Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins

76

Biochimica et Biophysica Acta 846 (1985) 76-84

Elsevier BBA 11517

Liposome uptake by cultured macrophages mediated by modified low-density lipoproteins Vadim O. Ivanov, Sergey N. Preobrazhensky *, Vladimir P. Tsibulsky, Vladimir R. Babaev, Vadim S. Repin and Vladimir N. Smirnov Laboratory of Cell and Tissue Culture, Institute of Experimental Cardiology, USSR Cardiology Research Center, 3rd Cherepkovskaya Street 15,4, Moscow 121552 (U.S.S.R.)

(Received December 14th, 1984) (Revised manuscript received April 17th, 1985)

Key words: Liposomeuptake; LDL; Carboxyfluorescein;(Macrophage)

We have investigated effects of native low-density lipoproteins (LDL) and malondialdehyde-treated LDL on the interaction of 5(6)-carboxyfluorescein-labeled liposomes bearing antibodies to LDL with cultured J774 macrophages. It was found that an addition of modified LDL to the incubation medium resulted in 15-20-fold increase of carboxyfluorescein binding to cells, whereas native LDL did not produce such effect. The increase of carboxyfluorescein binding to macrophages in the presence of modified LDL was not due to an enhanced leakage of the label from liposomes. The modified-LDL-mediated binding of carboxyfluorescein to cells was reduced to 20-30% of the initial level in the presence of cell-respiration inhibitors (NaF and antimycin A). Fluorescent microscopy data also indicate the modified-LDL-induced incorporation of liposome contents into cells. The results obtained in this study make it possible to assume that in the presence of mallondialdehyde-treated LDL, liposomes with antibodies to LDL may be incorporated into macrophages via the receptor-mediated pathway for modified L D L

Introduction Investigation of liposome interactions with cell surface receptors is a relatively new line of liposome research. Multiple studies of this kind deal with macrophages. Thus, tissue culture studies have shown that the macrophage receptors recognizing Fc fragments of IgG [1,2], C a component of the complement [3], and fibronectin [2] can be used to

* To whom correspondence should be addressed. Abbreviations: LDL, low-density lipoproteins; DPPE, dipalmitoylphosphatidylethanolamine; SPDP, N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate; MLV. multilamellar vesicles; SUV, small unilamellar vesicles; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonicacid.

induce the incorporation of liposomes into macrophages. It has been found that macrophages possess receptors which can bind the so-called 'modified' LDL with high affinity and specificity and transport them inside the cells (see Ref. 4 for review). In vitro, the modified L D L can be obtained by acetylation [5], acetoacetylation [6], maleylation [5], c a r b a m y l a t i o n [7], t r e a t m e n t with malondialdehyde [8], etc. A possible mechanism of LDL modification in vivo has not been revealed yet, though current evidence suggests that this process may be induced by the interaction of LDL with platelets [8] or endothelial cells [9]. Besides macrophages, the receptors for modified L D L were detected in endothelial [10,11], Kuppfer [10,11], and possibly, parenchymatous

0167-4889/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

77 liver cells [12]. There are also some indications to the presence of such receptors on the surface of vascular endothelial cells [13,14]. Furthermore, macrophages with the receptors for modified LDL were found at the site of atherosclerotic lesions [151. The purpose of our work has been to induce liposome incorporation into macrophages via the receptors for modified LDL. Such approach may be useful both for induction of specific drug delivery into macrophages and for investigation of the LDL interaction with the receptors for modified lipoproteins. This paper reports some characteristics of the LDL-mediated liposome interactions with macrophages. It is demonstrated that malondialdehydetreated LDL can significantly induce the uptake of liposomes bearing the antibodies to LDL into macrophages. The results of fluorescent microscopy indicate that liposomes with antibodies to LDL can be also used for the visualization of the cells possessing receptors for modified LDL. Materials and Methods

Lipoproteins, lipoprotein-deficient serum, and antibodies. Human LDL were isolated from the blood of healthy donors by sequential ultracentrifugation [16]. After adjusting the density of plasma to 1.019 g / c m 3 with NaBr, it was centrifuged at 105 000 x g for 18 h. The resulting supernatant was removed, the density of infranatant was adjusted to 1.050 g / c m 3, after which it was centrifuged again at 105000 x g for 18 h. Flotated LDL were recentrifuged in a solution with a density of 1.050 g / c m 3 and dialyzed against 0.15 M NaCI/0.2 mM EDTA (pH 7.4). The isolated lipoproteins were sterilized by filtration through 0.45 #M MiUipore filters and stored at 4°C for not more than 3 weeks. Homogeneity and purity of the isolated fraction were controlled by analytical centrifugation and electrophoresis in polyacrylamide gel with addition of sodium dodecyl sulfate [17]. The LDL concentration was evaluated by the protein content determined by the method of Lowry et al. [18]. Malondialdehyde-treated LDL were prepared according to the method described by Haberland et al. [19]. LDL (5-10 mg/ml) were mixed with 0.2 M malondialdehyde (1:1, v/v), obtained by

acid hydrolysis from m a l o n d i a l d e h y d e • bis(dimethylacetal), incubated for 3 h at 37°C and dialyzed against 0.01 M phosphate buffer/0.15 M NaC1/0.01% EDTA (pH 7.4). Lipoprotein-deficient serum was prepared by repeated ultracentrifugation of a heat-inactivated (30 min at 60°C) pooled blood serum of healthy donors (density > 1.25 g/cm 3) at 105000 × g for 48 h. After centrifugation, the serum was dialyzed against 0.15 M NaCl (pH 7.4), sterilized by filtration through 0.22 /~m Nalgene filters, frozen, and stored at -20°C. Antibodies to human LDL were generated in rabbits. LDL (1 mg/ml) were emulsified with an equal volume of freunds complete adjuvant and injected subcutaneously with 0.1-ml aliquots. Simultaneously, 0.1-ml aliquots (without adjuvant) were injected in multiple intradermal sites. Booster injections were performed twice at 2-weekly intervals via the same route. The rabbits were bled 1 week after the last injection and antibodies were isolated by affinity chromatography on SepharoseLDL. Frozen antibodies were stored in 0.01 M carbonate-bicarbonate buffer (pH 9.6) at -20°C. On an immunoelectrophoregram, the antibodies formed one precipitation line (in the LDL mobility zone) with the human plasma and did not cross-react with high-density lipoproteins and human serum albumin. Iodination of antibodies was carfled out according to the iodine monochloride method of Glover et al. [20]. Preparation of liposomes. Lipids obtained from Sigma (U.S.A.) were used for liposome preparation. Dipalmitoylphosphatidylethanolamine (DPPE) was modified with N-hydroxysuccinimidyl-3-(2-pyridyldithio)propionate (SPDP) as described by Barbet et al. [21]. Silica-gel thin-layer chromatography of the modified DPPE gave one spot in the chloroform/methanol/water system (65:25:4, v/v). The mixture of lipids in chloroform/methanol ( 9 : 1 , v/v), containing dipalmitoylphosphatidylcholine, cholesterol and modified DPPE (64 mo1%/35 mo1%/1 mol%, total of 20 #mol lipid), was evaporated on the walls of a glass-tube during rotation, and after that 3 ml 0.2 M 5(6)-carboxyfluorescein solution (Eastman Kodak, U.S.A.) was added. Carboxyfluorescein was preliminarily recrystalized from ethanol and purified from hydrophobic impurities on a column of LH-20 Sephadex (Pharmacia, Sweden) accord-

78 ing to the method described by Leserman and Barbet [22]. Liposomes were obtained by impulseprobe sonication on a water-bath at 55°C for 15-20 min. After cooling down to room temperature, the liposome preparation was centrifuged for 10 min at 3000 rpm to sediment titanic particles. From nonincorporated carboxyflyuorescein, liposomes were separated on a column (0.9 cm × 30 cm) of Sephadex G-25 equilibrated with a liposome buffer (150 mM N a C I / 1 0 mM Hepes (pH 7.5)). To separate multilamellar vesicles (MLV), the liposomes were centrifuged for 1 h at 105 000 x g. The pellet containing MLV was resuspended in 1 ml liposome buffer and filtered through a polycarbonate 0.4/~m filter (Nucleopor) to remove large aggregates. According to electron microscopy data [23], the average diameter of small unilamellar vesicles (SUV) and MLV was 600 and 1500 ~,, respectively. The concentration of liposomes was evaluated by the content of cholesteryl[1-14C]oleate traces and expressed in mM of total lipid. Conjugation of liposomes with antibodies. For conjugation of liposomes with antibodies, we have used the method described in details by Barbet et al. [21]. Desalting of antibodies was performed on a 5-ml syringe with Sephadex G-25 coarse by centrifugation according to the method of Fry et al. [24]. 1 ml of antibodies (1.5-2.0 m g / m l ) was transferred into 0.1 M phosphate buffer (0.1 M NaCI, pH 7.5) and incubated with a 20-fold SPDP excess (40 mM in ethanol) for 30 min at room temperature. The reaction was arrested by transferring modified antibodies into 0.1 M acetate buffer (0.15 M NaC1, p H 4.5). To activate the modified antibodies, 50 /.tl 1 M dithiothreitol was added and incubated for 20 min at room temperature. The degree of substitution of N H 2 groups, evaluated by spectrophotometry at 343 nm [25], was 8-10 residues/antibody molecule. Following a transfer into the liposome buffer, activated antibodies were added to a liposome preparation ( ( 4 - 5 ) . 10 -4 mol antibodies/mol total lipid) and incubated for 24 h at room temperature. Antibody-liposome complexes were purified by gel filtration on a column of Sepharose CL-4B (0.9 cm x 30 cm). The purified complexes were stored in the dark at 4°C and used within 1 week. Binding of liposomes to cells. J774 Macrophages were grown in 75-cm 2 culture flasks in medium

RPMl-1640 containing 4 mM glutamine, 100 /~g/ml canamycin, 50 ~M/3-mercaptoethanol and 10% fetal calf serum. All components of the culture media were obtained from Flow (U.K.), plastic-ware for cell culture from Falcon (U.S.A.) and Flow (U.K.). For experiments, cells were seeded into the wells of a 96-well plate at a density of 10000 cells/well. At the beginning of the experiment on the 3rd day, cells were rinsed twice with 200 /.tl phosphate buffer saline. Then, liposomes were added in 50 /zl of the growth medium containing 10% lipoprotein-deficient serum instead of fetal calf serum. Liposomes and lipoproteins were introduced into the medium immediately before the addition to cells. Liposomes were usually incubated with cells for 3 h at 37°C in a CO~ incubator. At the end of the incubation, cells were washed five times with 200/,tl liposome buffer and dissolved in 200 ~1 0.5% Triton X-100. Liposome binding to cells was evaluated by the intensity of carboxyfluorescein fluorescence measured on a SPF-500 spectrofluorometer (Aminco Bowman). In some wells, the amount of protein was determined according to Lowry et al. [18] after dissolving cells in 0.1 M N a O H for 30 min at room temperature. The deviation of protein content in different wells did not exceed 10%. The data on liposome binding are expressed in nmol of carboxyfluorescein/mg cell protein. All the experiments were carried out in duplicate and each experiment was repeated at least three times. The results of the most representative experiments are listed in the report.

Results and Discussion

Conjugation of antibodies with liposomes To induce the interaction of liposomes with macrophage receptors for modified LDL, we have used liposomes conjugated with antibodies to LDL. Such liposomes, unlike liposome-LDL conjugates, make it possible to avoid an additional modification of L D L in the course of their coupling to liposomes, and use the same liposome preparations in experiments with both native and modified LDL. To conjugate the antibodies with liposomes, a heterofunctional coupling reagent SPDP was used [21]. The results obtained at gel filtration of lesI-

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EFFLUENT (ml)

Fig. 1. Chromatography of the antibody-bearing liposomes on a column of Sepharose 4B. Liposomes with t251-1abeled antibodies in 1.5 ml liposome buffer were applied to a 0.9 × 30 cm column of Sepharose 4B and eluted with the liposome buffer at a rate of 18 m l / h . Before and after addition of Triton X-100, the intensity of fluorescence was measured in aliquots of the effluent. A, =25I-labeled antibodies; e, intensity of fluorescence before the addition of Triton X-100: C), intensity of fluorescence after the addition of Triton X-100.

labeled antibodies complexes with liposomes containing 0.2 M carboxyfluorescein in the aqueous phase are given in Fig. 1. The elution of ~25I-labeled antibodies in the void volume indicates the formation of antibody-liposome complexes. In our experiments, the efficiency of the antibody coupling b y this m e t h o d was ( 8 - 1 2 ) - 10 -5 m o l antibodies/mol lipids, on average, which corresponds to 5-10 antibody molecules per small unilamellar liposome. These results agree well with the data of Barbet et al. [21]. After the conjugation of liposomes with antibodies, a considerable part of carboxyfluorescein remained inside the liposomes, as indicated by an increase in the intensity of fluorescence of carboxyfluorescein incorporated into the liposomes (by 10-15-fold) following the breakdown of liposomes by a detergent. To test the ability of antibody-bearing liposomes to bind LDL, we used native and malondialdehyde-treated L D L adherent to the surface of plastic plates [26]. An increase in the carboxyfluorescein binding which paralleled a rise in L D L concentration points to the ability of these liposomes to bind L D L (Fig. 2). A lower level of liposome binding to modified L D L indicates that the modification of L D L by malondialdehyde

z

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g. 5 LDL CONCENTRATION ( j u g / m l )

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Fig. 2. Binding of liposomes to LDL. LDL in 200 p,l phosphate-buffered saline were added to the wells of a 96-well plate and incubated for 1 h at room temperature. After washing (five times) with 0.2% bovine serum albumin in phosphate-buffered saline, antibody-bearing SUV (0.3 mM of total lipid) in 50 ~tl medium RPMI-1640containing 10% lipoprotein-deficientserum were added to the wells and incubated for 1.5 h at 37°C. O, Native LDL; z~, malondialdehyde-treated LDL. CF, carboxyfluorescein. reduces the ability of L D L to bind antibodies. The control experiments with 125I-labeled L D L and 125I-labeled LDL treated with malondialdehyde (the results are not shown) demonstrated that the decreased liposome binding is not due to a lower efficiency of modified L D L adhesion to plastic, compared to native LDL.

Binding of liposomes to macrophages Addition of malondialdehyde-treated L D L to the medium during the incubation of antibodybearing liposomes with macrophages produced a 10-20-fold increase in the amount of cell-associated carboxyfluorescein. The efficacy of the liposome binding evaluated by the intensity of carboxyfluorescein fluorescence in the presence of modified LDL rapidly increased within the 1st h of incubation, and then slowed down and reached a plateau by the end of the 2nd h (Fig. 3). The dependence of the antibody-bearing liposome binding to macrophages upon L D L concentration is shown in Fig. 4. The binding of liposomes to macrophages grows proportionally to the concentration of modified LDL, reaching the saturation point at a lipoprotein concentration of about 25 ~ g / m l . In the presence of native LDL, the binding of antibody-bearing liposomes practically remained unchanged compared to the control

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INCUBATION TIME (hours)

Fig. 3. Time-course of liposome binding to macrophages in the presence of modified LDL. SUV conjugated with antibodies (0.3 mM of total lipid) were added to the wells with cultured macrophages in 50 /~l medium RPMI-1640, containing 10% lipoprotein-deficient serum in the absence (O) or presence of 20 v g / m l malondiaidehyde-treated LDL (O), and incubated at 37°C for the indicated periods of time. Binding of liposomes to cells was assessed according to the standard protocol. CF, carboxyfluorescein.

(without LDL). The saturation of the liposome binding may be due to saturation of the liposome-associated antibodies by the lipoproteins. At the modified LDL and liposome concentrations of 25/~g/ml and 0.3 mM lipid, respectively, there were 10-12 LDL particles per one small unilamellar liposome. Excess of unbound modified LDL

can inhibit interaction of the liposome-bound malondialdehyde-treated LDL with the cell receptors. A relatively low level of the antibody-bearing liposome-binding cells in the presence of native LDL indicates that the effect of modified LDL is specific, and allows to assume that in the presence of malondialdehyde-treated LDL, liposomes interact with the receptors for modified LDL. This assumption agrees well with the data obtained by the investigation of the concentrationdependent binding of antibody-bearing liposomes to macrophages in the presence or absence of modified LDL (Fig. 5). The liposome binding in the presence of modified LDL, just as in the previous series of experiments (Fig. 4), exceeded that in the absence of it. Within the examined range of liposome concentrations, binding did not reach the saturation point.

Effect of native and modified LDL on leakage of liposome contents Interaction of iiposomes with certain apoproteins and lipoproteins may lead to the damage of their membranes and leakage of the aqueous phase contents [27,28]. If modified LDL degrade liposomes more effectively than native LDL, this may account for the increased carboxyfluorescein binding to cells in the presence of malondialdehyde-treated LDL which was reg-

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LDL CONCENTRATION ()ug/ml)

Fig. 4. Dependence of liposome binding to macrophages upon the concentration of native and modified LDL. SUV conjugated with antibodies (0.3 mM of total lipid) were added to the wells with macrophages in 50 V1 medium RPMI-1640 containing 10% lipoprotein-deficient serum and the indicated amount of malondialdehyde-treated LDL (O) or native LDL (O), and incubated for 3 h at 37°C. Binding of liposomes to cells was evaluated according to the standard protocol. CF, carboxyfluorescein.

0'.5 LIPOSOME CONCENTRATION (mM of total

1.0

lipid)

Fig. 5. Carboxyfluorescein (CF) binding by cultured macrophages after exposure to increasing amounts of liposomes. SUV conjugated with antibodies were added to the wells with macrophages at the indicated concentrations in 50/Ll medium RPMI1640, containing 10% lipoprotein-deficient serum, in the absence (O) or presence of 20 /Lg/ml malondialdehyde-treated LDL (O), and incubated for 3 h at 37°C. Liposome binding to cells was evaluated according to the standard protocol.

81

istered in our experiments. To rule out such a possibility, we compared effects of native and malondialdehyde-treated LDL on the leakage of carboxyfluorescein from liposomes using the method of Guo et al. [27]. Not more than 20% of the incorporated carboxyfluorescein was released from small unilamellar liposomes during incubation of native LDL (100 ~tg/ml) with liposomes (5 ttM of total lipid) for 3 h at 37°C, whereas the carboxyfluorescein yield during incubation with modified LDL was only 8%. The damaging effect of LDL on multilamellar liposomes was less pronounced: native LDL induced the release of approx. 13% of the incorporated carboxyfluorescein, and modified LDL induced 3.5%. During the incubation of cells with liposomes (0.3 mM of total lipid) and LDL (100 /~g/ml) in the medium containing 10% of lipoprotein-deficient serum, we failed to register a considerable leakage of carboxyfluorescein from liposomes. These data suggest that the modified-LDL-induced increase in carboxyfluorescein binding to

macrophages is not due to a more effective breakdown of liposomes in the presence of malondialdehyde-treated LDL. A comparatively slower leakage of carboxyfluorescein from liposomes in the presence of modified LDL makes it possible to assume that the charge of lipoprotein, altered as a result of modification [19], may play a certain role in their damaging effect on the liposome membranes.

Effect of the inhibitors of cell respiration on the binding of liposomes of different size to macrophages Table I shows the data characterizing interaction of liposomes of different size with macrophages in the presence of modified LDL. Different liposomes contained equal amounts of carboxyfluorescein, but carboxyfluorescein in multilamellar liposomes was bound to macrophages 2-3-fold more effectively than carboxyfluorescein in small unilamellar liposomes. Liposomes (both SUV and MLV), conjugated with antibodies, bound to macrophages approx. 2-fold more effectively than those without antibodies, which may be due to the bind-

TABLE I B I N D I N G O F LIPOSOMES O F D I F F E R E N T SIZE T O M A C R O P H A G E S Liposomes (0.2 m M of total lipid) were added to the wells of Microtest plate with macrophages in 50 /~l medium RPMI-1640 containing 10% lipoprotein-deficient serum in the absence or presence of 2 0 / z g / m l malondialdehyde-treated L D L and incubated for 3 h at 37°C. To some wells, liposomes were added in the medium containing N a F and antimycin A (10 m M and 1 /~g/ml, respectively). Liposome binding to cells was evaluated according to the standard protocol. The results listed are means _+S.E. of four determinations. Liposomes

SUV

Antibodybearing SUV

Antibodybearing SUV + modified L D L

MLV

Antibodybearing MLV

Antibodybearing MLV + modified L D L

Carboxyfluorescein added (pmol/well)

2280

2060

2060

2330

1970

1970

Without inhibitors (carboxyfluorescein p m o l / m g cell protein)

68.18 _+ 10.69

166.19 + 16.02"

1257.11 ± 44.09

108.24 ± 3.23

216.48 _+ 3.55

3376.71 _+ 79.58

With N a F and antimycin A (carboxyfluorescein p m o l / m g cell protein)

35.79 ± 9.35

90.38 +_ 12.93

286.37 ± 27.78

63.92 + 3.30

110.55 _+ 7.67

1970.32 ± 27.65

Energy independent binding (%)

52.49

54.38

22.78

59.05

51.07

28.74

82 ing of antibodies to the receptor for the Fc fragment. A lower level of induction, c o m p a r e d to the data of other authors [1,2], is apparently explained by the lower affinity of rabbit antibodies to the Fc-fragment receptors of mouse macrophages. To study the ratio of surface binding to internalization of antibody-bearing liposomes inside cells in the presence of malondialdehyde-treated L D L , we have investigated effects of the inhibitors of cell respiration [3] on binding of carboxyfluorescein to cells. Addition of antimycin A and N a F , inhibitors of mitochondrial respiratory chain and glycolysis, respectively, to the incubation medium equally decreased the binding of SUV and MLV, both in the absence and presence

of modified LDL. This points to an energy-dependent character of liposome binding and a possibility of liposome penetration inside cells by endocytosis. Thus, our data indicate that particles of relatively large size can penetrate inside cells via the receptors for modified LDL.

Visualization of carboxvfluorescein incorporation into macrophages Fig. 6 illustrates the results of fluorescent microscopy of macrophages incubated with antibody-bearing liposomes. Without modified L D L , a m i c r o s c o p i c e x a m i n a t i o n revealed a barely noticeable specific fluorescence on the periphery of cells. After an addition of modified (but not

Fig. 6. Incorporation of carboxyfluorescein into macrophages (fluorescent microscopy). SUV conjugated with antibodies (0.05 mM of total lipid) were added to macrophages, cultured on coverslips, in medium RPMI-1640 containing 10~ lipoprotein-deficient serum, in the presence (A, B) or absence (C, D) of malondialdehyde-treated LDL (20 ffg/ml), and incubated for 3 h at 37°C. After rinsing according to the standard procedure, the cells were examined on a fluorescent microscope. A and C, phase contrast ( × 320t" B and D, fluorescence.

83 native L D L ) to the i n c u b a t i o n m e d i u m , intensive specific fluorescence often in the form of s e p a r a t e granules a p p e a r e d in the cells, which indicates that the l i p o s o m e label p e n e t r a t e d inside the cells. The i n t e n s i t y of c a r b o x y f l u o r e s c e i n fluorescence varied from cell to cell but, after the a d d i t i o n of m o d i f i e d L D L , u n s t a i n e d cells were p r a c t i c a l l y absent. A s is shown in this report, i n c o r p o r a t i o n of the a n t i b o d y - b e a r i n g l i p o s o m e s into m a c r o p h a g e s is i n d u c e d only in the presence of modified, b u t not native L D L . It suggests that the i n c o r p o r a t i o n of l i p o s o m e s into m a c r o p h a g e s is d u e to their interaction with the m a c r o p h a g e receptors for m o d i f i e d lipoproteins. The l i p o s o m e s interacting with macr o p h a g e s via this p a t h w a y m a y be used to s t u d y the p r o p e r t i e s of receptors for m o d i f i e d L D L , e.g., for identification of cells possessing this type of receptors, which is p a r t i c u l a r l y i m p o r t a n t for e l u c i d a t i o n of the n a t u r e of f o a m cells typical in a t h e r o s c l e r o t i c vascular lesions [15]. Utilization of lipoproteins, specifically L D L , for selective delivery of drugs is lately b e c o m i n g m o r e a n d m o r e feasible [29]. T h e existence of r e c e p t o r p a t h w a y s of L D L m e t a b o l i s m m a k e s it p o s s i b l e to regard L D L as p o t e n t i a l vectors for d r u g t r a n s p o r t [30]. Besides, L D L can be used as d r u g containers. T h e c a p a c i t y of such containers, however, is rather limited, a n d they can hold o n l y h y d r o p h o b i c c o m p o u n d s with a certain structure [31]. T h e results of o u r s t u d y d e m o n s t r a t e that it is p o s s i b l e to t r a n s p o r t the c o n t e n t s of l i p o s o m e s of different size inside cells via the receptors for m o d i f i e d L D L . T h e use of l i p o s o m e s for drug delivery via the l i p o p r o t e i n - r e c e p t o r p a t h w a y cons i d e r a b l y enlarges the p o t e n t i a l of this m e t h o d , since these particles m a y serve as universal highc a p a c i t y c o n t a i n e r s b o t h for h y d r o p h o b i c a n d hyd r o p h i l i c substances.

Acknowledgements T h e a u t h o r s wish to t h a n k Dr. G . A . E r m o l i n for v a l u a b l e help in the p r e p a r a t i o n of a n t i b o d i e s , Dr. L.D. L e s e r m a n a n d Dr. V.P. Torchilin for the c o n s u l t a t i o n s on the l i p o s o m e m e t h o d o l o g y , Dr. N.B. I v a n o v for help in the l i p o s o m e electron m i c r o s c o p y . W e also t h a n k I.I. T o v a r o v a , L.R. Skugarevskaya, a n d A . G . K h v o s t o v for the prep a r a t i o n of the m a n u s c r i p t .

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