Grafting of different glycosides on the surface of liposomes and its effect on the tissue distribution of 125I-labelled γ-globulin encapsulated in liposomes

562

Biochimica et Biophysica Acta, 632 (1980) 562--572

© Elsevier/North-Holland Biomedical Press

BBA 29402 G R A F T I N G O F D I F F E R E N T GLYCOSIDES ON THE S U R F A C E OF LIPOSOMES AND ITS E F F E C T ON THE TISSUE DISTRIBUTION OF I~SI-LABELLED ~-GLOBULIN ENCAPSULATED IN LIPOSOMES

P. GHOSH and B.K. BACHHAWAT Indian Institute of Experimental Medicine, 4 Raja S.C. Mullick Road, Jadavpur, Calcutta 700032 (India)

(Received February 29th, 1980) (Revised manuscript received June 12th, 1980) Key words: Liposome encapsulation; "y-Globulin; Glycoside grafting; Tissue distribution

Summary Different glycosides were grafted on the surface of liposomes containing 12SI-labelled 7-globulin by two ways: (1) by using glycolipid and (2) by covalent coupling of p-aminophenyl-D-glycosides to phosphatidylethanolamine liposomes using glutaraldehyde. The distribution of ~2sI-labelled 7-globulin was determined in mouse tissues from 5--60 min after a single injection of these liposomes. The liver uptake of encapsulated ~2SI-labelled 7-globulin was highest from liposomes having galactose and mannose on the surface. Competition experiments and cross-inhibition studies indicate that this uptake are mediated by specific recognition of the surface galactose and mannose residues of liposomes by the receptors present on the plasma membrane of liver cells. Stearylamine-containing liposomes were found to be more efficient in mediating the uptake of ~2SI-labelled 7-globulin by the lung, whereas in the case of spleen, phosphatidylethanolamine liposomes were more efficient. The extent of uptake of 12SI-labelled 7-globulin from all types of liposome decreases as the amount of given liposomes increases. The uptake of 12SI-labelled 7-giobulin from liposomes containing asialogangliosides depends upon the phospholipid/ giycolipid ratio. These experiments clearly demonstrate that enhanced liposome uptake by liver cells could be achieved by grafting galactose and mannose on the liposomal surface.

Introduction

Liposomes have been used as vehicles for transporting drugs and enzymes in vivo in experimental animals [1--5]. The successful application of liposome-

563 entrapped enzymes and drugs in the control of cell behavior is d e p e n d e n t upon the target specificity of liposomes. The problem of target selectivity of liposome-entrapped drugs and enzymes can be approached by creating liposomes possessing a specific affinity towards the target tissue. Some modulation of the in vivo distribution of liposomes can be achieved by altering the size, charge and composition of liposomes [6--8]. Earlier reports have shown that glycolipid-containing liposomes can specifically recognise and bind to lectins [9], antibodies [10] and cells [11]. It has also been shown that monosialoganglioside- and asialofetuin-containing liposomes can specifically bind to liver parenchymal cells containing lectin-like molecules on their membranes [ 12,13]. Recently, lectin-like molecules in Kupffer cells which recognise terminal mannose residues of biopolymers have been demonstrated [14--18]. This lectin-like molecule may permit liposomes carrying mannose residues on their surfaces to be specifically targeted to Kupffer cells. In this communication, we describe the effect of varying the surface glycosides of liposomes on their uptake by various tissues in mice and compare them with neutral and charged liposomes. Materials and Methods Egg lecithin, cholesterol, phosphatidylethanolamine, beef brain gangliosides and galactocerebrosides were obtained from CSIR Centre for Biochemicals, Dehli. Stearylamine was purchased from Fluka, Switzerland. Fetuin, yeast mannan, p-nitrophenyl-~-D-galactoside, p-nitrophenyl-a-D-mannoside, p-nitrophenyl-N-acetyl-/3-D-glucosaminide, bovine v-globulin were obtained from Sigma Chem. Co., U.S.A. Carrier-free Na12SI was obtained from BARC, Bombay. Glutaraldehyde was obtained from Koch-Light Laboratories Ltd., U.K. Asialofetuin and asialoganglioside were prepared as mentioned earlier [12]. Ricinus communis agglutinin was prepared according to the m e t h o d of Appukuttan et al. [19] and concanavalin A was prepared as per the m e t h o d of Surolia et al. [20]. All other chemicals used were of analytical grade.

Radioiodination o f ~[-globulin Radioiodination of 7-globulin was done by the chloramine-T method of Hunter [21] using Na12SI (carrier-free). The iodinated protein was separated from free iodine by gel filtration through a Sephadex G-25 column. The specific activity of iodinated protein was 1.5 • 106 cpm/pg. Conversion o f the p-nitrophenylglycosides into the corresponding p-aminophenylglycosides Reduction of the p-nitrophenylglycosides was carried out as described by Bloch and Burger [22]. The c o m p o u n d s p-nitrophenyl-fl-D-galactoside, p-nitrophenyl-a-D-mannoside and p-nitrophenyl-N-acetyl-/~-D-glucosaminide (100 mg each ) were dissolved in 50 ml 0.5 M sodium bicarbonate containing 0.1 M sodium dithionate, separately and stirred vigorously for 3 h at room temperature. It was then dried under vacuum and extracted thrice with 50 ml methanol each time. The total methanol extract was then dried under vacuum. Reduction of the NO2 group to the amino group and the purity of the p-aminophenylglyco-

564 sides were ascertained by the ratio of amino groups to neutral sugar determined by diazotisation and neutral sugar measurements [23], respectively. The conversion of the nitro group to amino group was 90--95%.

Preparation of liposome containing ~2SI-labelled 7-globulin 12si_labelled 7-globulinentrapped liposomes were prepared with egg lecithin, cholesterol and phosphatidylethanolamine in the molar ratio 7 : 2 : 2 according to the m e t h o d of Gregoriadis and R y m a n [1]. In short, a thin dry film consisting of lipid mixture was dispersed in 0.025 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaC1 and 10 mg/ml 7-globulin mixed with trace a m o u n t of 12SI-labelled 7-globulin. The dispersion was completed by brief sonication (30 s) in an MSE ultrasonicator. The unentrapped protein was separated by repeated washing in buffer by ultracentrifugation at 105 000 × g for 60 min. The asialoganglioside- galactocerebroside- and stearylamine-containing liposomes were made with a mixture of egg lecithin/cholesterol/glycolipid (or stearylamine) in a 7 : 2 : 2 molar ratio. Neutral liposome was prepared with a mixture of egg lecithin/cholesterol in a 7 : 2 molar ratio. In some experiments, designed to test for the effect of nonspecific association of protein with liposomes, liposomes were prepared in the absence of protein. ~25I-labelled 7-globulin was then mixed with the liposome 15 min prior to injection.

Covalent coupling of p-aminophenylglycosides to phosphatidylethanolamine liposome Covalent coupling of p-aminophenylglycosides to phosphatidylethanolamine liposomes was done according to the m e t h o d of Torchillin et al. [24]. Phosphatidylethanolamine liposomes (1 ml) suspension (30 mg lipid/ml) in 0.025 M sodium phosphate buffer, pH 7.2, containing 0.15 M NaC1 was mixed with 20 mg (contained in 2 ml)p-aminophenylq3-D-galactoside, p-aminophenyl-a-Dmannoside and p-aminophenyl-N-acetyl-[J-D-glueosaminide, separately. Glutaraldehyde was added slowly to the liposome suspension upto 15 mM final concentration and the mixture was incubated for 5 rain at 20°C. Uncoupled glycosides and glutaraldehyde were removed by dialysis against the same buffer. The coupling of the glycosides on liposomes was monitored by two ways: (1) titration of the liposomal phosphatidylethanolamine amino groups with trinitrobenzene sulfonic acid according to the m e t h o d of Torchillin et al. [24]; (2) agglutination of liposomes with the appropriate lectin according to the m e t h o d of Surolia et al. [9]. The titration of the liposomal phosphatidylethanolamine amino groups with trinitrobenzenesulphonic acid in the presence of 0.1% Triton X-100, demonstrated that about 80--85% of the amino groups were modified, in all the cases, by the above t r e a t m e n t . The p-aminophenyl-~-D-galaetoside, p-aminophenyl-a-D-mannoside and p-aminophenyl-N-acetyl.[J-D-glueosaminide coupled liposomes are henceforth designated as /3-galaetose liposomes, a-mannose liposomes, and GlcNAc liposomes, respectively.

Animal experiments Male Swiss Albino mice (IIEM strain) weighing approx, 25--30 g were used t h r o u g h o u t the experiment. Each mouse received a single intravenous injection

565 of 0.5 ml liposome (1.0--1.5 mg lipids) suspension containing 3--6 • 104 cpm ~2SI-labelled 7-globulin. As a control, free ~zSI-labelled 7-globulin ( 3 0 . 1 0 3 cpm) and ~2SI-labelled 7-globulin mixed with liposomes were also injected. After time intervals of 5, 15, 30 and 60 min respectively, groups of three mice were killed and their livers, kidneys, spleens and lungs were removed. Each tissue was washed with 0.9% NaC1 solution and blotted with filter paper. Radioactivity was then measured in the tissues after homogenization.

Subcellular fractionation of liver After the appropriate treatment, the mice were killed and their livers were removed and washed with 0.9% NaC1 solution and blotted with filter paper. The whole liver was then homogenised in 0.32 M sucrose solution and subsequently fractionated by differential centrifugation into nuclear, mitochondriallysosomal and soluble fractions [25].

Measurement of radioactivity 12sI was measured in a Prias Scintillation gamma counter. Liver homogenate (0.5 ml), whole nuclear and mitochondrial-lysosomal fractions and 2 ml of the soluble supernatant were taken for counting. The whole kidneys, spleens and lungs of each mouse were digested in 2 ml 30% KOH solution and were taken for counting. Results and Discussion The rates of uptake of ~2SI-labelled ~,-globulin entrapped in various types of liposome are presented in Fig. 1A and B. In most of the cases, the maximum uptake of 12SI-labelled ~'-globulin was at 15 min after the administration of lipo-

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Fig. i . R a t e o f u p t a k e o f v a r i o u s t y p e s o f l i p o s o m e b y m o u s e liver. Male Swiss A l b i n o m i c e ( 2 5 - - 3 0 g ) w e r e i n j e c t e d w i t h 12 S i . l a b e l l e d 3,.globuli n ( 3 - - - 6 . 1 0 4 c p m ) e n t r a p p e d in d i f f e r e n t t y p e s o f l i p o s o m e ( 1 . 0 - - 1 . 5 m g l i p i d s ) . T h e t o t a l v o l u m e o f l i p o s o m e s u s p e n s i o n in all t h e cases w a s 0 . 5 m l . T h e m i c e w e r e killed at t i m e intervals b e t w e e n 5 a n d 6 0 rain a n d t h e a m o u n t o f 12 Si_labeUe d -),-globulin in t h e i r h o m o genate was measured. Each point represents the mean value f~om three mice. (A) Asialoganglioside lipos o m e (A *); ~ - g a l a c t o s e l i p o s o m e (A ~); g a l a c t o c e r e b r o s i d e l i p o s o m e (o - o ) ; neutral lipos o m e (© ©). (B) cx-mannose l i p o s o m e (© ---~); G I c N A c l i p o s o m e ( a a); phosphatidylethan o l a m i n e l i p o s o m e (¢ ~.).

566

TABLE I DISTRIBUTION NOUS INJECTION

OF

125I-LABELLED

")'-GLOBULIN IN MICE TISSUES

OF LIPOSOME-ENTRAPPED

15 min AFTER

INTRAVE-

5,-GLOBULIN

V a l u e s are m e a n p e r c e n t of injected 1 2 5 i . l a b e l l e d 7 . g l o b u l i n ± S . D . Each group c o n t a i n e d three mice. P C ,

p h o s p h a t i d y l c h o l i n e ; Chol, cholesterol; PE, p h o s p h a t i d y l e t h a n o l a m i n e ; PE-Gal, p h o s p h a t i d y l e t h a n o l a m i n e c o v a l e n t l y c o u p l e d w i t h p-aminophenyl-fl-D-galactopyranoside; PE-Man, p h o s p h a t i d y l e t h a n o l a m i n e covalently coupled with p-aminophenyl-~-D-mannopyranoside; PE-GlcNAe, phosphatidylethanolamine c o v a l e n t l y c o u p l e d w i t h p-aminophenyl-2-acetamido-2-deoxy-fl-D-glueopyranoside; Gal-Cer, galactocerebroside; S A , s t e a r y l a m i n e , n . d . , n o t detected. L i p o s o m a l lipid c o m p o s i t i o n

Molar ratio o f lipid

Liver

PC/Chol/PE PC/Chol/PE-Gal. PC/Chol/PE-Man. PC/Chol/PE-GIcNAc. PC/Chol]Asialoganglioside PC/Chol/Gal-Cer PC/Chol PC/Chol/SA/Asialoganglioside PC/Chol/SA ~/-Globulin mixed with asialoganglioside liposome

7:2:2 7:2:2 7:2:2 7:2:2 7:2:2 7:2:2 7:2 7:2:2:2 7:2:2

46.0 63.1 72.9 50.3 85.0 56.0 45.0 45.0 39.0

Free ~/-globulin

± ± ± ± ± ± ± ± ±

2.1 2.5 5.4 1.5 1.1 1.0 1.0 5.0 4.5

1 5 . 0 _+ 1 . 2 1 2 . 0 ± 1.1

* ** ** * ** * * *

Spleen

Kidney

Lung

6.3 3.1 2.7 1.7 1.7 4.1 2.6 1.3 1.3

0.9 0.5 0.6 0.7 0.8 1.0 0.9 0.6 0.6

0.7 2.1 2.0 2.1 1.6 0.9 0.6 1.1 16.0

n.d. n.d.

± 1.2 ± 0.7 -+ 0 . 6 _+ 0 . 2 ± 0.7 ± 0.7 ± 0.4 ± 0.6 ± 0.4

n.d. n,d,

± ± ± ± ± ± ± ± ±

0.1 0.2 0.2 0.1 0.2 0.3 0.1 0.1 0.2

+ 0.2 ± 0.4 + 0.4 ± 0.5 -+ 0 . 2 ± 0.3 + 0.1 ± 0.2 -+ 1 . 5

n.d. n.d.

* T h e difference in the uptake o f these l i p o s o m e s in c o m p a r i s o n to o n e a n o t h e r is n o t significant; P 0 . 1 f o r 4 degrees o f f r e e d o m .

** T h e difference in the u p t a k e o f these l i p o s o m e s as c o m p a r e d to o t h e r l i p o s o m e s * is highly significant; P < 0 . 0 0 1 f o r 4 degrees o f f r e e d o m .

somes except in the cases of asialoganglioside liposomes where the maximum uptake was observed at 5 min. However, in all the cases there was a gradual decrease in the amount of 12SI-labelled ~,-globulin in the liver 60 min after injection of liposomes. The amount of 12SI-labelled ~/-globulin taken up by the liver and other tissues after 15 min is dependent on the types of the liposomes employed as shown in Table I. The tissue levels of 12sI marker reflect the difference between uptake and possible catabolic degradation and loss of marker. The possible role (if any) of various liposomes on the catabolic process has not been investigated. The extent of uptake by the liver of liposomal 125I-labelled ~-globulin was more in the cases of asialoganglioside liposome, fl-galactose liposome and a-mannose liposome. This indicates the involvement of a specific interaction of these sugar-containing liposomes with the liver. During the last few years, Morell and coworkers have emphasised the importance of free terminal galactose moieties on desialylated glycoprotein for their rapid uptake by the liver. They have shown further that there are specific galactose binding receptors exclusively on the parenchymal cells of the liver [26--28]. Stahl et al. [14] and Hughes [16] have also emphasised the presence of terminal mannose residue on lysosomal enzymes for their rapid uptake by the liver. They have concluded that these enzymes are specifically taken up by the Kupffer cells of the liver [17,18]. Our observation of high uptake of ~2SI-labelled ~/-globulin from asialoganglioside liposomes, fl-galactose liposomes and a-mannose liposomes, confirms the presence of similar recognition mechanisms for these liposomes. The pres-

AND MANNAN

ON THE UPTAKE

OF LIPOSOMAL

! 25I_LABELLED

"y-GLOBULIN

BY LIVER

AT 15 MIN

alone + mannan + asialofetuin

63.1 + 2.5 6 1 . 7 ± 2".5 50.3 ± 2.1 *

PC/Chol/ PE-Gal

Types of llposome

72.9 ± 5.4 49.6 ± 3.3 ** 68.7 ± 4.0

PC/Chol/ PE-Man

** The difference in the uptake Student's t-test.

of ~-mannose

liposomc

as compared

to mannan-dependent

56.0 ± 1.0 -5 3 . 0 _+ 4 . 0

PC/Chol/ Gad-Ccr.

inhibition

of a-mannose

to asialofetuin-dependent

84.9 + 1.5 82.6 ± 2.0 60.4 ± 2.9 *

PC/Chol/ Asialoganglioside

as c o m p a r e d

4 5 . 8 _+ 1 . 5 44.2 ± 2.0 43.1 ± 2.8

PC/Chol/PE

* The difference in the uptake of asialoganglioside and ~-galactose liposomes liposomes were highly significant; P ~ 0.001, Student's t-test.

Liposome Liposome Liposome

Experiments

liposome

inhibition

45.0 + 1.0 -49.6 ± 1.6

PC/Chol/SA/ Asialoganglioside

was highly significant; P (

0.001,

of asialoganglioside and ~-galactose

50.3 ± 1.5 51.2 ± 1.0 49.3 ± 1.2

PC/Chol/PEGlcNAc

Liposome-entrapped 12 S i . l a b e l l e d 7 - g l o b u l i n ( 3 - - 6 • 1 0 4 c p m ) w i t h o r w i t h o u t 8 - - 1 0 m g a s i a l o f e t u i n a n d m a n n a n w a s i n j e c t e d i n t h e m i c e t a i l v e i n a n d t h e u p t a k e o f 1 2 5 i . l a b e l l e d T . g l o b u l i n is e x p r e s s e d a s m e a n p e r c e n t a g e o f i n j e c t e d 1 2 5 i . l a b e l l e d T _ g l o b u l i n ± S . D . E a c h g r o u p c o n t a i n e d t h r e e m i c e . A b b r e v i a t i o n s u s e d a r e a s i n T a b l e I.

OF ASIALOFETUIN

TABLE II

EFFECT

G5

568 ence of sugar on the surface of these liposomes was confirmed by the aggregation of asialoganglioside liposome and ~-galactose liposome with R i c i n u s c o m m u n i s agglutinin and a-mannose liposome with concanavalin A as described by Surolia et al. [9]. It was also observed that the aggregation of asialoganglioside and fi-galactose liposomes with R i c i n u s c o m m u n i s agglutinin was inhibited by lactose and the aggregation of a-mannose liposomes with concanavalin A was inhibited by a-methylmannoside. These observations are persuasive enough to suggest that the surface sugars of these liposomes are accessible to their respective lectins. The influence of the surface galactose and mannose residues in determining the high uptake of these three types of liposome were confirmed by competition experiments using asialofetuin and mannan. Asialofetuin, which has a terminal ~-galactose residue, inhibits the uptake of asialoganglioside liposomes and fi-galactose liposomes by the liver. But it had no effect on the uptake of a-mannose liposomes, GlcNAc liposomes and phosphatidylethanolamine liposomes (Table II). Mannan inhibits the uptake of a-mannose liposomes b u t it had no effect on the uptake of asialoganglioside liposomes, fi-galactose liposomes, GlcNAc liposomes and phosphatidylethanolamine liposomes. These experiments suggest that the surface galactose residues of asialoganglioside liposomes and ~-galactose liposomes are recognised by the receptor on the plasma membrane of liver parenchymal cells and surface mannose residues of a-mannose liposome are recognised by the receptor on plasma membrane of Kupffer cells of liver. The low uptake of galactocerebroside liposomes in comparison to asialoganglioside liposomes and ~-galactose liposomes (in spite of having the requisite galactose residue) may be due to a restricted access of the hepatocyte receptor towards the galactose residue of cerebrosides. This finding is in agreement with the earlier observation that there was poor binding of lectins [29,30] and antibodies [10] to the galactocerebroside liposome. The uptake of free lzSI-labelled 7-globulin and 7-globulin mixed with asialoganglioside liposome by liver was 12 and 15%, respectively. These results show that entrapment inside liposome was essential for the enhanced uptake of protein. In lungs, the uptake of 12SI-labelled 7-globulin from stearylamine-containing liposomes (positively charged liposomes) was significantly higher than that from all other liposomes. The spleen accumulated the highest concentration of ~2SI-labelled ~,-globulin from phosphatidylethanolamine-containing liposomes (neutral liposomes). The uptake of 12SI-labelled ~/-globulin by kidney from all types of liposome studied was very poor. Since cell surfaces bear a net negative charge, greater uptake of a protein encapsulated in positively charged liposomes might be expected, as observed in the case of lungs, b u t this has not been observed consistently in other tissues in the present study as also reported by other workers [7,8]. Specific interaction of a tissue with liposomes of a defined composition may therefore be a specific property of particular cell surfaces and cell environments. Relatively low hepatic uptake of liposomal 12SI-labelled 7-globulin was observed from stearylamine-containing liposomes (Table I). This observation agrees with an earlier report by Gregoriadis et al. [7]. To investigate whether the uptake of asialoganglioside liposome can be inhibited by the presence of stearylamine and whether it can be diverted to other organs, mice were injected

569

with liposomes (with both asialoganglioside and stearylamine on their surfaces) containing 12SI-labelled 7-globulin and were killed after 15 min. It was observed that the uptake of asialoganglioside liposome by liver was inhibited b y the presence of stearylamine in liposomes (Table I). Inhibition studies with asialofetuin indicate that the uptake of these liposomes was nonspecific. It was also observed that the uptake of these liposomes by other tissues (spleens, lungs, kidneys) was slightly decreased. The mechanism by which stearylamine interferes with the rate of uptake of asialoganglioside liposomes is not well understood. Interactions between liposomes and plasma constituents are almost certain to occur in vivo and to influence liposomal uptake by tissues. It has been demonstrated that stearylamine liposomes are quantitatively attached to cells in culture in the absence of serum, presumably as a result of electrostatic attraction, b u t fail to do so in the presence of serum [31]. Indeed, the absence of an enhanced uptake in vivo of liposomes containing both stearylamine and asialogangliosides b y the liver could be the results of interaction of such liposomes with negatively charged plasma constituents, whereby the galactose moiety on the surface of these liposomes might be partially masked or n o t fully accessible to the h e p a t o c y t e receptors. The subcellular distribution studies of the liver homogenate were carried o u t on livers obtained 15 min after injection of liposomes (asialoganglioside liposome, fl-galactose liposome, a-mannose liposome and neutral liposomes). These studies revealed that the lysosomal-mitochondrial fraction contained about 30--40% of the incorporated radioactivity and the nuclear fraction contained a b o u t 10--18% of the incorporated radioactivity. The inhibition, by asialofetuin and mannan, of uptake of liposomes by liver does n o t alter the subcellular localisation of the marker taken up. It was observed that the percentage of uptake of ]2SI-labelled 7-globulin-

I00 90 80

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Amount of lipids in mg Fig. 2. D o s e - d e p e n d e n t u p t a k e o f l i p o s o m e s c o n t a i n i n g 1 2 5 i . l a b e l l e d 7 . g l o b u l i n b y m o u s e liver. Male Swiss A l b i n o m i c e w e r e i n j e c t e d w i t h v a r y i n g a m o u n t s of l i p o s o m e ( 1 . 0 - - 1 2 . 0 m g lipids) c o n t a i n i n g differe n t a m o u n t s o f 1 2 5 I - l a b e l l e d "),-globulin ( 0 . 0 2 5 - - 0 . 3 m g ) a n d killed a f t e r 15 m i n . As a c o n t r o l , d i f f e r e n t a m o u n t s ( 0 . 0 2 5 - - 0 . 3 rag) of 1 2 5 i . l a b e U e d 7 . g l o b u l i n m i x e d w i t h d i f f e r e n t a m o u n t s o f asialogangiioside l i p o s o m e as in t h e o t h e r cases, w e r e also i n j e c t e d . In all t h e cases, t h e v o l u m e o f l i p o s o m e s u s p e n s i o n i n j e c t e d w a s k e p t c o n s t a n t . R a d i o a c t i v i t y is given as a p e r c e n t a g e o f t h a t i n j e c t e d . E a c h p o i n t r e p r e s e n t s t h e m e a n v a l u e f r o m t h r e e m i c e . A s i a i o g a n g l i o s i d e l i p o s o m e (~ A); a - m a n n o s e l i p o s o m e (o o);

phosphatidylethanolamine l i p o s o m e (e

--); "},-globulin + a s i a l o g a n g i i o s i d e l i p o s o m e (A

A).

570

TABLE In D E N S I T Y - D E P E N D E N T U P T A K E OF A S I A L O G A N G L I O S I D E L A B E L L E D " ) ' - G L O B U L I N BY M O U S E L I V E R

LIPOSOMES

CONTAINING

125I-

Mice w e r e i n j e c t e d w i t h asialoganglioside l i p o s o m e ( 1 . 0 - - 1 . 5 m g lipids) w i t h v a r i o u s m o l % o f asialoganglioside ( 1 . 5 - - 1 8 m o l % ) , c o n t a i n i n g 1 2 5 i . l a b e l l e d ~ - g l o b u l i n a n d killed a f t e r 15 rain. T h e a m o u n t o f v - g l o b u l i n in t h e liver w a s m e a s u r e d . V a l u e s are m e a n p e r c e n t a g e of 1 2 5 i . l a b e l l e d ~/-globulin i n j e c t e d ± S.D. E a c h g r o u p c o n t a i n e d t h r e e m i c e . S t a t i s t i c a l analysis is t h a t of t h e u p t a k e of l i p o s o m e d u e to galactose r e s i d u e in c o m p a r i s o n to n e u t r a l l i p o s o m e . Asi.,doganglioside in l i p o s o m e

U p t a k e b y liver (%)

(mol%) 0 1.5 3.0 6.0 12.0 18.0

U p t a k e d u e to galactose r e s i d u e

Statistical analysis ( S t u d e n t ' s t-test)

(%) 45.0±I.0 45.0±0.0 51.0±2.0 57.0±2.6 60.0±1.6 85.0±1.1

--6.0±2.0 12.0±2.6 15.0±1.6 40.0±1.1

0.i 0.002 0.002 0.001

containing liposomes by liver within 15 min following their injection, decreases as the a m o u n t of liposomes increases, as shown in Fig. 2. But the percentage of uptake of 125I-labelled ~,-globulin mixed with asialoganglioside liposomes as well as of free 12SI-labelled ~,-globulin given at different dosages, remain fairly constant. The low uptake from high doses of liposomes could be the result of saturation of liver, i.e. the percentage of uptake in the liver within fixed intervals is inversely proportional to the a m o u n t of liposome given. But slight increase in uptake by spleen, kidney and lung was observed in high doses (data not shown). The uptake of asialoganglioside liposome undergoes a significant variation with the phospholipid/glycolipid ratio, as shown in Table III. There was no galactose-specific uptake of these liposomes at asialoganglioside content less than 3 mol%. The uptake of these liposomes by liver increases with increasing c o n t e n t of asialoganglioside. This suggests that the uptake of asialoganglioside liposomes is a function of the surface density of glycolipid receptor. A similar requirement for a threshold concentration of surface receptor has also been reported for antibody-induced agglutination of red blood cells [32,33] and lectin-induced aggregation of GM1 ganglioside liposome [9] and lactosylcerebroside liposome [30]. Recent studies of Rahman et al. [34] on the uptake of EDTA entrapped in GM1 liposome are at variance with the studies reported here as well as with our earlier report [12]. The difference in the extent of uptake of GM1 liposome appears largely due to a lower concentration of monosialoganglioside used by these workers for the uptake studies. And indeed our studies also reveal a difference of 35% in the uptake of asialoganglioside liposome when the concentration of the glycolipid was raised from 3 to 18 mol%. A higher uptake of galactocerebroside liposomes as compared to GM1 liposomes involving a specific interaction with hepatic galactose binding lectin as reported by Rahman et al., is inconsistent with the reported inaccessibility of the galactose moiety of the galactocerebroside incorporated in liposomes towards various receptors such as

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lectin [29,30], antibody [10], etc. Moreover, an unequivocal proof that the uptake of galactocerebroside liposome is mediated through hepatic galactose binding lectin requires a demonstration of the inhibitory effect on the uptake of these liposomes by asialoglycoprotein having terminal galactose moieties. Any interpretation of the molecular and cellular events for targeting of entrapped material utilising ligand-receptor interaction should take into account the density and availability of receptor on the surface of liposomes. These experiments clearly demonstrate that specific enhanced uptake of liposome by the liver hepatocytes and Kupffer cells can be achieved by the incorporation of galactose and mannose, respectively, on the liposomal surface either covalently or by using glycolipids possessing terminal galactose and mannose. Acknowledgments This work was supported by a grant from the Department of Science and Technology, India. It is a pleasure to acknowledge the participation of Mridul Ghosh in the early stages of this work. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

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