Clinical Science and Molecular Medicine (1975) 49 99-106.

Liposomes as vehicles for the local release of drugs




Division of Clinical Investigation, Clinical Research Centre, Harrow, Middlesex

(Received 27 November 1974)

Standish & Watkins, 1965). Their size can be reduced by sonication and their surfacechargeadjusted by the incorporation of charged phospholipids (Bangham et al., 1965). It has been proposed that liposomes be used as a carrier system to facilitate the parenteral administration of enzymes (Gregoriadis, Leathwood & Ryman, 1971) and drugs (Gregoriadis, 1973). Enzymes and drugs administered in this way would not be exposed directly to the circulation and could be 'homed' to specific target cells (Gregoriadis,

SummarY 1.The rat testicle was used in studying the release of radio-labelled compounds from locally injected liposomesof various sizes, charge and lipid composition. 2. Large unsonicated liposomes markedly delayed the release of entrapped ZsII-labelledalbumin. Delay was due to liposomal entrapment rather than the presence of lipid per se and it was greater with neutral than charged liposomes. The albumin left the testis after release from, and not in association with, liposomal lipid. 3. Large unsonicated liposomes also delayed the release of entrapped actinomycin D and 5-fluorouracil. The former retained its cytotoxic activity and resulted in focal, dosedependent tissue necrosis. 4. Small sonicated liposomes did not delay the release of entrapped 1251-labelled albumin, and enhanced release of actinomycin D, producing high concentrations of these compounds, which were released in association with Iiposomal lipid, in draining lymph nodes.

1974a). The present study was undertaken to investigate the possibility of employing liposomes as agents to delay and localize the release of compounds after injection into tissues.

Materials and methods Preparation of liposomes

Liposomes were prepared as previously described (Gregoriadis, 1973). In short, a thin Ilm of lipid is deposited on the walls of a round-bottomed flask by rotary evaporation of a solution of lipids in chloroform. The lipid a m spontaneously forms liposomes when dispersed in aqueous media. Small liposomes of minimum diameter 24 nm (Johnson, 1973) form after sonication of this suspension and are separated from the surrounding non-entrapped material by molecular sieve chromatography. Large unsonicated liposomes, of several pm diameter, were made by dispersing the lipid film in an aqueous solution by Vigorous shaking for 30 min (Griffin flask shaker), the suspension being allowed to stand at room temperature overnight and then

Key words: chemotherapy, drug therapy, liposomes.

Introduction Liposomes are minute phospholipid vesicles composed of a closed system of one or more lipid bilayers alternating with aqueous compartments (Bangham, Present address: Department of Medicine, Royal Postgraduate Medical School, Du Cane Road, London W12 OHS. Correspondence: Dr Gregory Gregoriadis, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ.



A. W. Segal, G. Gregoriadis and C. D . V . Black

centrifuged at 1000 g for 30 min. The pellet was washed twice with 5.0 ml of NaCl(l50 mmol/l) and finally suspended in 0.5-1.0 ml of NaCl(l50 mmol/l). All liposomes were prepared from solutions of egg lecithin (160 pmol) and cholesterol (46 pmol) in chloroform. Negative liposomes were made by the addition of phosphatidic acid (23 pmol) and positive liposomes by the addition of L-hexadecylamine (23 pmol). The concentration of lipids in liposome preparations, expressed as lipid-bound phosphorus (Baginski, Foa & Zak, 1967), varied between 1.0 and 5.0 g/l and 1.5 and 8.5 g/l for sonicated and unsonicated liposomes respectively. L-Hexadecylamine was obtained from Kodak Ltd, London. The source and grade of other lipids and of Triton X-100 were as described previously (Gregoriadis, 1973). Actinomycin D was from Merck, Sharp and Dohme, and 5-fluorouracil (base) was a generous gift from Dr Ian Lenox-Smith, Roche Products Ltd, Welwyn Garden City, Herts. [3H]Actinomycin D ( > 3000 Cilmol), 5-[3H]fluorouracil (500 Cilmol) and [4-14C]cholesterol( > 50 Cilmol) were obtained from The Radiochemical Centre, Amersham, Bucks. lZsI- and 1311-labelled human albumin were prepared by the iodine monochloride method (McFarlane, 1958). Compounds to be entrapped in the lipid phase of the liposomal membrane were mixed with the chloroform solution of lipids before evaporation, whereas compounds to be entrapped in the aqueous phase were mixed with the aqueous solution in which the dried lipid film was dispersed. Liposomes containing 1251-labelledalbumin. The lipid film was dispersed in 2.0 ml of NaCl (150 mmol/l) containing i2s1-labelled human albumin (0.04 pmol/ml, 50 pCi). Liposomes containing actinomycin D . Actinomycin D was entrapped in the lipid and in the aqueous phase of liposomes. For entrapment in the lipid phase, actinomycin D (0.39 prnol) was dissolved in 5.0 ml of chloroform containing [3H]actinomycin D (20 pCi), filtered (Whatman no. 1 filter paper) to remove insoluble mannitol (0.11 mmol, included in the preparation by the manufacturer), and mixed with the solution of lipids in chloroform. Non-entrapped actinomycin D was separated from sonicated liposomes by molecular sieve chromatography (Gregoriadis, 1973) and from unsonicated liposomes by centrifugation. The liposome pellet was suspended in a solution of mannitol (270 mmol/l) and centrifuged at 1000 g for 30 min. Liposomes

floating on the surface were removed and the process was repeated twice. Liposomes were then suspended in 1.0 ml of NaCl (150 mmol/l) and dialysed against 500 ml of NaC1 (150 mmol/l) to remove the mannitol. Actinomycin D was entrapped in the aqueous phase by dispersing the lipid film in 1.1 ml of an aqueous solution containing actinomycin D (390 nmol), [3H]actinomycin D (20 pCi) and mannitol (110 pmol). Liposomes containing 5-fluorouracil. Unsonicated positive liposomes containing 5-fluorouracil were made by dispersing the lipid film in 5.0 ml of an aqueous solution of unlabelled (0.38 mmol) and 3H-labelled (2 pCi) 5-fluorouracil. Animal experiments In the principal experiments in this study, albino rats (Sprague-Dawley strain) weighing 200-250 g were anaesthetized with ether, injected through the scrota1 sac into the body of the left testis with 50 pl of the liposome-entrapped radio-labelled material and into the contralateral testis with an equal volume of the same concentration of the nonentrapped material. Pairs of animals were killed by cervical dislocation at timed intervals after injection, their abdominal walls opened and the testes removed through the abdomen, dissected free of the epididymis, excised and assayed for total radioactivity and in some experiments for trichloroacetic acid-precipitable radioactivity (Gregoriadis, Swain, Wills & Tavill, 1974). Identification of lymph nodes draining the left testicle was achieved by the injection of 10 pl of a 1: 10 dilution of Pelikan ink (carbon particles suspension, 10% carbon black, particle size 2 6 5 0 nm) in NaCl (150 mrnol/l) 2 h before removal of the testes. The dark-coloured lymph nodes usually removed were situated above the left renal vein at its junction with the inferior vena cava. Gamma radioactivity was counted with a well-type sodium iodide detector, the background being 12 c.p.m. For the measurement of beta radioactivity in a liquid-scintillation system (background 40 c.p.m.), 0.25433 ml of serum or tissue homogenate in water was made up to 1.0 ml with water and added to 10.0 mi of the scintillant containing 33% (v/v) Triton X-100 (Patterson & Greene, 1965). Lymph nodes were homogenized in 1.0 ml of water and the whole homogenate was added to the scintillant.

Release of drugs from liposomes

Corrections for quenching were carried out by the use of internal standards ([U-14C]cyclohexane, 18 156 d.p.m., or tritiated water, 55 500 d.p.m.). Radioactivity of samples was counted for 100 s with a cut-off at 10000 c.p.m. Counting efficiency was approximately25 and 80 % for 3H and 4Crespectively. Effect of liposomal entrapment on the rate of elimination of 251-labelledalbumin from the testes Effect of the charge and size of liposomes. The elimination of free albumin and of albumin entrapped in sonicated negative and positive liposomes and in unsonicated negative, positive and neutral (in the absence of charged lipid) liposomes was determined. Effect of the volume of injection of liposomes. Rats were injected with 100, 80, 60, 40 and 20 p1 of the same preparation of albumin, either free or entrapped in unsonicated neutral liposomes. Tissue distribution of liposomes after injection

1z51-labelledalbumin (as a marker of the aqueous

phase) was entrapped in liposomes containing [4-14Clcholesterol(10 BCi, 160 nmol, as a marker of liposomal lipid), which was incorporated into the lipid phase by addition to the solution of lipids before evaporation of the chloroform. Animals were injected with 50 PI of the above preparation in the form of unsonicated neutral liposomes or sonicated negative liposomes. Pairs of animals injected with each preparation were killed at 10 min and 24 h after injection, their organs removed and homogenized in water. 14C and 1251radioactivity and trichloroacetic acid-precipitable 5I radioactivity was counted in serum and tissue homogenates. The observed beta counts were corrected for the contribution due to lZ5I(5-10% of total beta counts). Results Model system

The coefficient of variation for mean testicular radioactivity for each pair was 13.5% and 9.6% respectively for animals injected with liposomeentrapped and free 251-labelled albumin (thirty-

Time (h)

FIG. 1. Percentage of residual lZ5Iradioactivity in the rat testes at timed intervals after the injection of free 1251labelled albumin (0, 7 . 2 ~10j-1.7~lo5, mean = 6 . 6 ~ lo4 c.p.m.) or the same compound (3x 103-1-7x lo5, mean = 5.4x lo4 c.p.m.) entrapped in sonicated negatively (A) or positively (A) charged liposomes, and unsonicated neutral (0), negatively (a)or positively ( 0 )charged liposomes. Mean values ( ~ S E M and ) the number of animals studied at each time-interval are shown.




A . W. Segal, G. Gregoriadis and C. D. V. Black TABLE 1. Percentage of radioactivity remaining in the testis at various timeintervals after injection of unsonicated neutral liposomes

Either unsonicated neutral liposomes containing 12sI-labelled albumin and mixed with unentrapped 13sI-labelledalbumin (preparation A: lZsI, 1.2 x lo 5 c.p.m.; l J I I , 1 . 7 lo4 ~ c.p.m.) or unsonicated neutral liposomes mixed with unentrapped 1251-labelledalbumin (preparation B; 4.3 x lo5 c.p.m.) were injected. Individual values from paired studies in a single experiment are shown. Preparation B

Preparation A

Time '''I radioactivity 1311 radioactivity lZsI radioactivity (h) (entrapped albumin) (unentrapped albumin) (unentrapped albumin) 0.17 1.oo 4.00

91.5, 96.5 87.9, 86.6 81.8, 82.9

66.1, 71.4 33.8, 28.3 5.9, 7.8

seven and thirty-six pairs of observations) and 5.9% and 6.8% for animals injected with entrapped and free actinomycin D (nineteen and eighteen pairs of observations respectively). I00

10 rnin

73.0, 89.5 55.1, 45.4 8.0, 9.5

Elimination of 2sI-labelledalbumin Eflect of entrapment in Iiposomes. Free albumin was eliminated from the testis at a reproducible rate 24 h















C 0.5 c 0 m c

W c



0.01 D W

D a l



FIG.2. Organ distribution of 14C radioactivity 10 min and 24 h after injection into rat testicles of [14C]cholesterollabelled sonicated (250 pg of P, 1 . 2 lo6 ~ d.p.m.; stippled bars) and unsonicated (85 pg of P, 5 . 0 lo5 ~ d.p.m.; open bars) negatively charged liposomes containing 1251-labelledalbumin. Individual values and mean values (% of injected radioactivity per whole tissue or serum) from two animals injected with each preparation are shown. The volume of serum was taken as 9.7 m1/200 g body wt. (Gregoriadis & Ryman, 1972b).


Release of drugs from liposomes

(Fig. 1). The rate of elimination of entrapped albumin varied with the charge and size of the liposomes. Albumin entrapped in sonicated negatively or positively charged liposomes was eliminated at a similar rate to that of free albumin. In contrast, entrapment of albumin in unsonicated liposomes greatly delayed its elimination (P< 0.001 at all timeintervals studied) and it was slowest with neutral and most rapid with negatively charged liposomes. The difference between these two preparations of unsonicated charged liposomes (P< 0-002 at 1, 4 and 24 h after injection) was a result of the different surface charges and not of different concentrationsof injected liposomal lipids (2.0-4.5 pg of phosphorus//rl injected volume). When the same experiments were repeated with the same concentration of lipids (2.5 pg of phosphorus/pl), similar results were obtained. Delayed elimination of albumin entrapped in unsonicated neutral liposomes was the result of entrapment rather than that of the physical presence of liposomes (Table 1). When both entrapped ( z51-labelled)and free ( lI-labelled) albumin were injected together in a single preparation (A), the rates of elimination of the two albumins were respectively the same as when the preparations were injected independently (Fig. l), elimination of the entrapped compound being much slower than that of free albumin. In addition, when 1251-labelledalbumin was mixed with, but not entrapped in, similar liposomes the rate of its elimination was again that of free albumin (preparation B, Table 1). Effect of the volume of injection. The volume of injection (20-100 pl) did not affect the elimination of free or entrapped albumin measured at a time (4 h) after injection when the residual radioactivity in the testes varied between 77 and 86% and 68 and 76% of the injected dose for unsonicated positive and negative liposomes respectively. Tissue distribution of injected liposomes. (i) Unsonicated liposomes. Ten minutes after the injection of [4-14C]cholesterol-labelledunsonicated neutral liposomes containing lzSI-labelled albumin, most 14C (74.5%, Fig. 2) and lZ5Iradioactivity (82.7% of the injected dose, not shown in Fig. 2) remained localized to the testis. No 14Cand minimal lZ5I radioactivity (0.02%) was observed in the draining lymph node. After 24 h, 84.8% of the 14C radioactivity (Fig. 2) and 49.8% of the lZ5Iradioactivity (not shown in Fig. 2) was still present in the testis, 5.7% of the lZ5Iradioactivity and none of the

14C radioactivity was present in the serum, and 0.5% of each was found in the liver. In another

experiment the 14Cand lZ5Iradioactivity in testes of pairs of rats injected with 50 pl of the same liposome preparation was measured at 0, 20 and 40 h. The 14C radioactivity was found to be 101.4 and 88.6% of the injected dose at 20 and 40 h respectively and the ratio of beta to gamma radioactivity (taking the ratio in the injected material as 1.0) was 1.29 at 20 h and 1.52 at 40 h. Of the lZ5I radioactivity, 97% was trichloroacetic acid-precipitable. (ii) Negative sonicated liposomes. Most of the 14C radioactivity (and of lZ5I radioactivity as inferred from Fig. 3) had been eliminated from the testes 24 h after injection of [14C]cholesterollabelled sonicated negative liposomes containing 9-labelled albumin and was observed predominantly in the liver, serum, spleen and lungs (Fig. 2). 25

10 rnin



15 m N




0 c





B FIG.3. Radioactivity(l4C/lZ5I)ratio in rat tissues 10 min and 24 h after injection into rat testicles of [14C]cholesterollabelled (1.2x lo6 d.p.m.) sonicated negatively charged l i p somes (250 pg of P) containing 1251-labelledalbumin (1.37~ lo5 c.p.m.). Individual values and mean values from two animals are shown.

A . W. Segal, G. Cregoriadis and C. D . V. Black








Time (h)

FIG.4. Elimination of radioactivity from rat testicles injected with [3H]actinomycin D, either free ( 0 , 2.3 jig, 4 . 7 lo5 ~ d.p.m., n = 8) or entrapped in unsonicated positively charged liposomes(O,15Opg of P, 2.0pg of actinomycin D, 4 . 0 ~10' d.p.m., n = 6), and 5-[3H]fluorouracil, free (B, 136 pg, 2 . 3 ~ 10' d.p.m., n = 2) or entrapped in unsonicated positively charged liposomes(n, 175 p g of P, 127 p g of 5-fluorouracil, 4 . 6 ~lo4 d.p.m., n = 2). Mean values ( f s e ~for [3H]actinomycin D studies) are shown.

At 24 h the ratio of I4C to lzSIradioactivity in these organs showed a five- to twenty-five-fold increase over the ratio in the injected material (Fig. 3). A relatively high proportion of the injected radioactivity (1.2 and 2.0% of the injected 14C and l z 5 I radioactivity respectively) was found in a single draining lymph node 10 min after injection but it had almost completely disappeared 24 h later.

high concentrations of the drug in the draining lymph nodes. Thus, 4 and 24 h after injection, the


4 h



c s!

80 -


70 -

24 h


-.60 0


._ 2 > .e

Elimination of drugs Actinomycin D . Entrapment of [3H]actinomycin D in unsonicated positive liposomes markedly delayed the elimination of the drug from the testes (P< 0.001 at all time-intervals studied; Fig. 4). There was no difference between the rate of elimination of actinomycin D entrapped in either the lipid or in the aqueous phase of unsonicated positive liposomes. The entrapment of actinomycin D in sonicated negative liposomes (in the aqueous phase) not only enhanced the rate of the loss of the drug from the testis (residual radioactivity at 4 and 24 h amounting to 68.0 and 54.7% respectively of that observed after the injection of a similar amount of free drug, Fig. 5), but also resulted in the accumulation of

._ 50U




-- 30 a 0



$i2 0 -



: 10 ,0-" 0 L E I a

Testes Lymph nodes

Testes Lymph nodes

FIG.5 . 3H radioactivity (% of the injected dose per mglof fresh tissue) in the left testicles and lymph nodes of rats 4 and 24 h after injection of the testicle with free (hatched bars; 1.4 p g of [3H]actinomycin D, 1 . 4 10' ~ d.p.m.) and liposomeentrapped (open bars; sonicated, negatively charged, 450 ,ug of P, 1.5 p g of [3H]actinomycin D, 1 . 5 10' ~ d.p.m.) actinomycin D. Mean values from two animals are shown.

Release of drugs from liposomes

concentration (per mg of tissue) of the drug in draining lymph nodes was 17 and 64 times greater than the concentration in the injected testes, and 7 and 11 times respectively greater than in lymph nodes of animals injected with a similar amount of free drug (Fig. 5). 5-Fluorouracil. The rate of elimination of 5fluorouracil from the testicle was diminished by entrapment in unsonicated positive liposomes (P

Liposomes as vehicles for the local release of drugs.

1. The rat testicle was used in studying the release of radio-labelled compounds from locally injected liposomes of various sizes, charge and lipid co...
583KB Sizes 0 Downloads 0 Views