Molecular and CellularBiochemistry 120: 119-126, 1993. 9 1993 KluwerAcademic Publishers. Printedin the Netherlands.
Influence of liposome charge and composition on their interaction with human blood serum proteins Trinidad Hernfindez-Caselles, Jos6 Villalafn and Juan C. Gdmez-Fernfindez Departamento de Bioquimica y Biologia Molecular A, Facultad de Veterinaria, Universidad de Murcia, Apartado Postal 4021, E-30080 Murcia, Spain Received 11 May 1992; accepted 14 October 1992
Abstract Lipid composition and specially their electrostatic properties, were found to greatly influence the stability of liposomes in human blood serum. The amount and type of serum proteins bound to the liposomes were also clearly influenced by lipid composition and charge of liposomes. A good correlation was found between the amount of serum proteins adsorbed to a given type of liposome and its instability as measured by the release of an encapsulated fluorescent probe. Liposomes that bind the highest amount of protein were the least stable, except for the case of liposomes containing gangliosides, which were fairly stable even at a high amount of bound protein. Liposomes with neutral charge containing phosphatidylcholine were the most stable and bound the lowest amount of protein. Liposomes with positive charge behaved similarly to those with neutral charge. However, the stability of negatively charged liposomes was very dependent on their composition. Those liposomes containing only one class of negatively charged phospholipids bound a great amount of protein and were very unstable. However, those liposomes containing also phosphatidylcholine bound less protein and were more stable. The examination of the electrophoresis patterns of serum proteins bound to the different types of liposomes indicated the presence of specific proteins which correlated with liposome instability. (Mol Cell Biochem 120: 119-126, 1993)
Key words." liposome stability, liposome-blood interaction Abbreviations: CF - 6-carboxyfluorescein, Chol - Cholesterol, DCP - Dicetylphosphate, DPPC - Dipalmitoylphosphatidylcholine, G C - Galactocerebroside, GM1 - Monosyaloganglioside, PC - Phosphatidylcholine, PG Phosphatidylglycerol, PI - Phosphatidylinositol, PS - Phosphatidylserine, RES - Reticuloendothelial system, SA Stearytamine, SM - Sphingomyelin
Introduction Liposomes have been used as delivery systems by in-
travenous injection into the circulation due to their
Address for offprints: J.C. G6mez-Fernfindez,Dpto. Bioquimicay Biologfa Molecular A, Fac. Veterinaria, Universidad de Murcia, Apartado Postal 4021, E-30080 Murcia, Spain
120 capacity to encapsulate different types of therapeutic agents [1-5]. However, their use as drug carriers in vivo is limited, firstly, by the stability of liposomes in the blood and secondly, by their removal by the reticuloendothelial system (RES) cells. Nevertheless, if these two limitations were abolished or reduced, liposomes could be used actively as a dosage form. The problem of interaction of liposomes with blood components and their stability has been extensively reviewed [4-8]. It has been found that liposome stability in blood can be enhanced by cholesterol [9], gangliosides [10, 11], structural modification of the phospholipids [12] or lipid polymerization [13]. But in spite of these and other studies, the interaction of liposomes with blood components and its mechanism is far from clear. Moreover, different blood proteins, including lipoproteins, can bind to liposomes in whole blood or serum [13-15]. For example, plasma high density lipoproteins remove phospholipids from the liposome bilayer unstabilizing liposomes [16] and opsonins enhance the uptake of liposomes by the RES cells [17]. However, very few studies have correlated protein binding, liposome stability and phospholipid composition. In the present study, human serum has been used as model of a biological fluid [18] to study liposome stability. Binding of serum components to neutral, positive and negatively charged liposomes with different phospholipid composition has been correlated with its stability in serum. In addition, the amount and type of specific proteins bound to the surface of liposomes were related to liposome instability.
Materials and methods Materials Dicetylphosphate (DCP), 6-carboxyfluorescein (CF), bovine brain monosyaloganglioside (GMI) , egg yolk phosphatidylethanolamine (PE), bovine brain phosphatidylserine (PS), egg yolk sphingomyelin (SM), galactocerebroside (GC), stearylamine (SA) and molecular weight standards for electrophoresis were obtained from Sigma, Poole, Great Britain. Egg yolk phosphatidylcholine (PC), wheat germ phosphatidylinositol (PI) and phosphatidylglycerol (PG) were obtained from Lipid Products, South Nutfield, Great Britain. L-c~-dipalmitoylphosphatidylcholine (DPPC) was obtained from Avanti Polar Lipids, Birmingham, Alabama and cholesterol (Chol) from Merck, Darmstadt,
Germany. CF was purified according to Ralston et al. [19] and cholesterol was recrystallized twice from ethanol before use. All inorganic reagents were of analytical grade and aqueous solutions were made up with double distilled and deionized water.
Blood serum preparation Human serum was obtained from clotted transfusion blood obtained from the Seccidn de Inmunologia of the Arrixaca Hospital, Murcia. Before use the serum was clarified by centrifugation for 2 rain in a Heraeus microfuge.
Liposome preparation Standard solutions of lipids were stored as chloroformmethanol solutions at - 40~C and samples were taken for preparations as required. Chloroform solutions of lipids were dried under a stream of O2-free N 2 and the last traces of solvent were eliminated by desiccation under vacuum for more than three hours. Multilamellar liposomes were prepared in Tris 10raM, NaCI 0.9%, pH 7.4 buffer to give a final concentration of 10 mg lipid/ml by vortexing above the temperature of the gel to liquid-crystalline phase transition of each sample. Mixing was continued until a homogeneous and uniform suspension was obtained. To study the integrity of liposomes in the presence of serum, multilamellar liposomes containing encapsulated CF were formed in the same way by using a buffer containing Tris 10mM, NaC1 50 mM and CF 100 mM pH 7.4. Extraliposomal CF was removed by washing with Tris 10mM, NaC1 0.9%, pH 7.4 buffer three times and sedimenting at 30000 x g for 15 min at 4 ~C. Liposomes were resuspended to give a final 10 mg lipid/ml solution. Lipidic phosphorus was determined as described by Bartlett [201.
Effect of serum on liposome integrity Liposome fractions containing CF (0.3 ml containing 3 mg total lipid) were mixed with 0.3 ml human serum and incubated for 3 h at 37~C. Aliquots were diluted 500 times and the fluorescence (?,ex= 492 nm and k~m= 518 rim) measured using a Shimadzu RF-540 spectrofluorophotometer before and after liposome lysis by the
121 addition of 100/xL 10% (w/v) Triton X-100. Percentage of encapsulated CF was calculated from the expression: % C= 200-((Fi-
Fo)* 100)
7=,--?;
where F~and F t represent the fluorescence of the diluted sample before and after detergent lysis, and Fo represents the fluorescence of a diluted aliquot of the same sample immediately after liposome preparation but before incubation with serum. Background leakage of CF was studied as a function of time in Tris 10 mM, NaCI 0.9%, pH 7.4 buffer at 37~ To measure serum protein binding to liposomes, liposomes without encapsulated CF were incubated with serum as before but 1 h. Then, the suspension was diluted to 10ml with buffer and washed three times at 30000 x g for 15rain. Pellets were resuspended with buffer to a final volume of 200/xL. Protein concentration was determined using the Lowry method [21] as modified in [22] for use in the presence of detergents. The interference of the lipids on the Lowry assay was taken into account when liposome-bound proteins were assayed. The absorbances of the blanks were always lower than 20% of the problems. In order to check sedimentation of soluble serum proteins, serum was diluted and washed as described above and protein was determined in both pellets and supernatants with no significative quantity of protein found in the pellets. Proteins bound to liposomes were solubilized as described in [13] and subjected to electrophoresis in the presence of sodium dodecyl sulfate under non-reducing conditions according to Laemmli [23]. Equal amounts of protein were added per lane and the gels were stained with Coomassie brilliant blue R [24].
Results and discussion Figure 1 shows the influence of charge and lipid composition of liposomes on their stability in human serum. Neutral liposomes (Fig. 1A) can be divided in two groups depending on their stability, those containing phosphatidylcholine (PC) and those containing sphingomyelin (SM) but not PC. PC containing liposomes and liposomes with dipalmitoylphosphatidylcholine (DPPC) alone retained much of the encapsulated CF even at 3 h of incubation at 37 ~C. It is also interesting to note that PC and DPPC liposomes have similar stability in serum at 37 ~C (Fig. 1A), i.e., in the liquid-crystalline state for PC liposomes and in the gel phase state for
DPPC liposomes [25]. However, liposomes containing SM, pure or plus cholesterol (Chol), were much less stable. After a short time of incubation, nearly all encapsulated CF was liberated to the medium from SM liposomes, whereas the addition of Chol slightly reduced the liberation of CF. By contrast, incorporation of PC to SM liposomes increased considerably the liposome stability in serum (Fig. 1A) as it has been shown before [26]. Although both types of phospholipids bear the same head-group, they show a significative difference in stability. It is important to note that SM has the ability to form intermolecular hydrogen bonds [27] whereas PC has not. The rigidification induced in SM molecules by hydrogen bonding may be responsible for this difference in liposome stability. Incorporation of stearylamine (SA) to PC or PC/ Chol liposomes converts neutral liposomes to positively charged liposomes, and increases slightly the stability of liposomes (Fig. 1B) as it has been found before [28]. Negatively charged liposomes showed a great variability in their stability as shown in Fig. 1C and 1D. Of those liposomes containing only one type of phospholipid, the most stables were composed of phosphatidylinositol (PI) and phosphatidylserine (PS) whereas the less stable were those composed of phosphatidylglycerol (PG) (in the order PI = PS > PG). Incorporation of Chol in these liposomes increased their stability by approximately the same extent, but the stability was maximal when PC was incorporated into liposomes composed of Chol and either PI, PS or PG. Incorporation of dicetylphosphate (DCP), which gives negative charge to PC liposomes, did not change significatively the stability of liposomes, but the incorporation of negatively charged sphingolipids, such as monosyaloganglioside (GMI), decreased slightly their stability (Fig. 1C). Liposomes formed by PC, SM, Chol and GM1 mimic the external monolayer of the erythrocyte membrane [21] and showed a slightly lower stability when compared to PC, PC/Chol or PC/SM/ Chol liposomes (compare Fig. 1A and 1C). As a general trend, it can be said that liposomes formed by only one type of phospholipid, except PC, showed the lowest stability, independent on surface charge. Incorporation of Chol increased the stability of liposomes by approximately 10-20% in all of them, but maximal stability was obtained when PC was incorporated into the liposomes. The increase in stability upon Chol incorporatio n may be related to the changes in the physical properties of the membrane, which are known to take place [29] and which will prevent, to some
122 i
i
i
9
i
]3
A
1.0 0.9 0.8
Z I.,--I C) 09
m~ 0
0.7 0.6 0.5
~
0.4 0.5 0.2 0.1
X
' " -..... : . . . . . . . . . . . . . . .
0
I
7
I
I
i
I
l
I
I
I
I
I
I
D
m~ .<
~
0.0
~
o. 9
......
,
0
Z
O. 6
\~ \
0.5 E~
.<
o.4 ' *
,>..
-.
0.3 0.2 0.1
[
0.0 t
0
1
2
TIME
3
0
(H
i
i
1
I
2
t
5
OURS)
Fig. 1. Stability of liposomes in serum at 37 ~ C as a function of time. (A) Neutral liposomes composed of ( 0 ) PC, ( A ) PC/Chol 5 : 1, ( T ) SM, ( ~ ) SM/Chol 5 : 1, (11) SM/PC/Chol 5 : 5 : 1 (E]) DPPC, (B) positively charged liposomes composed of (O) PC/SA 9 : 1 and (D) PC/SA/Chol 5 : 1 : 0.1 and negatively charged liposomes composed of (C) ( 9 PC/Chol/DCP 5 : 1 : 0.1, (*) PS, ([]) PS/Chol 5 : 1, (O) PS/PC/Chol 5 : 5 : 1, ( V ) PC/GM19 : 1, ([3) PC/SM/Chol/GM11 : 1 : 1 : 0.14 and (D) ( 0 ) PG, ( 9 PG/Chol 5 : 1, (~I~) PG/PC/Chol 5 : 5 : 1 (O) PI, ( A ) PI/Chol 5 : 1, (@) PI/PC/Chol 5 : 5 : 1. Lipid ratios are expressed on a molar basis and the curves represent the ratio of liposome leakage in serum to liposome leakage in buffer. Values are shown + S.D. for three experiments.
extent, the interaction of serum proteins with the liposomes. It has been shown, for example, a reduction of lipoprotein interaction with phospholipids [30]. Incorporation of negative (DCP) molecules to PC liposomes did not influence significatively their stability, but incorporation of positive (SA) molecules to PC liposomes increased their stability. It is known that serum proteins bind to the surface of liposomes and this binding changes with liposome composition affecting liposome stability [7]. We have studied liposome stability and protein binding by quantify-
ing proteins bound to liposomes and determining differences in types of bound proteins in order to correlate liposome stability and protein binding. Figure 2 shows the relationship between the amount of bound protein and stability of different types of liposomes. It can be observed that the most stable liposomes in serum (neutral liposomes and liposomes containing either SA or DCP) are the liposomes that bind the smallest quantity of proteins, whereas the less stable types of liposomes bind bigger quantities of protein (Fig. 2), which correlates with their stability in serum
123 100 90
~_~i~[~ -
I
I
I
I
I
I
I
I
I
I
t---~--~
4
80 .,--4
70 13 r~
60 11
(1)
50
0
40
r~
0 C~
30
\
20
\
\
\
10 0
0
I
I
I
I
I
I
I
I
I
I
I
5
10
15
20
25
50
55
40
45
50
55
60
/xg b o u n d p r o t e i n / mg lipid Fig. 2. Stability of liposomes in serum at 37 ~ C as percentage of retained CF at i h with respect to bound protein for (11) neutral, ( 0 ) positively and negatively charged liposomes containing ( ~ ) D C P or (O) negative phospholipids. Liposomes were c o m p o s e d of (1) PC, (2) PC/Chol 5 : 1, (3) D P P C , (4) P C / P E / C h o l 5 : 5 : 1, (5) P C / G C / C h o l 5 : l : 1, (6) SM/PC/Chol 5 : 5 : 1, (7) PC/Chol/DCP 5 : 1 : 0 2 , (8) PC/Chol/DCP 5 : 1 : 0.5, (9) PG, (10) Pg/Chol 5 : 1, PG/PC/Chol 5 : 5 : 1, (12) PC/SM/Chol/GM11 : 1 : 1 : 0.14, (13) PC/GM19 : 1, (14) PS, (15) PS/Chol 5 : 1, (16) PS/PC/Chol 5 : 5 : 1, (17) PI, (18) PI/Chol 5 : 1, (19) PI/PC/Chol 5 : 5 : 1, (20) PC/Chol/SA 5 : 1 : 0.1, (21) PC/Chol/SA 5 : 1 : 0.5 and (22) PC/SA 9 : 1. Lipid ratios are expressed on a molar basis. Values are shown + S.D. for three experiments.
(li)
(see above). The incorporation of either Chol or PC reduces the amount of protein bound to liposomes as seen in Fig. 2, thereby increasing their stability. A special case is that of those liposomes incorporating GMI, which having a high stability in serum bind comparatively a great quantity of protein. It has been shown before that gangliosides, and specifically GMI, prolong liposome circulation half-life [11, 31]. Then, the proteins bound at the surface of these liposomes must be different from the proteins bound to liposomes containing negative phospholipids because they do not induce their instability. It has to be noted that bound proteins could not be determined in SM and SM/Chol liposomes because it was impossible to recover any appreciable amount of liposomes after centrifugation of the incuba-
tion mixture. Then, as a general trend of the systems studied here, a liposome will be more stable when it binds a smaller quantity of protein except for GM1 containing liposomes. However, quantity of protein is not the only factor to take into account when studying liposome stability but also the type of protein. Since there are many serum protein components which bind to liposomes either specifically or unspecifically [reviewed in 7], we have studied the type of protein which binds to each specific type of liposome by comparing the electrophoresis pattern of proteins with that of whole serum proteins (Fig. 3). As it can be seen in Fig. 3, serum albumin is bound in a great extent to all the different types of liposomes. In fact, it is the most abundant protein among neutral,
124
Fig. 3. Bindingof humanserumproteins on neutral and negativelychargedliposomesby polyacrylamidegel electrophoresis.(1) PC, (2) PC/Chol5 :
1, (3) PS, (4) PS/Chol5 : 1, (5) PS/PC/Chol5 : 5 : 1, (6) human serum, (7) PI, (8) PI/Chol5 : 1, (9) PI/PC/Chol5 : 5 : 1, (10) PG, (11) PG/Chol5 : 1, (12) PG/PC/Chol5 : 5 : 1, (13) PC/SM/Chol/GM11 : 1 : 1 : 0.14 and (14) PC/GM~9 : 1. Lipid ratios are expressed on a molar basis. positively and negatively charged liposomes. The exceptions among the negatively charged liposomes are those containing GM1 where there are two proteins of about 200 Kd of molecular weight which are also found to bind in a great amount. It has been demonstrated before that serum albumin may adsorb to the outer surface of liposomes but its binding has no effect on liposome integrity [32]. Since it would be interesting to correlate liposome stability in serum with the proteins bound to each type of liposomes, it is convenient to examine which protein specificities can be discerned from the electrophoretic patterns shown in Fig. 3. Neutral liposomes, characterized by low protein binding and high stability (see above), as those composed by PC and PC/Chol, bind only serum albumin (marked as F in Fig. 3) and small quantities of very high molecular weight proteins (molecular weight > 450Kd). The same pattern of binding was obtained when the liposomes contained SA, either with or without Chol (results not shown). On the other hand, negatively charged liposomes show different patterns depending on lipid composition. It should be kept in mind that within this type of liposomes, the amount of protein bound and the stability in serum were very dependent on lipid composition (Fig. 1 and 2). As a general trend some proteins of
52-55 Kd (labeled as G in Fig. 3) were found in all of them, except in those containing GM1. There are however some other proteins which are found only in negatively charged liposomes, which show the lowest stability in serum, as it is the case of PS, PS/Chol, PI, PI/Chol, PG and PG/Chol. These proteins, whose molecular weight range from approximately 250 Kd to 480 Kd, have been labeled as A, B, C and D. It is interesting to note that these proteins do not bind to other negatively charged liposomes which are more stable in serum as PS/PC/Chol, PI/PC/Chol, and PC/Chol/DCP. It seems that the presence of PC in these liposomes prevents the binding of proteins A, B, C, and D. The reason may be that the density of negative charges given by PS and PI is decreased when other neutral lipids are intercalated among them. The electrophoretic pattern of proteins bound to liposomes containing PG is different from those found for liposomes containing PS and PI where many more types of proteins are found (Fig. 3). Again, liposomes containing PC (PG/PC/Chol), which are more stable than those containing PG and PG/Chol, are observed to bind considerable lower number of different serum proteins. Another protein which should be remarked is protein E ( = 7 5 K d ) which binds to all negatively charged liposomes (Fig. 3). It is known that Chol reduces the interaction of serum
125 proteins with liposomes [33] and inhibits the action of lipoproteins [30] and phospholipases [34]. It is also known that Chol inhibits the uptake of liposomes by RES cells [9, 35]. Changes in physical properties of membranes produced by Chol may be responsible for the higher stability, and this may be possible through difficulting the insertion of certain serum amphipathic proteins in the membrane. It can be observed in Fig. 3 changes in the patterns of protein bound upon inclusion of Chol, specially in some types of liposomes. This is the case of PS liposomes which bind proteins C and E upon inclusion of Chol; PI liposomes also bind more protein E and several other proteins and the same applies to PG liposomes. Most probably, proteins A - G are positively charged. We have not been able of identifying these proteins by comparison with other previously described serum proteins of similar molecular weight and positive charge at physiological pH. We then conclude that they are minoritarian serum proteins, since they are not observed in whole serum. Only protein E ( ~ 75 Kd) has a molecular weigth similar to that of transferrin, 76.5 Kd [36]. However, transferrin has a pI of 5.9 [37], i.e., it will have a negative charge at pH 7.4. Since protein E probably has a positive charge at this pH, we conclude that protein E and transferrin are different proteins. On the other hand, it has been described before that negatively charged liposomes are taken up more quickly than positive or neutral liposomes by peritoneal macrophages [38] and the incorporation of PS to liposomes increases phagocytosis by the RES cells [39]. It is thus tempting to speculate that one or some of the proteins which bind specifically to negatively charged liposomes may be implicated in their recognition by the RES cells. As seen in Fig. 2, GM1 containing liposomes are different from liposomes containing negatively charged phospholipids because, binding a relatively large amount of proteins, they are very stable. We have used liposomes containing GM1, either with PC alone or with PC, SM and Chol mimicking the outer erythrocyte membrane leaflet [11], and the results obtained are shown in Fig. 3. As it can be seen, they bind in relatively greater amounts serum protein components in the molecular weight range 185-450kd. These proteins, although specifically bound to these liposomes in relatively great amounts, do not decrease significatively their stability in serum as shown in Figs 1 and 2. Therefore they do not seem to be able of disrupting the liposome membrane. It has been described before that gangliosides stabi-
lize liposomes in plasma [31]. It can be speculated that this stabilization is due to the binding of the proteins observed in Fig. 3. These proteins should have positive charge and seem to be minoritarian ones in whole serum, with the exception of albumin (Fig. 3). In conclusion, liposome composition and charge seem to be of paramount importance in determining the stability of liposomes in serum, with neutral and positively charged liposomes being more stable, whereas those liposomes with a high negative charge density are the least stable. A good correlation between the amount of protein bound and instability has been observed with the exception of liposomes containing gangliosides which bind a great amount of proteins but still they possess a good stability.
Acknowledgements This work has been supported in part by grant No. PA86-0211-CO2-01 from CAICYT (Spain). We wish to thank Dr. Rocio Alvarez from Arrixaca Hospital, Murcia, for her generous help in obtaining serum samples and her useful comments.
References 1. GregoriadisG: Targetingof drugs. Nature 265: 407-411, 1977 2. Juliano RL, Stamp D: Pharmacokineticsof liposomeencapsulated anti-tumor drugs. BiochemPharmacol27: 21-27, 1978 3. Juliano RL: Renaissance of the magic bullet: an essay on the controlled deliveryof drugs. PharmacyInt 2: 41-45, 1980 4. Scherphof GI, Damen J, Wilschut J: Interactions of liposomes with plasma proteins. In: Gregoriadis G (ed) Liposome Technology. CRC Press, Boca Rat6n, Florida, Vol 3, 1984, pp 205224 5. Senior J, Gregoriadis G: Methodology in assessing liposomal stability in the presence of blood, clearancefrom the circulation of injected animals,and uptake by tissues. In: GregoriadisG (ed) Liposome Technology.CRC Press, Boca Ratdn, Florida, Vol 3, 1984, pp 263-282 6. Juliano RL: Interactionof proteins and drugswith liposomes.In: Ostro MJ (ed) Liposomes.Marcel Dekker, New York, 1983, pp 53-86 7. Bonte F, Juliano RL: Interaction of liposomes with serum proteins. Chem Phys Lipids40: 359-372, 1986 8. BlackCDV, GregoriadisG: Interaction of liposomeswith blood plasma proteins. BiochemSoc Trans 4: 253-256, 1976 9. KirbyC, GregoriadisG: The effect of the cholesterol content of small unilamellarliposomeson the fate of their lipidcomponents in vivo. Life Sci 22: 2223-2230, 1980 10. Allen TM, Ryan JL, PapahadjopoulosD: Gangliosidesreduce
126
11.
12.
13.
14.
15.
16.
17.
18. 19.
20. 21.
22. 23. 24.
25.
leakage of aqueous-space markers from liposomes in the presence of human plasma. Biochim Biophys Acta 818: 205-210, 1985 Alien TM, Hansen C, Rutledge J: Liposomes with prolonged circulation times: factors affecting uptake by reticuloendothelial and other tissues. Biochim Biophys Acta 981: 27-35, 1989 Agarwal K, Bali A, Gupta CM: Effect of phoepholipid structure on stability and survival times of liposomes in circulation. Biochim Biophys Acta 883: 468-475, 1986 Bont6 F, Hsu MJ, Papp A, Wu K, Regen SL, Juliano RL: Interactions of polymerizable phosphatidylcholine vesicles with blood components: relevance to biocompatibility. Biochim Biophys Acta 900: 1-9, 1987 Juliano RL, Lin G: The interaction of plasma proteins with liposomes: protein binding and effects of the clotting and complement systems. In: Tom BH, Six HR (eds.) Liposomes and Immunology. Elsevier, Amsterdam. pp 49-66, 1980 Moghimi SM, Patel HM: Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol rich liposomes. FEBS Letters 233: 143-147, 1988 Scherphof G, Roerdink F, Waite M, Parks J: Disintegration of phosphatidylcholine liposomes in plasma as a result of interactions with high density lipoproteins. Biochim Biophys Acta 542: 296-307, 1978 Moghimi SM, Patel HM: Different properties of organ-specific serum opsonins for liver and spleen macrophages. Biochim Biophys Acta 984: 379-383, 1989 Altman PL, Dittman DS: Blood and other Body fluids. Fed Am Soc Exp Biol, Washington DC, 1961 Ralston E, Hjelmeland LM, Klausner RD, Weinstein JN, Blumenthal R: Carboxyfluorescein as a probe for liposome-cell interactions. Effects of impurities and purification of the dye. Biochim Biophys Acta 649: 133-137, 1981 Bartlett GR: Phosphorus assay in column chromatography. J Biol Chem 234: 466-471, i958 Lowry OK, Rosebrough N J, Farr AL, Randall RJ: Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265275, 1951 Wang C-S, Smith RL: Lowry determination of protein in the presence of Triton X-100. Anal Biochem 63: 414-417, 1975 Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685, 1970 Weber K, Osborn M: The reliability of molecular weight determinations by dodecyl sulfate polyacrylamide gel electrophoresis. J Biol Chem 244: 4406-4419, 1969 Marsh D: Handbook of Lipid Bilayers, CRC Press, Boca Rat6n, Florida, 1990
26. Allen TM, Chonn A: Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEBS Letters 1: 4246, 1987 27. Boggs JM: Intermolecular hydrogen bonding between membrane lipids. In: Kates M, Manson LA (eds) Membrane fluidity. Plenum Publishing Corp, New York, 1984, pp 3-53 28. Kirby C, Clarke J, Gregoriadis G: Effect of the cholesterol content of small unilamellar liposomes on their stability in vivo and in vitro. Biochem J 186: 591-598, 1980 29. Oldfield E, Chapman D: Dynamics of lipids in membranes. Heterogeneity and the role of cholesterol. FEBS Letters 23: 285-297, 1972 30. Kirby C, Clarke J, Gregoriadis G: Cholesterol content of small unilamellar liposomes controls phospholipid loss to high density lipoproteins in the presence of serum. FEBS Letters 111: 324328, 1980 31. Gabizon A, Papahadjopoulos D: Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc Natl Acad Sci USA 85: 6949-6953, 1988 32. Van Rooijen N, Van Nieuwmegen R: Liposomes in immunology: multilamellar phosphatidylcholine liposomes as a simple, biodegradable and harmless adjuvant without any immunologic activity of its own. Cell Immunol 49: 402-407, 1980 33. Tall I J, Lange Y: Incorporation of cholesterol into high density lipoprotein recombinants. Biochem Biophys Res Comm 80: 206212, 1978 34. Op den Kamp JAF, Kauerz MT, Van Deenen LLM: Action of pancreatic phospholipase Az. Biochim Biophys Acta 406: 169177, 1975 35. Patel HM, Tuzel NS, Ryman BE: Inhibitory effect of cholesterol on the uptake of liposomes by liver and spleen. Biochim Biophys Acta 761: 142-151, 1983 36. Bing DH, Rosenbaum RA: Plasma and cellular modulatory proteins, Center for Blood Research, Boston, 1981 37. Malamud D, Drysdale JW: Isoelectric points of proteins: A Table. Anal Biochem 86: 620-647, 1978 38. Hsu M J, Juliano RL: Interaction of liposomes with the reticuloendothelial systems. II. Non-specific and receptor-mediated uptake of liposomes by mouse peritoneal macrophages. Biochim Biophys Acta 720: 411-419, 1982 39. Allen TM, Williamson P, Schlegel RA: Phosphatydylserine as a determinant of reticuloendothelial recognition of liposome models of the erythrocyte surface. Proc Natl Acad Sci USA 85: 806%8072, 1988