to incorporated molecules. Perhaps one of the most promising immunological properties of liposomes is their action as adjuvants [68]. Adjuvants previously available, such as Freund's complete adjuvant, have been unsuitable for use in man because of adverse reactions at the injection site. Liposomes, however, may be formed from lipids occurring naturally in the body, and are therefore less likely to produce undesirable effects. This may permit the production of active immunity against pathogens which, in the past, have been non-immunogenic when injected in the absence of adiuvant. The production of the "virosome" [831 may well herald the advent of a new generation of vaccines. The development of the therapeutic applications of liposomes is likely to lead to a wide variety of possible uses, especially in the delivery of drugs to specific target tissues. This is a matter of special interest in the field of cancer chemotherapy where the aim is to prevent potentially toxic drugs from reaching tissues other than the tumour. Liposomes could also find therapeutic use in the introduction of enzyme proteins into cells in various pathological storage conditions such as the glycogenoses, for which they were originally proposed [4]. The uptake of liposomes and their contents from the gastro-intestinal tract has great therapeutic potential [9], since a method of administering insulin orally has been in demand for many years. Before this could be fully acceptable as a method of treatment, some guarantee of reproducible uptake of liposomes from the gut must be produced, so that dosage can be accurately regulated. Although insulin in liposomes would be more expensive to produce than the insulin preparations now used, the trauma of repeated injections would be avoided, as would the cost of syringes and needles. Similarly, other drugs which are not normally amenable to oral administration may also be capable of oral use in liposomes. Yet another therapeutic application of liposomes could be their use to form intramuscular depots of water-soluble compounds [139]. All the applications suggested above have some basis in experimental results already obtained; there are other possible applications which could be envisaged despite there being little work in these fields at present. If the uptake of liposomes into the tissues can be almost totally prevented, they could be used to create a circulating, intravascular depot of drugs, or an intravascular store of a missing enzyme (as was originally envisaged by Chang [1,2]). Hormones, either polypeptides in the aqueous phase or steroids within the lipid bilayers, could also be administered liposomally, by oral or parenteral routes, and then directed to specific target tissues. One effect of the administration of small molecular weight, water-soluble drugs intravenously in liposomesis a decrease in the excretion rate of the drugs in the urine, thereby leading to maintenance of bloodl evels for longer periods of time than obtained using the free drug.

297 A l t h o u g h all the aspects o f l i p o s o m e s discussed in this review are o f c o n s i d e r a b l e interest a n d will u n d o u b t e d l y r e p a y further study, before l i p o s o m e s can be accepted as t h e r a p e u t i c agents in man, m o r e m u s t be k n o w n a b o u t the factors g o v e r n i n g the u p t a k e o f l i p o s o m e s when given in vivo. Until this aspect a n d the o t h e r m a t t e r s outlined a b o v e are clarified, the p a e a n s o f praise for the m u l t i p o t e n t i a l l i p o s o m e m u s t r e m a i n slightly muted.

ACKNOWLEDGEMENTS S u p p o r t f r o m the W e l l c o m e T r u s t a n d the M e d i c a l a n d Science Research Councils is gratefully a c k n o w l e d g e d b y the authors, as is the c o - o p e r a t i o n o f investigators in the l i p o s o m e field who have k i n d l y sent us reprints a n d p r e p r i n t s o f their work. The skilful secretarial assistance o f Miss R o s e m a r y D u n c o m b e is also a c k n o w l e d g e d , t o g e t h e r with the assistance received f r o m o u r Library,

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