Liposomal targeting and drug delivery: kinetic consideration

advanced

drug delivery reviews

ELSEVIER

Advanced

Liposomal

Drug

targeting

Delivery

Reviews

Sciences,

425-444

and drug delivery: kinetic consideration

H. Harashima”, Faculty of Pharmaceutical

19 (1996)

The University

H. Kiwada

of Tokushima, I-78-1, Shomachi. Tokushima 770. Japan

Abstract

As drug carriers, liposomes have great potential in that selective targeting and release rate control of drugs can be performed by appropriate modifications to the carrier itself, without altering the structure of original drugs. In this paper, factors determining the disposition of liposomes were discussed in relation to the underlying mechanisms. Selective targetings of liposomes to specific tissues such as hepatocytes, macrophages and tumors were also evaluated based on the in vitro as well as in vivo data. Finally, the perspective of intracellular drug delivery was also discussed from the mechanistic as well as kinetic points of view for the development of intracellular targetable drug carriers. Keywords:

Liposomes; Pharmacokinetics; Drug delivery Long-circulating liposomes; Intracellular targeting

system; Targeting;

Clearance;

Phagocytosis;

Opsonin;

Contents 1. Introduction .......................................................................................................................................................................... 2. Space for liposome distribution.. ........................................................................................................................................... 2.1. Vascular space and transcapillary passage.. .................................................................................................................... 2.2. Bi-phasic disappearance curve.. ..................................................................................................................................... drugs from liposomes.. ...................................................................................................... 3. Release kinetics of encapsulated 3.1. Interaction between liposomes and blood components.. ................................................................................................ 3.2. Hydrophilic compounds.. ............................................................................................................................................... 3.3. Lipophilic compounds.. .................................................................................................................................................. 4. Factors affecting the clearance kinetics of liposomes ............................................................................................................ 4.1. Uptake mechanisms of liposomes .................................................................................................................................. 4.2. Lipid composition .......................................................................................................................................................... 4.3. Surface charge ............................................................................................................................................................... 4.4. Size ................................................................................................................................................................................ 4.5. Dose .............................................................................................................................................................................. 4.6. Species.. ......................................................................................................................................................................... 5. Selective targeting of liposomes............................................................................................................................................ 5.1. Targeting to hepatocytes.. .............................................................................................................................................. S.2. Targeting to macrophages .............................................................................................................................................. 5.3. Long-circulating liposomes ............................................................................................................................................ 5.4. Targeting to tumors ....................................................................................................................................................... 6. Intracellular disposition of liposomes.. .................................................................................................................................. 6.1. Intracellular pathways after receptor-mediated endocytosis.. ........................................................................................ 6.2. Acidic compartment ......................................................................................................................................................

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6.3. Lysosomal compartment _....... ,,,,.....___.... .._.._...... 6.4. Antigen presentation .._.._.__........._................................ 7. Perspectives .,,,,,..,,......._..................,.,,.,............................... 8. Notation _.....,.,,,,,.__._.......................,.,,.,,,...........................,.. Acknowledgments ..____........,.._...........,................,.,,.............. References _......__,. .._________.... ___....._.____. I.._____.___... .__...._.......

1. Introduction

Selective targeting of drugs using liposomes is expected to optimize the pharmacological effect and toxicities of the encapsulated drugs [l]. For the construction of an effective drug delivery system using liposomes, understanding the kinetics of drug carriers as well as encapsulated drugs is essential. However, the kinetics of free drugs released from liposomes is complex, especially in the in vivo condition, because the observed data results from the convolution of the disposition of liposomes, the rate of release of encapsulated drugs from liposomes and the disposition of free drugs [2-41. To understand the whole system, it is important to evaluate each process as a separate unit. In this paper. we have focused on the disposition of liposomes and the release process of encapsulated drugs, since the disposition of free drugs per se has been examined intensively in each case. The introduction of the clearance concept in the kinetic analysis of liposomes is effective when we analyze the in vivo data and consider the underlying mechanisms [S-7], since ‘clearance’ describes the rate process (such as elimination. release, uptake, etc.) based on the concentration and can link the data obtained in vivo and in vitro. The importance of understanding the intracellular fate of drug carriers was also stressed for the development of a rational drug delivery system.

2. Space for liposome

distribution

2.1. Vascular space and tramcapillary

passage

Capillaries are not barriers for lipophilic species and small molecular weight hydrophilic substances between blood and tissue. yet they are nearly impermeable to macromolecules. Pap-

penheimer et al. formulated the pore theory of capillary permeability [8]. It predicts the diffusion across capillary walls of small hydrophilic solutes through water-filled channels or pores of radius -4 nm. A recent study shows that a majority of microvascular walls seem to show a bimodal size selectivity [9] This implied the presence of a large number of functional small pores (- 4 nm) and an extremely low number of non-size selective pathways, permitting the passage of macromolecules from blood to tissue with large pores ( - 25-30 nm). Based on these pieces of anatomic and physiologic evidence [9], the distribution of principal components of commonly used liposomes are restricted in the vascular space due to their size ( 2 2.5 nm) except where blood vessels are leaky such as in inflamed tissues, tumor tissues and sinusoidal tissues such as liver, spleen and bone marrow. However, the transcapillary passage of small liposomes cannot be excluded. Hwang et al. examined the volume of distribution and the transcapillary passage of small unilamellar vesicles (SUV) made of sphingomyelin (SM): cholesterol (CH) which incorporated the complex of nitrilotriacetic acid with “‘In as radioactive marker for gamma-ray perturbed angular correlation [lo]. This method can distinguish the intact and degraded radioactive marker even in vivo. They suggested that initially the SM:CH SUV remained within the vascular system and occupied a volume of distribution approximately 1.28 times larger than that of erythrocytes in mice. However, they have also indicated that with time, the SM:CH SUV could exit the vascular system and were taken up by surrounding tissues over a period of 24 h. Therefore the vascular space is the minimum value for the volume of distribution of liposomes. The transcapillary passage of liposomes may contribute to the extravascular distribution of long-circulating liposomes (as described in Section 5.3).

H. Harashima, H. Kiwada I Advanced

2.2. Bi-phasic disappearance

curve

Bi-phasic disappearance curves are often observed in the blood concentration of many kinds of liposomes and those curves can be described as follows: C, = A exp( - LYE) + B exp( - fit) where C, represents the blood concentration of liposomes, A and B represent the initial blood concentrations for each of the exponents and (Y and p represent the disappearance rate constants. These observations indicate several possibilities in the distribution of liposomes. The first possibility is that there is a peripheral tissue compartment which slowly equilibrates to the blood compartment, as frequently observed in common drugs. This transport process could be a transcapillary passage through the fenestration

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Drug Delivery Reviews 19 (1996) 42.5-444

and/or the endocytosis or exocytosis of liposomes by endothelial or parenchymal cells. Another explanation for the bi-phasic disappearance curves is that liposomes are composed of heterogeneous components such as different diameters [ll]. In such case, the blood disappearance curve can be regarded as the sum of two which different pharhave exponents macokinetics. Usually the contribution of the second exponential is small ( 510%) for large liposomes and the majority of blood concentration can be explained by the first exponential term. This possibility should be taken into account in the analysis of disposition curves of liposomes. 3. Release kinetics of encapsulated

drugs from

liposomes 3.1. Interaction between liposomes and blood components

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T,,, WW Fig. 1. Relation of total amount of protein bound to liposomes and circulation half-life. Various compositions of liposome labeled with [‘HJcholesteryl hexadecyl ether (100 nm) were injected intravenously into CD1 mice at -20 pmol total lipid/l00 g body weight. At 2 min post injection, the blood was sampled and aliquots of the recovered liposomes were delipidated, and the extracted proteins quantitated using the micro bicinchoninic acid protein assay. The liposomes were composed of (v) PC:CH:PI (bovine liver, 354520) (0) PC:CH:DOPA (35:45:20), (W) PC:CH:CL (35:45:10). (0) PC:CH (5545) (A) PC:CH:PI (plant; 35:45:20), (0) SM:PC and (0) SM:PC:G,, (72:18:10). Cited from Ref. [22].

The mechanisms of interaction between liposomes and blood components were extensively studied [12-211. Lipid composition of liposomes is a critical factor in determining the release property of encapsulated drugs. The rigid membranes retained aqueous phase marker for a longer period in blood circulation [ 12,131. The addition of cholesterol to egg phosphatidylcholine (PC), and the replacement of PC by distearoylphosphatidylcholine (DSPC) can reduce the release rate of encapsulated carboxyfluorescein (CF) both in vitro [14] and in vivo [15]. High density lipoprotein (HDL) was shown to remove phospholipid molecules from liposomes and encapsulated drugs were released at rates depending on the extent of liposomal damage [16]. The complement system is also playing an important role in both the degradation and opsonization of liposomes [17,18]. The activation of complement system by liposomes has been reported in human (191, guinea pig [19] and rat [20,21] serum/plasma. Although there is some inconsistency in the exact mechanism of activation pathways, a negative charge, CH, saturated acyl chains and the size of liposomes are important factors in the activation of complement system.

4?X

II.

Harashima.

H. Kiwada

I Advanced

Drug

Chonn et al. introduced a parameter termed ‘P, value (protein binding value)’ to quantitate the surface adsorption of proteins [22]. P, represent the total protein binding ability of liposomes, expressed in ‘g of protein/m01 of lipid’. As shown in Fig. 1, there is an inverse relationship between P, values and the circulation halflives of liposomes. In particular, liposomes that have greater than 50 g protein/m01 lipid associated with their membranes are cleared very rapidly from the circulation, whereas liposomes that have less than 20 g of associated protein/ mol lipid exhibit more extended circulation times. This shows the significant influence of blood proteins associated with liposomes in the clearance rate of liposomes in vivo. 3.2. Hydrophilic compounds The rate and extent of the release of encapsulated drugs from liposomes was measured quantitatively and continuously in rat serum [23] (Fig. 2). The release process of CF was described by the lag time (r). the first-order rate constant (k) and the maximum release ((Y). Both k and a increased with increasing liposomal size. The increased affinity of larger liposomes for comple-

Delivery

Reviews

IY (lYY6)

425444

ment was suggested to increase both k and (Y.On the other hand, the u decreased with increasing liposome concentration without altering k, which suggested the depletion of complement components in higher concentration of liposomes [23]. Quantitative evaluation of the release process of liposomally entrapped drugs in vivo is more complete than in vitro, because the blood concentration of liposomes changes due to the uptake by RES. [‘HJinulin is a suitable marker to evaluate the release process from liposomes in vivo, because [‘Hlinulin distributes into only extracellular space and is rapidly excreted from kidney via glomerular filtration [4]. The release process of [3H]inulin from liposomes was estimated by the fitting of the time course of [‘Hlinulin excreted into urine based on the pharmacokinetic model of free [ “HJinulin linked with the first-order degradation rate constant (k,) of the marker from liposomes. and the disappearance curves of liposomally encapsulated (Hlinulin [4]. The effect of dose of liposomes composed of hydrogenated egg phospha(HEPC):CH:dicetylphosphate tidylcholine (DCP) on the release process of [‘Hlinulin from liposomes was kinetically evaluated in vivo. The dose-dependent decrease in k, might result from B

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Fig. 2. Time-courses of liposomc degradation in vitro. The latencies of liposomes were continuously measured after incubation with serum. using CF as an aqueous phase marker. Typical curves arc shown to demonstrate the effect of size (A) and concentration (B) of liposomes. (A) The effect of liposome six was examined at low liposome concentration: (0) 800 nm; (W) 400 nm. (B) The effect of liposomc concentration was examined for large liposomes: (0) low concentration (190 nmol HEPCml ‘); (A) high concentration (750 nmol HEPCml ‘). The points represent the observed values and the solid lines represent the fitting curves according to the non-linear least-squarca method. Cited from Ref. [23].

H. Harashima. H. Kiwada

I Advanced Drug Delivery Reviews 19 (1996) 42-f-444

the depletion of complement components [4]. There was a good correlation in k, between in vivo and in vitro for the liposomes with diameters from 200 to 1000 nm [24].

3.3. Lipophilic compounds The transmembrane pH-gradient method is an efficient method for encapsulating weak bases such as doxorubicin (DXR) or vincristine [25,26]. Weak bases are concentrated in the acidic compartment according to the pH-partition theory in addition to the partition to membrane. Almost 100% of these drugs can be loaded into liposomes with various kinds of lipid composition. The half-life for the release of vincristine in buffer increased from approximately 1 h for dimyristoylphosphatidylcholine (DMPC) / CH to 12 h for diarachidoylphosphatidylcholine (DAPC) and dibehenoylphosphatidylcholine (DBPC) containing liposomes. Vincristine was retained in liposomes composed of DSPC/CH more than 90% in an internal pH (pHi) of 2.0, but was 50% at pH 4.0 in mouse serum [26]. Thus, the retention of encapsulated drugs principally depends on the initial pH-gradient as well as lipid composition in vitro [26]. This tendency was also shown in an in vivo study [27], where DSPC/CH/monosialoganglioside GM, (G,,) liposomes, prepared at pHi 2.0, exhibited less than a 20% decrease in drug to lipid ratio over 24 h in the circulation. This high retention of vincristine by DSPC/ CH / G M, liposomes was suggested to depend on how long the internal pH can be kept in the blood circulation. The relationship between release rate of antitumor drugs and antitumor effect will be discussed in Section 5.4.

4. Factors affecting the clearance kinetics of liposomes 4.1. Uptake mechanisms

of liposomes

In general, liposomes are taken up by RES and the rate of clearance of liposomes in blood circulation is determined principally by the

42’)

phagocytic or endocytic uptake ability of liver and spleen [28]. Most soluble compounds with high molecular weight are internalized by cells via pinocytosis or receptor mediated endocytosis [29]. On the other hand, particulate substances such as aged red blood cells, bacteria and cell debris are cleared from blood circulation by professional phagocytes via phagocytosis [30]. In phagocytosis, opsonins play an important role in enhancing the uptake of those particles through binding on the surface of foreign particles and bound opsonins are recognized specifically by the receptors on the surface of phagocytes [30]. Principal opsonins and dysopsonins (which inhibit the uptake) for liposomes were reviewed intensively by Pate1 such as IgG, complements, fibronectin and IgA [31]. Physicochemical factors such as lipid composition, surface charge, liposome size, dose of liposomes as well as species are deeply related in determining the affinity of liposomes to opsonins.

4.2. Lipid composition Gregoriadis and Senior examined the effect of lipid composition in the blood clearance of liposomes where they observed half-lives of liposomes composed of dilauroylphosphatidylcholine (DLPC) (0.1 h), dioleoylphosphatidylcholine (DOPC) (1 h), DSPC (1.5 h), PC (2 h), dimyristoylphosphatidylcholine (DMPC) (6 h), SM (16 h) (all liposomes except DSPC contained 50% CH) [12]. They also found the effect of SM in prolonging the circulation time of SUV, where the half-lives decreased from 11, 11, 7.0, 4.5 and 2.1 h for 100% SM, 77% SM/23% PC, 47% SM/53% PC, 23% SM/77% PC and 100% PC, respectively [13]. The inhibitory effect of SM on the uptake clearance was also observed in the cultured mouse bone marrow macrophages [32]. The uptake of LUV with a mean diameter of 100 nm was decreased remarkably by inclusion of SM in a concentration-dependent manner. These results in vivo as well as in vitro clearly indicated the importance of the composition of phospholipid in determining the rate of clearance of liposomes. The importance of CH was also shown in decreasing the uptake clearance in vivo. As for

430

H. Harahnta.

H. Klwada I Advanced

SUV composed of DSPC, half-lives increased from 1.3 to 20 h by incorporation of CH (50% ) in mice [33]. The effect of CH was also examined for both SUV and reverse phase vesicles (REV) [34]. In both types of liposomes. the hepatic uptake of CH-rich (47%) liposomes were lower than those of CH-poor (20%) liposomes after intravenous administration in mice. The inhibitory effect of CH was confirmed in the isolated rabbit peritoneal macrophages, where the uptake of PC:DCP liposomes of 1070 nm in diameter was decreased in the concentration-dependent manner of CH [35]. Moghimi and Pate1 postulated that serum contains opsonins specific for hepatic and splenic phagocytic cells and these opsonins have different affinities for CH-rich and CH-poor liposomes based on the in vitro uptake experiments [36]. Kupffer cells avidly took up CH-poor liposomes, whereas splenic phagocytic cells took up preferentially CH-rich liposomes in the presence of serum [36]. The inclusion of SM or DMPC inhibited the uptake in both Kupffer and splenic cells, which suggested that fluidity and hydrophobicity of liposomal membranes plays an important role in attracting the right opsonins which determine their phagocytic fate [37]. As for small liposomes, hepatocytes play an important role in the hepatic uptake of small liposomes [38,39]. however, no specific proteins for SUV to hepatocytes were identified.

Drug Delivery Reviews 19 (1996) 42-f-444

On the other hand, the opposite order was observed for large multilamellar liposomes (positive (CL,,,, =20)
4.3. Surface charge The effect of charge was examined using IrsIalbumin as an aqueous phase marker [40]. Negatively charged liposomes disappeared more rapidly than neutral ones and positively charged liposomes remained in blood much longer than negative or neutral ones in rats. Souhami et al. examined the effect of charge and size of liposomes on the blood disappearance curves in mice [41]. Positively charged SUV (33 nm, PC:CH:stearylamine (SA) = 7:2:1; total body clearance (CL,,,) =36 ml/h per kg) disappeared from blood more rapidly than negatively charged liposomes (PC:CH:DCP = 7:2:1; CL,,,, = 27). Neutral SUV (PC:CH = 8:2: CL,,, = 8.4) remained longer in blood circulation than negative ones.

OS! 10

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Size (nm) Fig. 3. The relation between size and total body clearance of liposomes. The data on the blood disappearance curves of liposomes after intravenous administration to mice or rats were cited from the literature [12,13,32,40,42-441 and analyzed by moment analysis. The size and CL,<,, were plotted on the logarithmic scale. The regression analysis of these data was statistically significant. CL,,,, =0.132 (size)’ ““, r* =0.478 (P~O.001).

H. Harashima, H. Kiwada I Advanced

based on the analysis of literature data [12,13,41,43-451. The time-courses of blood disappearance curves were analyzed by moment analysis and CL,,, was calculated by the dose divided by area under the curve (AUC). The studies for which liposome doses higher than 100 pmollkg were omitted from this analysis. The result are shown in Fig. 3 and the regression analysis indicates that log(CL,,,) is proportional to the log(size) of liposomes. The coefficient of variation (T*) was 0.478 and the correlation was significant (PsO.001). The variation which cannot be explained by this regression may result from several factors such as lipid composition, dose of liposomes, surface charge of liposomes, species, or markers for liposomes. The CL,,, ranged from 1 to 900 ml/h per kg) and corresponding half-lives ranged from -2 min to -20 h. The variation explained by the size of liposomes ranged more than lOO-fold, while the variation due to other factors ranged within only lo-fold. Thus, this relation indicates the critical role of the size of liposomes in determining their clearance. The size of liposomes is closely related with the mechanism of liposome uptake by RES. The uptake of large liposomes is mediated via phagocytosis, that of small liposomes via pinocytosis [l&46,47]. The activation of complement system by liposomes were detected in human [19,48], guinea pig [19,49] and rat [20,21] serum/plasma, and complement mediated uptake of liposomes was reported [18,50]. The uptake mechanism of MLV made of HEPC:CH:DCP=5:4:1 were examined using isolated perfused rat liver [18]. The hepatic uptake of liposomes was enhanced by serum depending on the size of liposomes. As shown in Fig. 4, the opsonic effect was seen for the liposomes with a diameter larger than 400 nm. This enhancement was shown to result from the activation of complement system based on the inhibitory effect of opsonic activity by the pretreatment of serum with anti-C3 antiserum [18]. The enhanced uptake was also inhibited by K76-COOH, which inhibits the production of C5a in the cascade of complement activation. C5a activates macrophages that can take up liposomes via complement receptor mediated phagocytosis, while unstimulated macrophages

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Drug Delivery Reviews 19 (lY96) 425-444

*_ 0

0

Complemst

IndqendeDt 8

5

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Release (96) Fig. 4. The size-dependent enhancement of hepatic uptake of liposomes. The hepatic uptake clearance of liposomes are composed of size dependent, complement receptor mediated pathway and complement independent, non-specific pathway. The hepatic extraction of liposomes increased with the increase of liposome size (200, 400 and 800 nm), which was evaluated in the isolated perfused rat liver. This correlated well with the complement mediated release of CF in rat serum (data from Ref. [US])

only bind but do not take up liposomes [l&30,50]. Thus, the enhanced hepatic uptake of these liposomes was suggested to be taken up by complement receptor-mediated phagocytosis. The importance of the size of liposomes in the activation of complement system was also reported [21,51]. There is a gradient in both diameter and frequency of fenestrae along the hepatic sinusoid. Although the mean diameter decreases, the density of fenestrae increases along the sinusoid in the direction of blood flow, resulting in a greater porosity toward the hepatic venule [52]. Kupffer cells are also unevenly distributed within hepatic lobules with the majority being found in the periportal region where they are larger and have greater phagocytic activity than Kupffer cells located in the centrilobular region of the lobule [53]. Based on these physiological evidences, liposomes of the diameter larger than 100 nm could not reach hepatocytes and taken up principally via phagocytosis. On the other hand, small liposomes can be taken up by hepatocytes as well as Kupffer cells. Fractionation of the liver into a parenchymal and a non-parenchymal cell fraction revealed that 80-90% of the slowly cleared liposomes (SM:CH = 1:l) were

H. Harashima, H. Kiwada I Advanced

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taken up by the parenchymal cells while the rapidly eliminated liposomes (PC:CH = 1:1) were taken up more than 95% by nonparenchymal cells [38]. Thus, the size of liposomes also plays an important role in the sorting of liposomes to Kupffer cells or hepatocytes. 4.5. Dose Saturable kinetics is one of the typical characteristics of liposomes and the effect of liposome dose on their disposition has been reported by many researchers [54-561. There are at least two uptake pathways, one is saturable and the other is non-saturable for both small [46,55] and large [57] liposomes. Beaumier et al. observed saturable and non-saturable pathways for SUV (SM:CH=2:1). Chow et al. reported that SUV can be targetable to hepatic parenchymal cells by increasing the dose of liposomes due to the saturation in the uptake by Kupffer cells [47]. Abra and Hunt reported that the saturable pattern of hepatic uptake of liposomes (PC:CH:phosphatidic acid (PA):cY-tocopherol= 4:1:5:0.1) with different diameter could not be explained in terms of the number of liposomes, but could be explained by the total surface area of liposomes [54]. The saturation kinetics of liposomes has been often analyzed based on the Michaelis-Menten equation [55]. However, Kume et al. showed the inadequacy of this equation for the saturable hepatic uptake clearance of liposomes [58], where the relationship between averaged blood concentration and CL,, was separated depending on the infusion rate. Rather, the saturation kinetics of hepatic uptake clearance of liposomes was explained by AUC independent of the infusion rate [59]. This AUC-dependence of the saturable hepatic uptake clearance was mathematically derived under the assumption of no efflux [57]: CL, = X/AUC = X,,, /AUC( 1 - exp( - CL,AUC/X,,,)) where CL,,, and X,,, represent the maximum uptake clearance and the maximum capacity of uptake, respectively. X represents the uptake

Drug Delivery Reviews I9 (1996) 42-f-444

amount by the liver. This analytical solution for CL,, is determined by AUC and this equation was applied well with the observation data regardless of the mode of administration (intravenous rapid injection or constant infusion) [57]. This model is named the ‘satiation model,’ because the decreased hepatic uptake clearance of liposomes corresponds well with the decreased appetite for food in satiation (Fig. 5). The underlying mechanism is the same for receptor mediated endocytosis, where recycling of receptor can be neglected. Therefore, this AUC-dependent saturation kinetics can be applied for many kinds of receptor-mediated endocytosis, so far as efflux (dissociation after receptor binding or excretion after internalization) can be neglected. In the case of saturation phenomenon for the uptake of particulate substances, it should be distinguished whether it is related to the depletion of opsonins or to the CL,, saturation [60]. By increasing the dose of liposomes from 1 to 10 ,umol HEPC/kg, CL,, showed saturation without depletion of opsonins, and the opsonic activity as well as CL,, decreased at 100 pmol HEPCIkg, using both in vivo and in situ experiments [61]. Since the result may depend on liposome condition, a distinction should be made in each case of liposomes. 4.6. Species

The allometric relationship between pharmacokinetic parameters and body weight was

wll.x

wllsx

(1 - wn.x)

&l.X

Fig. 5. Schematic representation of the concept of ‘Satiation Model’. The saturation kinetics of hepatic uptake clearance (CLh) was explained by assuming that the decrease of uptake clearance is in proportion to the amount taken up (X):CL, = CL_(l -XIX,), where CL, and X, represent the maximum uptake clearance and the maximum uptake amount. Cited from Ref. [57].

H. Harashima. H. Kiwada I Advanced

extensively studied in the 1980s [62,63]. There are good allometric relationships between body weight and each pharmacokinetic parameter in small as well as large molecular weight compounds [64]. However, little has been done regarding the species difference in the disposition of liposomes. The species difference of pharmacokinetic parameters was examined for liposomes composed of HEPC:CH:DCP=5:4:1 with a mean diameter of 400 nm at three dose levels (l-100 pmol HEPC/kg), among mice, rats and rabbits [65]. The mean residence time (MRT) of liposomes increased with the increase of body weight and the dose of liposomes. This increase of MRT resulted from the decrease of CL,,,, which was principally explained by the decrease of CL,,. The CL,, of these animals was regressed well in a multiple regression analysis as a function of body weight and the dose of liposomes with an exponent for the body weight around 0.5. This clearly indicates that smaller animals have a higher CL,, per unit body weight. The higher CL,, could not be explained by the higher density of Kupffer cells in smaller animals, because immunohistochemical analysis revealed that there was no significant difference in the density of Kupffer cells among these species. The species difference in the hepatic uptake mechanism was observed between rats and mice using isolated perfused liver system. In rats, the hepatic uptake of liposomes was mainly explained by an opsonindependent uptake pathway. On the other hand, an opsonin-independent pathway was dominant in mice [65] These results indicate that species difference in the disposition of liposomes is important in extrapolating the experimental results in small animals to humans.

5. Selective

targeting

of liposomes

5.1. Targeting to hepatocytes As mentioned above, the liver sinusoid has fenestration with an average diameter of -100 nm [52], and hepatocytes are exceptionally suitable target for liposomes with small diameter.

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However, according to the investigation of intrahepatic cellular distribution, SUV was shown to localize Kupffer cells as well as hepatocytes in rats [66] and mice [46]. Selective targeting (80% of the administered dose) of liposomes was possible by increasing the dose of liposomes to saturate the uptake capacity of Kupffer cells [46]. Efficient delivery of liposomes to hepatocytes was also performed by targeting the galactose receptor on the surface of hepatocytes by lactosylceramide [66] and asialofetuin [67]. Spanjer and Scherphof introduced lactosylceramide to be recognized by galactose (or asialoglycoprotein) receptor on the surface of hepatocytes [66]. The control SUV composed of DMPC:CH:PS = 4:5:1 distributed 20% in hepatocytes and 20% in nonparenchymal cells after 1 h of administration, while lactosylceramide containing SUV distributed 48% in hepatocytes and 27% in non-parenchymal cells. It has been shown that a galactose receptor is also located on Kupffer cells and functions in endocytosis or phagocytosis of particles with galactosyl residues [68]. This receptor is not evenly distributed on the surface of Kupffer cells but clustered for the uptake of particles [69]. However, the galactose receptors on hepatocytes are evenly distributed [70] and can uptake ligand of up to 8 nm in diameter [71]. Thus, galactose receptor may not be a good target for the selective delivery of liposomes to hepatocytes. Another ligand for hepatocytes was investigated by incorporating 30-stearyl glycyrrhizin (GLOSt) into SUV composed of HEPC: CH:GLOSt = 4:4:1 [72]. The distribution of GLOSt-SUV to liver increased from 10 to 42% of control SUV (DCP instead of GLOSt). The uptake of GLOSt-SUV by the hepatocytes was also examined under the primary cultured hepatocytes. The binding of GLOSt-SUV to hepatocytes was inhibited by free glycyrrhizin and the uptake of GLOSt-SUV was saturable [73]. This result is consistent with the in vivo experiments where the distribution of GLOSt-SUV to hepatocytes increased by 3-fold compared to control SUV. Although further increase of the selectivity is important, the intracellular fate should also be kept in mind in relation to the uptake pathway.

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5.2. Targeting to macrophages

The activation of tumoricidal activity of liposomes macrophages by encapsulating lymphokines was successful by choosing the appropriate size and composition of liposomes [74,75]. The inclusion of PS into MLV enhanced the phagocytosis by rat alveolar macrophages and was shown as a requirement for the in vivo delivery to the lung in mice 1751. It was also shown that liposomally encapsulated mouse interferon gamma (IFN-y) can activate the tumoricidal activity of human monocytes, which could not be activated by free mouse IFN-y due to the species-dependent receptor binding of IFN-y [76]. These results indicate the potential usefulness of liposomal targeting to macrophages. Selective delivery of liposomes to macrophages was performed by targeting the mannose receptor on the surface of macrophages [77.78]. The uptake of mannosylated liposomes was saturable and was not inhibited by control liposomes [77]. Cetylmannoside modified (Man) MLV were shown to increase CL,, in rats without altering splenic uptake clearance [79] and to increase the uptake by human blood monocytes [80]. These results suggested the contribution of mannose receptor mediated phagocytosis of Man-MLV in the enhanced hepatic uptake clearance. However, according to the investigation on the hepatic uptake mechanism using the isolated rat perfused liver system, surprisingly, Man-MLV were shown to be taken up via complement receptor-mediated phagocytosis, not by the mannose receptor [81]. Man-MLV were also shown to activate complement system in human plasma [82]. The role of a triggering factor which activates complement system by Man-MLV was also pointed out [83]. These results show the role of complement system in clearing the Man-MLV from blood circulation, and should be taken into consideration in the development of drug carriers. SW?.Long-circulating

liposomes

New formulation of liposomes has been developed by introducing ganglioside G,, in 1987

[84] and hydrogenated phosphatidyl inositol in 1988 [85] to prolong the half-life up to 12 h after intravenous administration. In 1990, it was shown by several groups that the inclusion of a new class of synthetic diacyl lipids with hydrophilic polymer poly(ethylene glycol) (PEG) [86-881 or palmitoylglucuronide [89] resulted in a further prolongation of blood circulation times. PEGliposomes prolonged the circulation half-lives regardless of membrane fluidity [87,90], or surface charge [91]. In addition, the saturation effect of the dose of liposomes in the uptake by RES was the typical characteristics of common liposomes, however, G,, or PEG-liposomes show linear pharmacokinetics with an average half-life of 20 h in mice for doses from 0.1 to 10 pmol phospholipid/mouse [92]. The relationship between dose and CL,,, of conventional (PC:CH= 2:l) and PEG-liposomes (Fig. 6) suggests that PEG-liposomes escape the specific uptake by RES such as opsonin-dependent uptake or receptor-mediated pathways and are taken up via a non-specific uptake pathway. A hypothesis on the molecular mechanism of long-circulating liposomes was postulated that stabilization results from local surface concentration of highly hydrated groups that sterically inhibit both electrostatic and hydrophobic interactions of a variety of blood components at the liposome surface [93]. Computer simulation was 1 50

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Fig. 6. The relation between liposome dose and total body clearance of conventional and long-circulating liposomes in mice. Conventional liposomes were composed of PC:CH = 2:l (O), while long-circulating liposomes were composed of SM:PC:CH:GMl= 1:1:1:0.2 (A) or SM:PC:CH:PEG(1900)DSPE 2731:1:1:0.2 (0). Liposomes were extruded through IO0 nm Nuclepore filters. Cited from Ref. [92].

H. Harashima, H. Kiwada I Advanced

also performed to explain the hypothetical model on the effect of PEG-liposomes, where the protective layer of the polymer on the liposome surface is considered as a statistical ‘cloud’ of polymer possible conformations in solution [94]. Dysopsonin hypothesis was postulated for G,, liposomes based on the fact that molecular structure of G,, is important for its functional ability to prolong the liposome circulation time [95]. GM,-liposomes were shown not to be effective in rats [96,97]. Although G,, liposomes show long circulating properties, depending on the species as described above, PEG-liposomes prolong the circulation time of liposomes independent of species such as in mice [86-881, rats [91,98,99], dogs and humans [NO]. Although there seems to be a tendency of increasing the half-life of PEG-liposomes with increasing the body weight, no systematic analysis has been performed.

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5.4. Targeting to tumors

Fig. 7. Effects of size and membrane fluidity on tumor accumulation of liposomes. ‘H-labeled liposomes were injected at a dose of 100 pmol lipid/kg into rats bearing Yoshida sarcoma. Rats were killed 24 h after injection for sampling of blood and tumors. Lipid composition in molar ratio: (0) HEPC:DCP:CH =5:1:4, (0) PC:DCP:CH= 5:1:4. Cited from Ref. [7].

It is known that high-molecular-weight compounds such as albumin and fibrinogen are retained in tumor tissue [loll. The exact mechanism is not clarified yet, but several factors such as the leakiness of the capillary wall and the lack of a lymph system in tumor tissue are considered as the underlying mechanisms of this phenomenon. Liposomes, especially long-circulating liposomes, are also retained in tumor tissue and there is an optimum size (-100 nm) of liposomes in the passive targeting (Fig. 7) [7,85,102]. Whether liposomes are taken up by tumor cells or not was tested by microscopic analysis. It was demonstrated that sterically stabilized liposomes traversed the endothelium of small blood vessels and extravasated into extracellular spaces [103,104]. The increased microvascular permeability of long-circulating liposomes was also reported based on the in vivo fluorescence video microscopy by measuring both the vascular and interstitial amounts of labelled liposomes [105]. According to their analysis, the vascular permeability of long-circulating liposomes was two times higher than that of conventional liposomes in tumor tissues, while no difference was observed in non-tumor tissues. The mechanism of

how liposomes are retained in the interstitial space in tumor tissue is still unclear [7]. The understanding of this mechanism will provide further direction for efficient targeting of anticancer agents to tumor tissue. Doxorubicin is a AUC-dependent anticancer agent [106] and is often used for liposomal targeting to tumor [25,102,107-1091. Intensive study revealed that encapsulation of DXR into conventional liposomes increased the therapeutic index via decreasing the toxicity [107]. For longcirculating liposomes, DXR was delivered to the extracellular space of tumor tissue in the form of liposomes. The released DXR in the extracellular space of tumor exerts its effect and increased the therapeutic index substantially [109] (Fig. 8). The endocytosis of liposomes was not necessarily required for the cytotoxicity of DXR [llO]. Thus, it was indicated that the local sustained release of DXR from liposomes in tumor tissues must be the key mechanism. The local sustained release effect of anticancer drugs by liposomes is considered to work much efficiently for the cell-cyclespecific anticancer drugs such as vincristine, since the IC,, for these anticancer drugs strongly

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Fig. 8. Effects of epirubicin (A) and doxorubicin (B) formulations on survival of mice innoculated with C-26 tumor cells on day 0. Treatment began 10 days later and was repeated twice for a total of three injections at weekly intervals. The day of death of each mouse was determined and the percentage of surviving mice in each experimental group was plotted against time up to 120 days. Treatment groups are indicated in the figure. Cited from Ref. [109].

depends on the exposure time [106]. Vincristine was loaded into liposomes by the pH gradient method, which increased the retention of vincristine both in vitro and in vivo by choosing DSPC:CH liposomes [ 1111.Vincristine loaded by the pH-gradient method decreased the toxicity and increased the cytotoxicity as compared to free drug, which increased the therapeutic index remarkably [112].

6. Intracellular disposition

of liposomes

6.1. Intracellular pathways after receptormediated endocytosis

Early studies implied liposome-plasma membrane fusion [113], but it is recognized that liposomes do not fuse with the plasma mem-

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brane, while endocytosis and/or phagocytosis are the dominant mechanism for cellular uptake of liposomes [114,115]. Intracellular transport and processing vary markedly between different receptor-ligand systems and different cell types, but these systems are classified in four general categories [29]. Class 1: Receptors that recycle but target their ligand to lysosomes. This category includes the LDL, asialoglycoprotein, mannose 6-phosphate and mannose receptor systems. Class 2: Receptors that recycle but do not target their ligand to lysosomes. The ligand remains attached to the receptor during its transit through the cell. In polarized cells this may result in transport of ligands between the apical and basolateral surfaces. Examples include transferrin and IgG. Class 3: Receptors that do not recycle and target their ligand to lysosomes, including the EGF receptor, the insulin receptor and the Fc receptor. Class 4: Receptors that do not recycle and do not transport ligands to lysosomes. Transport of IgA represents a unique mechanism, transcytosis of IgA results in cleavage and loss of the IgA receptor. This category is flexible and depends on the cell type. For example, asialoglycoproteins and mannose receptors are classified into class 1, where ligands are targeted to lysosomes; however, significant amounts (-30% ) of internalized ligand were recycled to the cell surface [116]. EGF and insulin are classified into class 3, where receptors do not recycle, however the recycle of these receptors are also reported [117,118]. As for liposomes, there is not enough information to classify the intracellular disposition; however, key steps such as the transport to the acidic compartment and degradation in lysosomes are evaluated quantitatively and the importance of the regulation of intracellular transport and processing of liposomes was discussed. 6.2. Acidic compartment The acidification of liposomes can be evaluated quantitatively, using pyranine (HPTS; Shydroxy-1,3,6_pyrenetrisulfonate), a highly fluorescent dye. This dye is highly water soluble, membrane impermeable, shows a pH-dependent shift rather than quench of its fluorescence spec-

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trum and has an isosbestic point at 413 nm, permitting correction of the measurements to the total amount of dye present in the sample [119]. HPTS-containing liposomes were acidified by a cultured murine macrophage cell line (5774) with a half-life of 15-20 min. The acidification of liposomes exhibited bi-phasic kinetics and 5080% of the liposomes reached an average pH lower than 6 within 2 h [120]. Acidification usually occurs in endocytic vesicles, endosomes and lysosomes, with the pH values in early and late endosomes and lysosomes reported to be 6-6.6, 5-6 and 4-5, respectively [121,122]. The mechanism by which endocytic substances are transported from early to late endosomes and finally to lysosomes has not been clarified. Two models have been postulated. The ‘vesicle-shuttle model’ assumes that early and late endosomes are preexisting compartments that communicate through vesicle-mediated transport [123], while the ‘maturation model’ assumes that early endosomes mature gradually into late endosomes [124]. Yoshimura et al. measured the time course of acidification of liposomes (PS:PC:CH= l:l:l, 130 nm) in a single mouse peritoneal macrophage and found that acidification proceeded continuously and linearly to pH 5.5 in 30 min, not step wise, which suggested the maturation model [125] (Fig. 9). This observation corresponded to the pH profiles of endocytic pathways for receptor-mediated endocytosis, where internalized ligands reaches early endosomes acidified to pH -6-6.5 with a half-life of 2-5 min, late endosomes acidified to pH 5-6 with a half-life of lo-15 min and lysosomes acidified to pH 4-5 with a half-life of -35 min [121,122,126].

6.3. Lysosomal compartment Since the degradation of liposomes in lysosomes is a key step in designing intracellular targetable carrier by liposomes, kinetics of intracellular degradation of liposomes, factors affecting the rate as well as extent of liposomes degradation should be systematically evaluated. The metabolic fate of “C-cholesteryl oleate ( 14C-CHOA) and ‘2”I-bovine serum albumin

0.3

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Fig. 9. Time-courses of change in the Ruorescence excitation ratio (F,,,,/F,,,,) of liposome-encapsulated HPTS, that is, acidification in macrophages. (A) Fluorescence of ten single cells were randomly measured and expressed as the mean with standard deviation. (B) Macrophages were incubated with HPTS containing liposomes for 3 min, washed and fluorescence measured in a single cell at intervals of 3 or S min. The arrow and dashed line show the time when SO mmol NH,CI was added and the time-course after its addition. respectively. Cited from Ref. [12.5).

(‘*‘I-BSA) e n capsulated in large unilamellar vesicles (LUV) were measured in cultured rat Kupffer cells [127]. After 2 h of incubation, twothirds of the internalized CHOA has been hydrolysed. The cholesterol moiety released from the cholesteryl ester is recovered in the free cholesterol pool of the Kupffer cells. The major fraction of the 14C-oleate moiety that is removed from the 14C-CHOA is re-incorporated into phospholipids. The liposomal “SI-BSA was also readily degraded and this degradation was inhibited by the lysosomotropic agent chloroquin (40 pmol) [127]. These results indicated that liposomes were extensively degraded in lysosomes. The degradation of liposomes was also evaluated kinetically using 4-methylumbelliferyl P-D-

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glucoside (4MU-Glc) as an aqueous phase, degradable marker [125]. The degradation of liposomes can be measured by the fluorescence of 4MU, which was released by the enzymatic hydrolysis in lysosomes. The degradation started 30 min after the uptake of liposomes (PS:PC:CH=l:l:l, 130 nm), which suggested that the delivery of liposomes from cell surface to lysosomes required 30 min (Fig. 10). The degradation of liposomes was also analyzed kinetically in peritoneal macrophages using “‘I-BSA as aqueous phase, degradable marker, where two kinds of degradation processes were observed. one with a half-life of 13 min. and the other with a half-life of 7.5 h [128]. The degradation was kinetically evaluated in

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vivo by double labelling the liposomes with ‘?BSA (represents the degradation of liposomes) and “H-cholesteryl hexadecyl ether (3H-CHE: represents the uptake of liposomes). The intact liposomes were estimated by the ‘2”I-BSA/“HCHE ratio. The biphasic degradation was also observed in vivo, and the rapid degradation process was diminished by increasing the dose of liposomes [129]. Two kinds of model, the ‘sorting model’ and the ‘traffic jam model,’ have been postulated to explain the saturable kinetics of liposome degradation in macrophages [129]. The effect of CH on the degradation of liposomes was also evaluated in rats [130]. The inclusion of CH decreased the degradation of liposomes both in vivo and in vitro. The incubation of both liposomes with isolated lysosomal fractions showed a difference in susceptibility to lysosomal where CH-free liposomes were degradation, much more sensitive to lysosomal esterase than the CH containing liposomes. There seems to exist a similar tendency in the degradation of liposomes between in the blood circulation and in lysosomes. However, the information on the factors determining the intracellular disposition is still lacking and further studies are required.

6.4. Antigen presentation

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Tlnw (min)

Fig. 10. Time-courses of intracellular fates of 4MU-@-o-Cilccontaining (A) and calcein-containing (B) liposomes in macrophages. For (A), LUV were composed of PS:PC:CH= 1:l:l. containing IO mmol 4MU-p-o-Glc. Filters with excitation bands of 330-380 and emission barriers below 420 were used. For (B), LUV containing 60 mmol calcein and filters with excitation bands of 410-485 nm and emission barriers below 515 nm were used. Cited from Ref. [125].

Liposomes can be a potential immunological adjuvants and antigen presentation is one of a typical index for the intracellular disposition of liposomally encapsulated substances. The process of antigen presentation to cell surface in the immune system involves initial processing of the antigen by specialized antigen presenting cells, such as macrophages, B lymphocytes and dendritic cells. Liposomes containing a synthetic recombinant protein were phagocytosed by macrophages and the internalized protein was recycled to the cell surfaces where it was detected by enzyme-linked immunosorbent assay [131]. Pulse-chase experiments revealed that the antigen appeared on the macrophage surface within 15 min after initial incubation and continued to be expressed until 24 h, with maximum at -6 h, then decreased gradually.

H. Harashima, H. Kiwada I Advanced

Major histocompatibility complex (MHC) molecules exist in two forms, class I (MHC-I) and class II (MHC-II). The mechanisms of antigen processing differ for the two classes [132]. MHC-II molecules present antigens derived from extracellular proteins or proteins that target endocytic compartments, whereas MHC-I molecules present antigens that are synthesized in the cytosol or penetrate into the cytosol [132]. The pH-sensitive liposomes were developed [I331 to deliver foreign molecules to the cytosolic compartment from within the endosomes. This type of liposome is mainly composed of dioleoylphosphatidyl-ethanolamine (DOPE) and an acidic amphiphile. The usefulness of pH-sensitive liposomes was shown in the delivery of ovalbumin (OVA) to the class I pathway [134]. Most of the ingested liposomal OVA were rapidly catabolized and released into the extracellular medium. The residual processed antigen took about 2 h to reach the cell surface for recognition by cytotoxic T lymphocytes, The liposome-mediated antigen presentation exhibited a transient kinetics which was manipulatable with antigen dose [134]. The protein antigen hen egg lysozyme was targeted to endosomes or lysosomes by encapsulating it in liposomes of different membrane composition [ 1351. Acid-sensitive liposomes released their contents in early endosomes, whereas acid-resistant liposomes sequestered their contents from potential endosomal processing events and released their contents only after delivery to lysosomes. Antigen encapsulated in acid-resistant liposomes was processed in a chloroquine-sensitive manner and presented more efficiently than soluble antigen or antigen encapsulated in acidsensitive liposomes. Thus it was suggested that peptides were recycled from lysosomes, transported to endosomes to bind MHC-II, and then expressed at the cell surface [135]. Based on these pieces of information, it was suggested that selective antigen presentation can be possible via MHC-I or MHC-II by choosing appropriate type of liposomes. Although the information on the intracellular transport and degradation of liposomes are accumulating as described above, further studies on the intracellular disposition of liposomes are

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required, especially in relation to the mechanism of uptake.

7. Perspectives The development of long-circulating liposomes made it possible to target selected tissues, such as tumors and the potential advantage of liposomes as drug carriers is recognized. Although intensive studies were performed on the factors determining the disposition of liposomes in experimental animals, only a few have been with regard to species difference. Thus, further studies are required in the species difference of the pharmacokinetics of liposomes so that valuable information in experimental animals can be extrapolated to humans. Analysis of the intracellular disposition of liposomes should be extended for designing a new ‘intracellular targetable drug carrier’ based on the intracellular transport and processing mechanisms.

8. Notation alb, bovine serum albumin AUC, area under the curve CH, cholesterol CHE, cholesteryl hexadecyl ether CHOA, cholesterylolate CL,,, hepatic uptake clearance CL,“,, total body clearance DAPC, diarachidoylphosphatidylcholine DBPC, dibehenoylphosphatidylcholine DCP, dicetylphosphate DLPC, dilauroylphosphatidylcholine DMPC, dimyristoylphosphatidylcholine DOPC, dioleoylphosphatidylcholine DOPE, dioleoylphosphatidylethanolamine DSPC, distearoylphosphatidylcholine DXR, doxorubicin G monosialoganglioside GM i Hz&. hydrogenated egg phosphatidylcholine MHC, major histocompatibility complex MLV, multilamellar vesicles LUV, large unilamellar vesicles OVA, ovalbumin PC, egg phosphatidylcholine

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PA, phosphatidic acid PS, phosphatidylserine REV, reverse-phase vesicles RES, reticuloendothelial system SA, stearylamine SM, sphingomyelin SUY small unilamellar vesicles

Acknowledgments

The authors thank Mr. R. Cogley for his correction of the English of our manuscript.

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Gabizon, A.. Shiota. R. and Papahadjopoulos, D. (1989) Pharmacokinetics and tissue distribution of doxorubicin encapsulated in stable liposomes with long circulation times. J. Natl. Cancer Inst. 81. 14X4-1488. 131Chonn. A. Semple, S.C. and Cullis, P.R. (1991) Scparation of large unilamellar liposomes from blood components by a spin column procedure: towards identifying plasma proteins which mediate liposome clearance in viva. Biochim. Biopys. Acta 1070. 215-222. H., Kume. Y.. Yamane, C. and Kiwada. H. [41 Harashima. (1993) Kinetic modelling of liposome degradation in blood circulation. Biopharm. Drug Dispos. 14, 265-270. H. and Kiwada. H. PI Kume. Y.. Yamane, C., Harashima, (1991) Quantitative analysis of the stability of liposomes in blood in vivo. Xenobiot. Metab. Dispos. 6, 201-208. H.. Midori, Y., Ohshima, S., Yachi. K.. ffd Harashima, Kikuchi, Y. and Kiwada, H. (1993) Kinetic analysis of tissue distribution of doxorubicin incorporated in liposomes in rats (II) Biopharm. Drug Dispos. 14. 595-608. [71 Uchiyama, K.. Nagayasu. A., Yamagiwa, Y., Nishida. T. Harashima, H. and Kiwada, H. (1993) Effect of the size and fluidity of liposomes on their accumulation in tumors: A presumption of their interaction with tumors. Int. J. Pharm. 121. 195-203.

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