Molecular and Biochemical Parasitology, 12 (1984) 49-60 49 Elsevier

MPP 00439

QUANTITATIVE AND ULTRASTRUCTURAL STUDIES ON THE UPTAKE OF DRUG LOADED LIPOSOMES BY MONONUCLEAR PHAGOCYTES INFECTED WITH LEISHMANIA DONOVANI

STEPHEN HEATH, MICHAEL L. CHANCE and ROGER R.C. NEW

Department of Parasitology, Liverpool School of Tropical Medicine, Pembroke Place, Liverpool L3 5QA, England

(Received 28 September 1983; accepted 3 January 1984)

This study compared splenic and hepatic uptake of free and liposome-entrapped sodium antimony gluconate after i.v. administration to mice infected with Leishmania donovani. It was demonstrated that entrapment within liposomes greatly altered the kinetics of uptake of the drug. We were also able to show that liposomes composed of sphingomyelin, stearylamine and cholesterol were marginally better than any other preparation in delivering entrapped drug to liver and spleen. X-ray microanalytical studies on the uptake of liposomes by Kupffer cells infected with L. donovani have indicated that internalised liposomes probably fuse with parasitophorous vacuoles, transferring their contents into the immediate locality of the leishmanial parasites. It is proposed that this is the way in which liposome entrapped antileishmanial agents have an enhanced therapeutic effect over free drug therapy.

Key words: Leishmaniasis; Liposomes; Sodium antimony gluconate; X-ray microanalysis

INTRODUCTION

Previous studies have described the use ofliposome-encapsulated sodium antimony gluconate for the treatment of experimental visceral leishmaniasis [1-3]. Each of these independent studies demonstrated that the efficacy of antimonial treatment could be increased several hundred fold by incorporating the antileishmanial drug into the liposomal aqueous phase. The success of liposome therapy is thought to be due to the fact that both parasites and liposomes are taken up by the same cells (ie. Kupffer cells). However, until now, only circumstantial evidence has been presented to show that intact, drug loaded liposomes interact with parasitophorous vacuoles within host macrophages. Obviously, the optimisation of this form of lysosomotropic drug therapy must depend solely on the increased localisation of the liposome-entrapped

Abbreviations: SP, sphingomyelin; PC, phosphatidylcholine; SA, stearylamine; DCP, dicetyl phosphate; CHOL, cholesterol; DMPC, dimiristoylphosphatidylcholine.

0166-6851/84/$03.00 © 1984 Elsevier Science Publishers B.V.

50

agent in the vicinity of the parasites. To maximise the effects of liposome-entrapped agents, it is therefore essential to determine the effects of altered liposome composi- tion and charge on the cellular and subcellular localisation of liposomes and entrap- ped agents.

This study describes a new approach to the problem of localising intracellular liposomes and their entrapped agents utilising the heavy metal properties of the entrapped antileishmanial compound sodium antimony gluconate (Pentostam) and the non-therapeutically active intracellular marker, chromium. The requirement for unequivocal evidence of true localisation of liposomes is justified by the fact that myelin-like membrane whorls can be regularly seen in electron micrographs of liver cells that have not been exposed to liposomes. These myelin-like membrane whorls, which are probably fixation artefacts, are often wrongly interpreted as incorporated liposomes [4]. It was envisaged that the exact identity and location of the liposomes within infected macrophages could be achieved using the technique of X-ray micro- analysis, which is based on the process of electron-excited excitation of X-rays. This 1;echnique has been used successfully by other workers to demonstrate the uptake of an antimony containing compound by Schistosoma mansoni in mice [5].

During the course of this investigation, the total antimony uptake by mouse liver and spleen, infected with L. donovani, was quantitated at timed intervals after intrave- nous administration of either free or liposome-entrapped sodium antimony gluco- nate, using the technique of atomic absorption spectrophotometry. It was thought that any alteration in the pharmacokinetics of drug uptake could probably explain why i.v. administered liposome-entrapped drug is more effective in killing leishmanial parasites in the liver than free drug alone. This area of study was also guided by the fact that very few studies have been carried out comparing the pharmacokinetics of known antileishmanial agents entrapped inside liposomes with that of free drug, although a previous study by Ward et al. [6] did follow the distribution of liposome- entrapped antimony in plasma, spleen and liver from uninfected, healthy mice, using liposomes of a single composition. However, quantitative uptake studies ofliposomes composed of radiolabelled lipids have been undertaken although these have been subject to criticism due to the rapid exchange of phospholipids between liposomes and cells [7,8].

MATERIALS AND METHODS

Infection of animals. NMRI inbred mice (18-20 g) from the Liverpool School of Tropical Medicine, were injected intravenously with 0.2 ml of a crude spleen cell homogenate containing leishmanial amastigotes at a density of approximately 10 7

m1-1, from cotton rats used routinely to passage the parasite. Infections were allowed to progress for at least 10 days before any liposome preparations were administered.

Preparations ofliposome. Liposomes were prepared as described by New et al [9] by a

51

modification of the method of Bangham and Horne [10] and were composed of the following lipids: phosphatidylcholine (PC)/cholesterol (CHOL) (mol ratio 7:3); sphingomyelin (SP)/dicetyl phosphate (DCP)/CHOL (mol ratio 7:2:1); SP/stearyla- mine (SA)/CHOL (mol ratio 4:3:1); PC/DCP/CHOL (mol ratio 7:2:1); dimiristoyl- phosphatidylcholine (DMCP)/DCP/CHOL (mol ratio 7:2:1). All lipids were chromatographically pure as demonstrated by TLC on silica gel. Briefly, liposome components were dried down under reduced pressure from a chloroform solution and dispersed in an aqueous solution of the antileishmanial compound sodium antimony gluconate (300 mg ml -~) or potassium dichromate (50 mg ml-~), at 4°C using an MSE 150 Ultrasonic Probe Disintegrator. The dispersion was subjected to 15 s burst of sonication (8-10 pm peak to peak) with 45 s periods of cooling. A total sonication time of 5 min was used and the final concentration of lecithin was 20 mg ml-L Typically the percentage of starting material entrapped was 7% and 5% respectively. All liposomes were shown to be multilammellar by negative staining techniques. All liposome preparations were stored under nitrogen at 4°C until required.

Unentrapped material was removed by gel filtration on a 5 ml microcolumn of Sephadex G50 by centrifuging the column at 10X g for 10 min. All unentrapped solute remained on the column while nearly all the liposomes were eluted. All doses of liposomes were freshly prepared and used within 3 h of preparation.

Atomic absorption spectrophotometry. Equivalent doses of either free or liposome-en- trapped sodium antimony gluconate were administered i.v. to 5 large groups of mice after which 5 mice from each group were removed at timed intervals and exsanguina- ted by cardiac puncture. After the mice had been killed, their livers and spleens were removed, weighed and homogenised thoroughly in dilute Tris/HCl buffer (10 mM, pH 7.2. The homogenates were pooled and centrifuged at 400 X g for 5 rain. The resultant pellet was re-homogenised and centrifuged as before. This process was repeated 3 times. Finally, the combined supernatants were filtered through very fine nylon mesh. Each of the samples were diluted, where necessary, with distilled water to obtain the desired concentration range and then passed through an EEL atomic absorption spectrophotometer equipped with antimony hollow cathode lamp. Anti- mony calibration solutions were prepared by appropriate dilution of a stock solution of antimony potassium tartrate (1 mg m1-1) to the desired concentration range (ie. 1-100 lag ml-l).

Microanalysis. To study the interaction of i.v. administered liposomes and their entrapped agents with macrophages infected with leishmanial parasites, normal transmission electron microscopy and X-ray microanalysis were employed. Either neutral (PC/CHOL, 7:3), positive (SP/SA/CHOL, 4:3:1) or negative (PC/DCP/ CHOL, 7:2:1) liposomes containing either sodium antimony gluconate or potassium dichromate were injected intravenously (0.4 ml) into two large groups of infected NMRI inbred mice. Mice were removed from experimental groups at times intervals

52

after the administration of either l iposome-entrapped or free drug and anaesthetised with sodium pentobarbitone (May & Baker Ltd.). After the onset of anaesthesia, the hepatic portal vein was cannulated and the liver was perfused with ice cold fixative for

3 rain at a flow rate of 2 ml min -~. The fixative and the tissue processing protocol were the same as that used by Scallen and Dietert [I 1]. The reason for choosing this fixative

was because of the inclusion ofdigitonin which is thought to combine with cholesterol in membranes, making the cholesterol less susceptible to extraction by organic

solvents used in the processing of tissue. It was envisaged that this may increase the electron density of liposomal membranes, thus aiding their localisation. For normal

transmission EM, ultrathin serial section 60-80 nm thick were cut on an LKB ultratome 111 and mounted on 200 mesh copper grids (Agar Aids), stained with lead citrate and viewed on an AEI EM6B electron microscope. For X-ray microanalytical studies, serial sections 80-100 nm thick were cut and mounted on 200 mesh carbon

nylon grids (Agar Aids) and viewed on a Kratos Cora electron microscope fitted with a Link microanalysis microprocessor.

RESULTS

Liver and spleen uptake of free and liposome entrapped sodium antimony gluconate. Table I shows the average amount of antimony in individual mouse livers at timed

intervals after intravenous administration of either l iposome-entrapped or free so- dium antimony gluconate.

The kinetics of uptake of sodium antimony gluconate by liver and spleen was markedly altered by entrapping the drug within liposomes. It was demonstrated that after 5 mins there were 2-3 fold differences in the amount of ant imony in the livers of liposome treated mice in comparison to livers from mice that had received free drug.

At the end of the sampling period (ie. 24 h) this difference had increased to greater than 40 fold. Over the whole sampling period the amount of antimony in livers from mice receiving liposome-entrapped drug steadily increased, whereas the opposite occurred in mice that had received free drug. Splenic uptake of iiposome entrapped antimony peaked after 4 h, at which time there was between 3-5 times less antimony in spleens from mice that had received free drug. After 8 h the amount of antimony in the spleens of mice that had received only free drug was undetectable using the employed technique.

The liposome preparation delivering the greatest amount of antimony to both liver and spleen over a 24 h period was composed of S P / S A / C H O L , although there appeared to be only slight differences in uptake between each of the liposome preparations. Tissue antimony uptake data for neutral liposomes was not included in Table I as the amount of antimony retained within this type of liposome was very small. Consequently the amount reaching the liver and spleen was not detectable after i.v. injection up to 24 h. Intravenous injection of an equivalent level of free drug was also not detectable using the present regime.

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Ultrastructural localisation of liposomes and their entrapped agents. Initial experiments attempting to localise antimony-containing liposomes by X-ray microanalysis were not very successful due to the difficulty in resolving the main antimony peak (3.75 keV) from other background emission peaks, calcium in particular (3.8 keV).

After this initially disappointing result it was decided to investigate the possibility of using alternative intracellular probes that could be more readily detected by X-ray microanalysis. After a number of compounds were investigated it was found that chromium fulfilled the criteria of a good marker for the purposes of this study. It was found that detectable quantities of chromium remained inside liposomes after remo- val of unentrapped material (results not shown) and that it had a Kct peak in the most sensitive area of the X-ray emission spectrum (5.4 keV), and was not overlapped by other background peaks.

Figs. la and b demonstrate the intracellular localisation of chromium-containing liposomes composed of SP /SA/CHOL, within Kupffer cells infected with L. donovani amastigotes. In Fig. la, the liver was removed and processed 30 min after i.v. administration of liposomes. It can be clearly seen that the muitilamellar nature of the liposomes is still apparent, but has been degraded slightly, probably by lysosomal lipases. When this structure was microanalysed in corresponding unstained serial sections, the emission spectrum obtained possessed a substantial chromium peak

Fig. la. Legend opposite.

55

(Fig. 2). Analysis of the surrounding cytoplasm gave no detectable chromium peaks, demonstrating true localisation of the liposomal marker. Multilamellar figures of this nature were not observed in control animals that had received injections of either physiological saline or free chromium. Also, no chromium was detected within cells from mice that had received injections of free chromium. When liposome-treated animals were left for longer periods after administration of liposomes (ie. 2 h), more vesicles were seen in closer proximity to the parasitophorous vacuoles, and some had actually fused with the vacuole, transferring some of their contents into the immediate locality of the amastigotes (Fig. lb), as demonstrated by the presence of the liposomal marker. Chromium was detected within the parasitophorous vaculole and also within some amastigotes which appeared slightly damaged (Fig. 3). X-ray emission spectra from repeated analysis of adjacent cytoplasmic areas demonstrated complete absence of a Kct peak at 5.4 keV.

Fig. 1. Intracellular localisation of liposomes, a, NMRI mouse liver infected with L. donovani. Mice received

i.v. injections of liposomes (SP /SA/CHOL) containing chromium. Livers were removed and processed for

EM 30 min after liposome administration. An intracellular liposome (L) can be seen within a phagocytic

vacuole in the same cell as a number of leishmanial amastigotes (A) (X 15 000). b, Mouse liver infected with

L. donovani. Mice received i.v. injections of liposomes (SP /SA/CHOL) containing chromium 2 h before

removal of liver for EM processing. A multilamellar liposome (L) in the process of degradation can be seen

within parasitophorous vacuole (PV), adjacent to the remains of a leishmanial amastigote (A). ()< 40 000).

All serial sections for normal transmission EM were stained with uranyl acetate and lead citrate.

56

tOO

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Fig. 2. X-ray emission spectrum of the multilamellar structure in Fig. la from a corresponding unstained

serial section. Analysis was carried out at 60 kV with a 4.0 nA beam current. The sampling time was 100 s

with a spot size of 120 nm. Areas were analysed 3 times unless sections became damaged due to radiation

effects. A chromium K~t peak is present at 5.4 keV indicating true localisation of the liposomal marker.

25- Ai K~< ,

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Fig. 3. X-ray emission spectrum of the partially degraded membranous whorl in Fig. lb from a correspon-

ding unstained serial section. Analysis was carried out at 60 kV with a 4.0 nA beam current. The sampling time was 100 s with a spot size of 120 nm. Areas were analysed 3 times unless sections became damaged due to radiation effects. A chromium Ka peak is present at 5.4 keV indicating true localisation of the liposomal

marker.

57

Qualitatively, there appeared to be little difference in the uptake and localisation of either positively or negatively charged liposomes. However, neutral liposomes (PC/CHOL), were very rarely observed inside cells but were quite frequently found free in liver sinusoids. The failure to see liposomes inside cells may be due to a low rate of uptake of these neutral liposomes under the conditions employed. Neutral lipo- somes were also observed to be taken up much more slowly than charge liposomes in vitro using monolayer cultures of mouse peritoneal macrophages (unpublished obser- vations). It would appear, therefore, that the presence of charged components within the liposomal bilayer is a prerequisite for rapid uptake of liposomes and entrapped agents.

DISCUSSION

The experiments described here show that entrapping the antileishmanial drug sodium antimony gluconate inside liposomes of varying composition and charge, can radically alter its kinetics of uptake by mouse liver and spleen infected with L. donovani. The results obtained here are broadly in agreement with the earlier findings of Ward et al. [6] who were able to demonstrate different rates of hepatic and splenic uptake for free drug and drug entrapped inside negatively charged liposomes. This study goes further in that a number of liposomes of varying composition and charge are compared with a view to obtaining optimum conditions for drug delivery.

Since liver and spleen are rich in reticuloendothelial cells and blood flow is through open sinusoids rather that capillaries, it was presumed that the increased uptake of liposome-entrapped drug was due to a preferential uptake of particulate material by phagocytic cells. It has been shown by Juliano and Stamp [12], that the surface charge on liposomes influences the length of time i.v. administered liposomes remain in the blood stream. They demonstrated that positive and neutral liposomes were retained in the circulation for long periods whereas those that were negatively charged were taken up by liver and spleen very rapidly. In our study however, it was shown that positively charged liposomes were taken up by liver and spleen more rapidly than negatively charged ones, although the difference observed could also be due to slight differences in liposome composition. It has been postulated by a number of workers [12,13] that liposomes composed of different phospholipids and possessing different surface charges, bind different arrays of plasma proteins and that these bound proteins may be important in determining the rate and extent of liposome uptake in vivo.

This study demonstrated unequivocally that upon i.v. administration of liposomes to mice, localisation of liposomes and their entrapped agents in the liver occurred in Kupffer cells, which is where the leishman!al parasites reside in visceral infections. Although this has been the proposed way in which liposome.entrapped antileishma- nial agents have an enhanced killing effect on leishmanial parasites, until now this was based solely on circumstantial evidence.

Initial experiments attempting to localise antimony-containing liposomes using the

58

technique of X-ray microanalysis proved unsuccessful. This was due principally to the inability of the solid state detector in the electron microscope, to separate the antimo- ny Kct line at 3.75 keV from the calcium Kct line at 3.8 keV. Alternative antimony peaks do exist, for example the KI3~ peak at 3.9 keV, but the greater antimony concentration required to obtain this peak would probably be toxic to mice. Detection of antimony on the basis of the presence of a primary peak could also be limited due to the chelating effect of the antimonial salt on calcium ions. This effect has been used in the past by a number of workers to detect calcium levels in various tissues (pyroantimoniate technique) [14,15].

It was also shown in this study, that not only were liposomes taken up by infected macrophages, but they also fused with vacuoles containing leishmanial parasites transferring their contents into the parasite's immediate locality. It had originally been postulated by us and other workers [16] that when liposomes were taken up by phagocytic cells, they fused with lysosomes. It was though that the acidic pH of the lysomal environment in some way caused the liposomes to become leaky, introducing their entrapped agents into the cytoplasm of the macrophage, thus making the cell ' immune' to infection by parasites. On no occasion was the liposome entrapped marker used in this study (chromium), found free in the cytoplasm, which is in agreement with the recent findings of Straubinger et al. [17], who were able to demonstrate the presence of unilamellar liposomes containing colloidal gold in secon- dary lysosomes, but on no occasion found free gold in the cell cytoplasm. Moreover, in our study, when liposome-like structures were observed adjacent to parasitophorous vacuoles, small quantities of the liposomal marker were only detected inside the vacuole, suggesting that fusion had taken place. Thus it is feasible to assume that fusion of intracellular liposomes with parasitophorous vacuoles is the way in which liposome-entrapped antileishmanial agents are introduced directly into the locality of the parasites.

Qualitatively, both positively and negatively charged liposomes were found to be taken up rapidly by cells infected with leishmanial parasites, unlike neutral liposomes which were very rarely observed inside cells, although they were frequently found free in the liver sinusoids. At no time during this study were liposomes found in parenchy- mal cells, which is contrary to the much earlier findings of Gregoriadis and Ryman [18], who found that 3 min after injecting albumin-containing liposomes into mice, liposomal markers appeared in parenchymal cells. These observations were based solely on autoradiographic visualisation of [3H] cholesterol-labelled liposomes which has been shown in the past to produce spurious labelling patterns due to the extraction and redistribution of radiolabelled lipids by solvents used in the processing of speci- mens for electron microscopy [19].

Thus, it is apparent that knowledge of the distribution of liposomes in the liver is still incomplete and much of the previously published work is confusing and open to criticism. In order to study more closely the relationship between drug-loaded lipo- somes and leishmanial parasites within parasitophorous vacuoles, we are at present

59

investigating liposome uptake by isolated Kupffer cells, infected with Leishmania, prepared by selective destruction of parenchymal cells by pronase digestion.

ACKNOWLEDGEMENT

This investigation received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases.

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17 Straubinger, R.M., Hong, K., Friend, D.S. and Papahadjopoulus, D. (1983) Endocytosis of liposomes and intracellular fate of encapsulated molecules: encounter with a low pH compartment after internalisation in coated vesicles. Cell 31, 1069-1079.

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