Life Sciences, Vol. 27, pp. 2223-2230 Pergamon P~ess

Printed in the U.S.A.

THE EFFECT OF THE CHOLESTEROL CONTENT OF SMALL UNILAMELLAR LIPOSOMES ON THE FATE OF THEIR LIPID COMPONENTS IN VIVO

Christopher Kirby and Gregory Gregoriadis

Division of Clinical Sciences, Clinical Research Centre, Harrow, Middx HA1 3UJ, UK

(Received in final form October 2, 1980)

Summar I

We have investigated the effect of the cholesterol content of small unilamellar liposomes composed of egg phosphatidylcholine (PC) and containing 6-carboxyfluorescein (6-CF) on the in-vivo fate o~ their radiolabelled PC (3H-PC) and tracer [1-14C]-cholesteryl oleate (14C-cholesteryl oleate) components. Chromatography of the blood plasma of mice at various times after injection with liposomes composed of equimolar amounts of PC and cholesterol (PCCHOL liposomes) showed a main peak (peak A) containing most 3H-PC, 14C-cholesteryl oleate and 6-CF and representing intact liposomes. With cholesterol- free liposomes (PC liposomes) on the other hand, there was increasing transfer of the two radiolabelled lipids from peak A to the subsequently eluted high density lipoproteins (HDL) (peak B) paralleled by increasing loss of ILposomal stability as evidenced by 6-CF release. Studies on the rate of clearance of PCCHOL liposomes showed half-lives of II0 min (3H-PC) and 120 min (14C-cholesteryl oleate marker). Similar studies with PC liposomes revealed complex patterns of clearance evaluation of which was hampered by a number of observed or anticipated concurrent events: removal of liposomes by tissues, transfer of PC and cholesteryl oleate to HDL, clearance of HDL and donation of the two lipids by HDL to, or their exchange with lipids of, tissues.

Rational use of liposomes as drug carriers (1,2) requires understanding of their behaviour within the biological milieu. Early observations (3,4) revealed that after intravenous injection, drugs are released from liposomes in- the circulation much more rapidly than is expected (5,6) from normal solute diffusion through the bilayers. This was subsequently attributed (7-9) to the transfer of much of the liposomal component PC to the plasma HDL in turn leading to the loss of the carrier's integrity and ability to retain solutes. We have shown that in the presence of whole blood plasma or serum in vitro and in the intravascular space of injected animals permeability to solutes of small multilamellar (5) or small unilamellar liposomes (6) is directly proportional to their cholesterol content. A similar effect of cholesterol on bilayer permeability has been observed in vitro by Finkelstein and Weissmann (i0). More recently we found (II) that cholesterol stabilises liposomes in the presence of ser~n by reducing PC transfer to HDL, presumably as a result of sterol-induced packing (12) of phospholipid molecules in the bilayer. In the present report we }lave studied the effect of the cholesterol content of small unilamellar liposomes on the fate o~ their PC component and cholesteryl oleate marker after intravenous injection into mice. Our results show that (a) the presence of cholesterol in the structure of phosphatidylcholine liposomes reduces their in vivo HDL-induced PC (and cholesteryl oleate) depletion and ensuing destabilization thus providing a means of controlling solute retention by

0024-3205/80/492223-08502.00/0

2224 Fate of Liposomal Lipids In Vioo Vol. 27, No. 23, 1980

liposomes i~i vivo; (b) PC and cholesteryl oleate cannot be used as markers for clearance studies of liposomes unless these are rich in cholesterol.

Materials and Methods

The sources and grades of egg PC, cholesterol, 6-CF, [1-14C]cholesteryl oleate (34 mCi/mmol) and Ultrogel AcA 34 (swollen, bead size 60-140 ~m) have been described elsewhere (6,[[). Tritiated PC (1.05 mCi/~mol) was radiolabelled (13) by catalytic hydrogenation of the fatty acid ester groups. All other reagents were of analytical grade.

Preparation of liposomes: Small unilamellar liposomes containing 0.25M 6-CF were prepared (6,11) from CHCI 3 solutions of 40 ~moles PC (PC liposomes) tracers o~ 3H-PC (2.3 ~Ci) and [4C-chole~t,~ryl oleate (0.06 ~Ci or I nmol) and were, when apgropriate , supplemented with 40 ~moles of cholesterol (PCCHOL liposomes; 1:1 molar ratio). The size of sach liposomes as measured by electron microscopy (6) was 30-60 nm [n diameter. Assay of 6-CF in suitably diluted liposomal or biological samples was carried out in the absence (free dye) and presence (total dye) of Triton X-IO0 (1% final concentration) in a Perkin-Elmer 204A fluorimeter using excitation and emission wavelengths of 490 and 520 nm respectively. The minimum amount of 6-CF which could be measured accurately was 4 ng/ml (6). Values of free and total 6-CF were used for the estimation (6,[I) of latent (i.e. entrapped) dye which serves as a convenient index of the stability of liposomes in terms of their solute retention within a given environment, biological or otherwise (5,6,11): at the concentration used (0.25M), liposomal 6-CF quenches fully but when liposomes become leaky to the dye, its escape (in proportion to the loss of liposomal stability) and dilution in the surrounding medium enables it to fluoresce (14).

In-vivo experiments: T.O. mice weighing 20-25 g were injected in the tail vein wfth-d~2-ml--of~ubly radiolabeILed (6 x 106 - 8 x 106 dpln 3H and 4 x

/ • 105 - 6 x 105 dpm I~C) [iposomes (3 mg phospholip[d) containing o-CF (67-173 ~g). Plasma (40 ~I) from the heparinized blood of animals bled at time intervals was diluted in 0.5 ml O.IM sodium phosphate buffer supplemented with 0.8% NaCI and 0.02% KCI (phosphate buffer), pH 7.4 (6,[I). Of this, 0.45 ml was passed at 20°C through an Ultrogel AcA 34 column (I x 25 cm) equilibrated with phosphate buffer. The column was eluted with the same buffer at a flow rate of 0.4 ml/min and the 1.0 ml fractions collected were analysed for free, total and latent 6-CF (II). For the measurement of 3H and 14C, double isotope

o ~ c ~ 14p counting conditions were set to take into c nsid.rati)n crossover or u into the 3H channel (11). In some experiments 0.5 ml pooled plasma from three mice injected with PC liposomes and killed 65 min later was chromatographed as above and 0.2 ml of pooled fractions of the eluate (see Results) was reinjected intravenously into intact X.O. mice. Animals were then bled at time ~ntervals and 3H and |4C measuc~d [n tile plas,na.

Results and Discussion

We have investigated the effect of the cholesterol content of small unilamellar liposomes on the extent to ~ich these retain their PC and (tracer) cholesteryl oleate lipid components and bilayer stability (in terms of 6-CF release) in the circulation of injected mice. After ~assage of freshly prepared PC or PCCHOL liposomes radiolabelled with 3H-PC and 1~C-cholesteryl oleate and containing 6-CF through an Ultrogel AcA 34 column, practically all (95-104%) of the lipid phase (3H and 14C) and aqueous phase (6-CF) markers recovered from the columns are eluted with fractions 10-13 (peak A) (see legend to Fig. i) as intact liposomes. However, when plasma from mice injected with similarly labelled cholesterol-free PC liposomes is chromatographed, a considerable portion of the two radiolabels is now recovered in fractions 14-18 (peak B) as

Vol. 27, No. 23, 1980 Fate of Liposomal Lipids f~ VCVo 2225

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FIG. L

The effect of the cholesterol content of small unilamellar liposomes on the fate of their lipid markers and stability in vivo. In a typical ~periment PC liposomes containing 6-CF and labelled with 3H-PC and

C-cholesteryl oleate were injected intravenously into mice which were then bled 3 (a), 45 (b) and 65 (c) min later. Cholesterol- rich PCCHOL liposomes were also injected into mice but as patterns of radioactivity and 6-CF were virtually identical for all three time intervals, only the pattern at 45 min is shown (d). Blood plasmas in ph~sphat~4buffer containing 2.2 x 105-106 dpm 3H, 6.9 x 103-28 x I0 dpm C and I-6 pg 6-CF were passed through3 Ultrogel AcA 3414 columns and fractions collected analysed for H (broken line), C (solid line) and 6-CF (dotted line). When 0.I ml of PC or PCCHOL liposomes was mixed with 1.0 ml phosphate buffer and 0.45 ml of the mixture passed through the column, 95-104% of the two radioactivities and of 6-CF were recovered in fractions 10-13. All values are expressed as % of total radioactivity and dye recovered. Recoveries were 93-107% of the quantities applied. 6-CF latencies in fractions 10-13 for all time intervals (both types of liposomes) were higher than 96% (for other details see Materials and Methods).

2226 Fate of Liposomal Lipids In Yivo Vol. 27, No. 23, 1980

well (rig. [, a-c). Previous work ([I) with sim[lar liposo~nes mixed with serum and then chromatographed, has established already that the two lip[ds in the second peak are largely associated with the HDL to which, it would appear from Fig. 1 (a-c) they are transferred (8,9) as early as 3 min after injection (35.9% of PC and 23.7% of cholesteryl oleate in peak B; Fig. la). Transfer of lipids increases with time to reach values of 75.5% and 66.4% for PC and cholesteryl oleate respectively after 65 min (Fig. Ic). At the same time, an increasing proportion of circulating 6-CF is recovered in fractions 26-31 (peak C) as free dye (Fig. I, a-c). As the rate of clearance of free 6-CF from the circulation is more rapid than that of the entrapped dye (6), the ratios of liberated (fractions 26-31) to entraDped 6-CF (fractions [0-13) must be even greater than appear (Fig. I, a-c). In contrast, when plasma from mice injected with the cholesterol-rich PCCHOL [iposomes and bled 3, 45 and 65 !nin later is passed through the columns, the two radioactive markers and the 6-CF ace

recovered quantitatively in peak A with only minor portions of the PC (7%) and cholesteryl oleate (5%) found in peak B (Fig. I,d). These results were confirmed in a similar experine,lt a,ld seem to indicate that for PCCHOL liposomes, 6-CF and most of the PC and cholesteryl oleate circulate in the blood as intact liposomes for the period of time studied.

Figure 2 shows the rate of clearance of 3H-PC from the circulation of mice injected with radiolabelled PCCHOL or PC liposomes as well as the portions of the label in the blood corresponding to peak A and peak B. For PCCHOL liposomes, PC associated with peak A (Fig. 2a) exhibits 3 mia after injection a linear rate of clearance with a half-life of approximately [I0 min. Since loss of phospholipid from PCCHOL liposomes to HDL is only minor and occurs within a few minutes after injection (see Fig. Id and legend), such a rate is likely to represent that of the clearance of PCCHOL liposomes. Indeed, in previous work (6) a half-life of about If0 m~n was al~o observed for a solute (6-CF) entrapped ia the aqueous phase of similar small unilamellar PCCHOL liposomes. On the other hand, for PC liposomes, the kinetics of PC clearance are more co nple~ (Fig. 2b). Since el[re[nation of PC from the blood in tlle fomn of liposomes is simultaneous to the eons£derable transfer of the phospholipid (see Fig. I, a-c and ref. 11) to circulating HDL (peak B), the net result is that PC of peak A appears to be removed from the blood at a rate (Fig. 2b) which is much more rapid (half-life 17 min) than that (II0 min) observed with the PC of the same peak (Fig. 2a) for the virtually intact PCCHOL liposomes. Furthermore, as PC clearance for peak B is expected to reflect to some extent the latter's clearance from the blood as HDL, the enrichment of the lipoprotein in PC from peak A as well as the anticipated donation of PC by HDL to, or exchange with lipids of, tissues (15-17), simultaneous occurrence of these events, probably at rates which vary with the course of time, results in an apparently low (and ano~nalous) overall rate of PC clearance as peak B (Fig. 2b).

With regard to cholesteryl oleate, patterns of its elimination as peaks A and B from the blood of mice injected with PCCHOL and PC liposomes labelled with 14C-cholesteryl oleate are, shortly after injection, essentially similar (legend to Fig. 2) to those of the phospholipid (Fig. 2a and 2b). For instance, half-lives of cholesteryl oleate for peak A were 120 (PCCHOL) and 18 min (PC liposomes) (not shown). However, judging from the ratio of the two isotopes in the blood of injected mice (Table I), transfer of PC to peak B is more rapid than that of colesteryl oleate. Thus, 3H:14C ratio values for peak A (both types of liposomes) become in 3 min and remain for at least up to 105 min, considerably lower (0.35-0.88) than the ratio value (i.0) in the injected preparation. Peak B 3H:14C ratios (Table I) which, because of PC acceptance by HDL, are relatively high at 3 min, also decline with time possibly as a result of more rapid PC loss to, or exchange with, tissues. The latter possibility was supported in an experiment in which the true rates of clearance of PC and cholesteryl-oleate associated with peak B (i.e. without the obscuring

Vol. 27, No. 23, 1980 Fate of Liposomal Lipids In Vivo 2227

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FIG. 2

Clearance of liposomal 3I}-PC from tile blood of injected mice. In an e~periment identical to that described in Fig. I, animals were injected with do~hly radiolabelled small unila~nellar PCCHOL (a) and PC (b) liposo:nes- Bl,~od plasmas from mice bled at 3, 60 and 105 rain were analysed for radioactivity and subsequently passed (9 x 104-7 x 106 dpm 3H and 2.5 x [03- 17 x 103 dpm 14C in 0.5 ml) through Ultrogel AcA 34 columns. As clearance patterns for t~ two radioactivities were similar, only 3H patterns are shown here. 3H values for whole plasma (solid line), pooled fractions 10-[3 (peak A) (broken line) and pooled fractions 14-18 (peak B) (dotted line) are expressed as % of injected dose per total mouse plasma (6). For other details see Materials and Methods. Repetition of the experiment in animals bled at time intervals (up to ii0 min) other than those shown in the Figure, gave rates of 3H-PC and 14C-choelsteryl oleate clearance strikingly similar to those shown above.

effects on such clearances of lipid movement from peak A to peak B) could be followed: peak B was isolated as in Fig. I from the pooled blood plasma of three mice injected with PC liposomes and killed 65 min later, It was anticipated that, by this time, much of the liposomal PC and cholesteryl oleate transfer to HDL (peak B) would have occurred with enough 14C and 3H radioactivity remaining in the blood for peak isolation and further use (see Fig. 2). Peak B was the~ injected into intact mice which were bled at time intervals. Results in Fig. 3 show that the two lipids of the injected peak B (which in this case cannot be enriched with the respective lipids from peak A) are removed from the blood differently, with PC exhibiting a more rapid rate of clearance.

2228 Fate of Liposomal Lipids In Vivo Vol. 27, No. 23, 1980

3 H:

TABLE I 14

C ratios in the Blood Plasma of Mice injected with Small Unilamellar Liposomes labelled with 3H-PC and 14C-cholesteryl oleate

3 14 H: C ratio

3 min 60 min 105 min

PC liposomes Plasma radioactivity 0.69 0.57 0.58 Peak A 0.55 0.35 0.88 Peak B 1.03 0.62 0.56

Plasma radioact [~[ty 0.7[ 0.64 Peak A 0.67 0.61 Peak B " 1.37 1.26

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Ratios of 3H-PC and 14C-cholesteryl oleate have been derived from experiments shown in part in Fig. 2 and legend.

0.59 0.56 1.05

In conclusion, the present studies together with previous findings (6-11) indicate that intravenous injection of cholesterol-free small unilamellar PC liposomes is followed by bulk transfer of their phospholipid component and cholesteryl oleate marker to HDL. T|m two lipids are subsequently cleared from the blood at (different) rates, probably determined by the clearance rate of the HDL itself and by its interaction with tissues in terms of lipid donation or exchange (15-17). Our findings also indicate that neither PC nor cholesteryl oleate are appropriate markers for studies of the fate of cholesterol-free liposomes in vivo. Therefore, as retention of 6-CF (6) and other small molecules (our unpublished observations) by the leaky PC liposomes in vivo is poor, the rate of clearance of the latter From the circulation can only be ascertained by markers (perhaps large molecular weight water soluble markers or other lipid markers) which remain quantitatively associated with such liposomes. On the other hand, incorporation of cholesterol into PC liposomes prevents the transfer (8,9) of PC and cholesteryl oleate to HDL (Fig. id). The way by which cholesterol acts so is not as yet clear although, as discussed elsewhere in more detail (11) it could be that phospholipid packing induced by excess cholesterol (12) prevents the lipids from being removed by HDL. It is of interest that cholesterol failed to reduce the loss of cholesteryl oleate when PCCHOL liposomes were incubated in the presence of human serum (II) and this is at present under investigation. It would thus appear that PC and cholesteryl oleate provide a legitimate means of following the rate of intravenously injected cholesterol- rich liposomes. ~lls is mo~e so for cholesteryl oleate which remains associated with the liposomal carrier to an even greater extent than PC does (Table l). Regardless of the mechanism by ~lich cholesterol prevents PC (and cholesteryl oleate) transfer to HDL and ensuing bilayer destabillzation, it seems that the sterol can, in proportion to its presence in the bilayers (6,11), delay solute loss from liposomes in vivo. As pointed out already (5,6,11,18), with drug-containing liposomes this could lead to improved drug action. Indeed, in recent work (18) the anti-tumour effect of a liposome- entrapped cytotoxic agent was promoted by the increased cholesterol content of its carrier.

Vol. 27, No. 23, 1980 Fate of Liposomal Lipids fn V~Vo 2229

100 - 8 0 -

6 0 -

-8 40 4 - - '

c -

O 2 0 -

1 0 -

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FIG. 3

Clearance of 3H-PC and 14C-cholesteryl oleate associated with injected peak B. Pooled fractions 14-18 (peak B) obtained from Ultrogel AcA 34 columns loaded with blood plasma from mice 65 min after their injection with small unilamellar PC liposomes labelled with 3H-pc and 14C-cholesteryl oleate (see legend to Fig. 1c), were injected (4 x 105 dpm 3H and 18 x 103 dpm 14C in 0.2 ml) intravenously into intact mice which were bled at time intervals. Plasma values (means ~rom three animals) for 3H (filled circles) and 14C (empty circles) are expressed as % of the injected dose per total mouse plasma (6).

Acknowledsements

We thank Miss Jacqui Clarke for technical assistance and Mrs. Dorothy Seale for secretarial work. These studies were supported in part by a NIH National Cancer Institute contract (NO1-CM-87171).

References

I. G. GREGORIADIS in "Drug Carriers in Biology and Medicine", G. Gregoriadis ed. pp 287-341, Academic Press, London, New York, San Francisco (1979).

2. J.F. FENDLER and A. ROMERO, Life Sci. 18, 1453-1458 (1976). 3. G. GREGORIADIS, FEBS Lett. 36, 292-296 (1973). 4. H.K. KIMELBERG, E. MAYHEW and D. PAPAHADJOPOULOS, Life Sci. 17, 715-724

(1975). 5. G. GREGORIADIS and C. DAVIS, Biochem. Biophys. Res. Comm. 89, 1287-1293

(1979). 6. C. KIRBY, J. CLARKE and G. GREGORIADIS, Biochem. J. 186, 591-598 (1980). 7. L. KRUPP, A.V. CHOBANIAN and P.l. BRECHER, Biochem. Biophys. Res. Comm.

72, 1251-1258 (1976). 8. G. SCHERPHOF, F. ROERDINK, M. WAITE and J. PARKS, Biochim. Biophys. Acta

542, 296-307 (1978). 9. J.V. CHOBANIAN, A.R.TALL and P.l. BRECHER, Biochemistry 18, 180-187 (1979). I0. M.C. FINKELSTEIN and G. WEISSMANN, Biochim. Biophys. Acta 587, 202-216

(1979). ii. C. KIRBY, J. CLARKE and G. GREGORIADIS, FEBS Lett. 111, 324-328 (1980). 12. R.A. DEMEL and B. KRUYFF, Biochim. Biophys. Acta 457, 109-132 (1976). 13. S. WHARTON, PhD Thesis, University of Liverpool (1979).

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14. J.N. WEINSTEIN, S. YOSHIKAMI, P. HENKART, R. BLUMENTHALL and W.A. HAGINS, Science 195, 489-492 (1977).

15. J.A. GLOMSET, Prog. Biochem. Pharmacol. 15, 41-66 (1979) 16. C.J. FIELDING, J.P., RENSTON and P.E. FIELDING, J. Lipid Res. 19, 705-711,

1978. 17. N.M. PATTNAIK and D.B. ZILVERSMIT, J. Biol. Chem. 254, 2782-2786 (1979) 18. E. MAYHEW, Y.M. RUSTUM, F. SZOKA and D. PAPAHADJOPOULOS, Cancer Treat.

Rept. 63, 1923-1928 (1979)