- Email: [email protected]
Metabolism of liposome-encapsulated heparin
THROMBOSIS RESEARCH 56; 369-376, 1989 0049-3848/89 $3.00 t .OO Printed in the USA. Copyright (c) 1989 Pergamon Press plc. All rights reserved.
METABOLISM OF LIPOSOME-ENCAPSULATED HEPARIN
T.D. Kim, J. Kambayashi, M. Sakon, T. Tsujinaka, T. Ohshiro, T. Mori Hematology Research Unit, The Second Department of Surgery, Osaka University Medical School, Fukushima l-l-50, Fukushima-ku, Osaka 553, Japan. (Received 6.12.1989; accepted in revised form 9.8.1989 by Editor H. Yamazaki)
ABSTRACT In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or "C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of "C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.
INTRODUCTION Although it has been well known that liposome encapsulation of a certain drug or agent results in the prolongation of the drug effect and reduction of side effect due to selective uptake by specific tissue or organ, the exact metabolism of the liposome encapsulated agent has not been fully elucidated yet. We have reported in the foregoing paper that encapsulation of heparin in multilamellar liposomes yields the prolongation of in vivo anticoagulant
Key words:
liposome, heparin, metabolism, rats
369
370
LIPOSOME-HEPARIN
Vol. 56, No. 3
effect (1). In order to study the mechanism of the prolongation, we prepared liposome-encapsulated heparin (LIPO-HEP) containing 3H-labelled heparin (3H-HEP) and/or '\C-labelled phosphatidyl-choline (14C-PC). Upon intravenous administration of the radiolabelled liposome or heparin into rats, the radioactivity as well as the biological activity in blood and various organs was serially determined.
MATERIAL AND METHODS Preparation of liposomes: In order to radio-label liposome, L-a'4C-Phosphatidylcholine (PC) (0.02 mCi/0.0132 mg, Amersham Japan, Tokyo) was mixed with 1 ml chloroform solution containing 50 mg yolk phospholipid (The Green Cross Co. Ltd., Osaka), which was composed of PC (79%), phosphatidylethanolamine (13%), sphingomyeline - lyso PC (4%) and alpha-tocopherol (1%). The mixture was placed in a round bottomed flask and it was evaporated for about 2 hours in nitrogen gas until a thin lipid film was formed. Then the mixture of heparin calcium (3,000 u, Mitsui Pharm. Co.Ltd., Tokyo) and 3H-heparin (0.33 mCi, 506 u/3.3 mg, Amersham Japan, Tokyo) was placed over the lipid film and encapsulation was completed by vigorous shaking of the flask for one hour at room temperature. All the lipids collected from the wall of flask were subjected to the repeated washing and centrifugation at 10,OOOxg for 10 min unti thrombin clotting time of the resultant supernatant returned to normal. Then an aliquot was taken for scintillation counting and assay of anticoagulant activity and the remainder was kept at 4°C under nitrogen in a sealed polyethylene tube until use. Animal experiments: Male Wistar rats weighing 180-230 g were obtained from Nihon Animals Co. Ltd., Osaka and were fed on standard diet for at least two weeks before the experiments. Rats were anesthetized by intramuscular injection of ketamine hydrochloride and jugular vein was cannulated using Silastic@ tubing (Dow Corning, MI). Through the catheter, LIPO-HEP (40 u/kg) was administered and titrated blood was drawn in plastic syringe at 5, 10, 30, 60, 90, 120, 150 min after the administration. After the completion of the last blood collection, the animal was sacrificed by injecting an excessive dose of pentobarbiturate intravenously and the liver, spleen, lung, kidney were harvested. Measurement of anticoagulant activity: To determine the anticoagulant activity of the prepared LIPO-HEP, one ml of chloroform was added to 0.1 ml of LIPO-HEP and the mixture was vortexed vigorously for about 2 min to disrupt liposomes. After 1 ml of saline was added further, the mixture was thoroughly mixed and centrifuged at 17OOxg for 10 min at 4"C, yielding three (aqueous, interface, and chloroform) phases. Twenty microliter of the aqueous phase was incubated with 100 pl of human pooled titrated plasma for 2 min at 37°C. Then, 50 1.11 of 0.05 M CaClz solution was added to the mixture and the clotting time was recorded in a Clotem system. Samples were usually diluted with saline so that thrombin clotting time (TCT) became in the range between 10 and 27 set, in which clotting time was well correlate with heparin concentration. The amount of heparin was then calculated, using a standard curve of TCT obtained in the presence of the known amount of heparin. Anticoagulant activity in vivo was determined by the plasma recalcification clotting time (RCT) as described before (1). Measurement of radioactivity: In order to measure radioactivity of 3H and/or 14C of liposome encapsulated heparin, 0.05 ml of sample was solubilized in
Vol. 56, No. 3
LIPOSOME-HEPARIN
371
Univer-Gel@ (10 ml) and the radioactivity was measured in a liquid scintillation counter (Packard Tri-CAEB, IL). As for the preparation of blood sample, 0.1 ml of plasma prepared from titrated whole blood was likewise solubilized in Univer-Gel@. The freshly harvested organs were treated as follows for radioactivity determination. After each organ was weighted, 1 ml of saline was added per 1 g of tissue. The mixture was thoroughly homogenized and 0.1 ml 50% KOH and 0.1 ml Hz02 were added to the homogenate. The mixture was heated for 30 min at 70°C for digestion and it was neutralized by adding 0.1 ml 2 N HCl. Finally 0.05 ml of the resultant mixture was solubilized in 10 ml Univer-Gel@. Analysis of the degradation of 14C-PC by thin layer chromatography (TLC): At designated times after intravenous administration of LIPO-HEP containing 14C-PC, the blood was collected and plasma was prepared as described earlier. The plasma was subjected to lipid extraction by the method of Bligh & Dyer (2) and the lipid was separated on TLC (solvent system, as described previously (3)) and was visualized by autoradiography.
FRONT _.
i
I
i
i
,\
/
,::j / , . I
PC-
“*
LYSO-PC-
ORIGIN-
0
2
5 TIME
10 60 150 (min>
TLC autoradiogram of 14C-PC labelled LIPO-HEP in blood after its FIG. 1 intravenous injection into rats.
372
Vol. 56, No. 3
LIPOSOME-HEPARIN
RESULTS Properties of the prepared liposomes: The final preparation of LIPO-HEP contained 35-50 U heparin/ml. The radioactivity of 3H or 14C ranged from 2 to 8 million dpm/ml. The amount of phospholipid in the final preparation was estimated to be 15-25 mg/ml. Other characteristic as stability, size distribution were essentially identical with those reported previously (1). Possible degradation of PC of LIPO-HEP: 14C-PC labelled heparin (40 u/kg) was intravenously administrated into rats and 1 ml of titrated blood was collected at the designated times. Then plasma obtained from each sample was subjected to lipid extraction and to lipid analysis by TLC. The chromatography was finally analyzed by autoradiography. As shown in FIG. 1, there was no significant radiolabelled metabolites except a trace amount of lyso-PC, indicating no significant hydrolysis of PC at least in plasma. Interrelationship between anticoagulant activity in blood of rats to which either untreated 3H-HEP or LIPO-HEP was intravenously administered. When untreated heparin (40 u/kg) was injected intravenously, RCT was significantly prolonged until 30 min after the injection and it returned to normal 60 min after. Upon injection of the same unit of LIPO-HEP, the anticoagulant effect judged by RCT was significantly prolonged as shown in FIG, 2. The amount of 3H-radioactivity in blood was significantly higher in rats receiving LIPO-HEP than those receiving untreated 3H-HEP. There was a rapid decline in the 14C-radioactivity until 30 min after the injection, and thereafter the activity remained almost unchanged at 5% of the total injected dose. This elimination pattern was significantly different from that of 3H-HEP, indicating an apparent dissociation of heparin and liposome in blood.
1
‘c. .N-L_____I~H-HE
118 510
I
I
30
60
TIME AFTER
90
120
150 (ml”)
INJECTION
FIG. 2 Recalcification clotting time and the radioactivity of LIPO-HEP (solid lines) and untreated 3H-HEP (broken lines), which were intravenously administered into rats (40 u/kg). The results were expressed as mean & SD (n-5). *p
Vol. 56, No. 3
LIPOSOME-HEPARIN
373
The amount of 14C-radioactivity in liver, spleen, lung and kidney FIG. 3 after intravenous injection of radiolabelled LIPO-HEP (40 u/kg). The results were expressed as mean ? SD (n=5).
LPO-HEPARIN 3 z
The amount of 3H-radioactivity in various organs after an intravenous FIG. 4 injection of untreated heparin (a) and of LIPO-HEP (b) into rats (40 u/kg). The results were expressed as mean 2 SD (n=3).
374
LIPOSOME-HEPARIN
Vol. 56, No. 3
14C-radioactivity of in organs: 14C-labelled liposome solution (2 ml/kg) was into 15 rats and each 5 rats were sacrificed at 10, 60, 150 min after the administration of the liposome. The radioactivity in liver, spleen, lung, and kidney was determined as described earlier. The results were summarized as % injected dose per gram tissue in FIG. 3. There was a significant accumulation of radioactivity in spleen, liver and lung, while almost no radioactivity was detected in kidneys. In both liver and spleen, the value at 150 min was significantly lower than that at 60 min, indicating liberation of liposome from those organs.
injected
3H-radioactivity in organs: Untreated 3H-heparin (40 u/kg) or the same unit of liposome encapsulated 3H-heparin was injected intravenously into 9 rats and each 3 rats were sacrificed at 10, 60, 150 min after the injection and the radioactivity in the four organs was determined. In rats receiving untreated 3H-heparin, there was some accumulation up to 5% of the total amount per gram tissue, as shown in FIG. 4-a. In cases when lipo-3H-heparin was injected, a significantly higher amount of 3H-radioactivity was detected especially in liver and spleen as shown in FIG. 4-b. Distribution of 3H- & r4C-radioactivity in various organs and plasma of rats receiving doubly radio-labelled Lipo-HEP: Doubly radio-labelled Lipo-HEP (40 u/kg) was injected intravenously in 9 rats and 3 each was sacrificed at 10, 60, 150 min after the injection and each radioactivity in organs and plasma was examined as described earlier. As shown in FIG. 5, the 3H-radioactivity in plasma was significantly higher than "C-radioactivity, which was compatible with the earlier results (FIG. 2). However in organs, almost an equal amount of the radioactivity was detected.
The amount of radioactivity [3H (a) & 14C (b)] invarious organs FIG. 5 and plasma after the administration of LIPO-HEP (40 u/kg). The results were expressed as % injected dose per whole tissue (mean f SD, n=5).
Vol. 56, No. 3
LIPOSOME-HEPARIN
375
DISCUSSION The metabolism of heparin administered intravenously to rats has been well elucidated by the use of 3H-labelled heparin (4) and the elimination rate of the untreated heparin in the present study was in accordance with that reported previously (4). When LIPO-HEP was injected, the amount of 3H-heparin in blood remained significantly higher between 30 to 150 min after the injection, which may account for the prolonged biological activity of LIPO-HEP. However, as far as the early phase (up to 10 min after administration) elimination rate is concerned, there was no significant difference between the two. Also, there was a dissociation between the radioactivity in blood of 14C-PC and 3H-HEP in rats receiving LIPO-HEP. This might indicate liberation of 3H-HEP from LIPO-HEP deposited in tissues. However, the ratio of 3H-HEP and 14C-PC was reversed at 90 min after the administration, the reason of which has yet to be elucidated. The liberation of heparin from liposome in circulating blood is unlikely, as there was no significant hydrolysis of lipids constituting the liposome used. The rapid elimination of LIPO-HEP from circulating blood may be partly due to the size and charge of the liposomes we used, as large multilamellar vesicles are cleared off much rapidly than small unilamellar vesicles (5,6). The determination of radioactivities in various organs in the present study confirmed highly specific uptake of liposomes into reticuloendothelial system (RES) (7-9), as a significantly smaller amount of untreated heparin was deposited in these organs. It has been reported that larger liposomes were selectively incorporated into RES (9,lO) and therefore marked uptake into RES was seen in our study as large multilamellar liposomes were used. However, no time dependent changes in the deposition of 3H-HEP in RES of rats receiving LIPO-HEP, which may not be compatible with the gradual liberation of heparin from the liposomes trapped in RES into circulating blood (11). The amount of liberated heparin might be too small to affect the total amount of deposition. These observations suggest that the prolonged biological activity of liposome-encapsulated heparin is not due to retarded elimination of the liposome from the circulating blood but to gradual liberation of heparin from liposome entrapped in RES.
REFERENCES 1.
KIM, T.D., SAKON, M., KAWASAKI, T., KAMBAYASHI, J., OHSHIRO, T., and MORI, T. Studies on liposome-encapsulated heparin. Thrombos. Res. 43, 603-612, 1986.
2.
BLIGH, E.G. and DYER, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917, 1959.
3.
KAWASAKI, T., KAMBAYASHI, J., MORI, T. and KOSAKI, G. Analysis of platelet phospholipids by high performance liquid chromatography. Thrombos. Res. 36, 335-344, 1984.
4.
KAMBAYASHI, J. Fate and anticoagulant activity of tritium labelled heparin in rats. Acta Haematol. Jpn. 43, 131-144, 1980.
5.
JDLIANO, R.L. and STAMP, D. The effect of particle size and charge on the clearance rates of liposomes and liposome encapsulated drugs. Biochem. Biophys. Res. Commun. 63, 651-658, 1975.
Vol. 56, No. 3
376
LIPOSOME-HEPARIN
6.
ABRA, R.M. and HUNT, C.A. Liposome disposition in vivo - III. dose and vesicle-size effects. Biochim. Biophys. Acta 666, 493-503, 1981.
7.
ALLEN, T.M., MURRAY, L., MacKEIGAN and SHAH, H. Chronic liposome administration in mice: effects on reticuloendothelial function and tissue distribution. J. Pharmacol. Exp. Ther. 229, 267-275, 1984.
8.
SPANJER, H.H., MORSELT, H. and SCHERPHOF, G.L. Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo. Biochim. Biophys. Acta 774, 49-55, 1984.
9.
RAHMAN, Y.E., CERNY, E.A., PATEL, K.R., LAU, E.H. and WRIGHT, B.J. Differential uptake of liposomes varying in size and lipid composition by parenchymal and Kupffer cells or mouse liver. Life Sci. 2, 2061-2071, 1982.
10.
HWANG, K.J., PADKI, M.M., CHOW, D.D., ESSIEN, H.E., LAI, J.Y. and BEAUMIER, P.L. Uptake of small liposomes by non-reticuloendothelial tissues. Biochim. Biophys. Acta 901, 88-96, 1987.
11.
GABIZON, A., DAGAN, A., GOREN, D., BARENHOLZ, Y. and FUKS, Z. Liposomes as in vitro carriers of Adriamycin: Reduce cardiac uptake and preserved antitumor activity in mice. Cancer Res. 42, 4734-4739, 1982.
METABOLISM OF LIPOSOME-ENCAPSULATED HEPARIN
T.D. Kim, J. Kambayashi, M. Sakon, T. Tsujinaka, T. Ohshiro, T. Mori Hematology Research Unit, The Second Department of Surgery, Osaka University Medical School, Fukushima l-l-50, Fukushima-ku, Osaka 553, Japan. (Received 6.12.1989; accepted in revised form 9.8.1989 by Editor H. Yamazaki)
ABSTRACT In order to elucidate the metabolism of liposome encapsulated heparin (LIPO-HEP), LIPO-HEP containing 3H-heparin (3H-HEP) and/or "C-phosphatidylcholine (14C-PC) was intravenously administered into rats and the radioactivity as well as the biological activity in plasma and certain organs was investigated. The amount of 3H-radioactivity in plasma was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin. The amount of "C-radioactivity in plasma of rats receiving LIPO-HEP, however, was not proportional to the amount of 3H-radioactivity in the same rats, indicating the dissociation of liposome and heparin in plasma. Incorporation of 3H-radioactivity into various organs examined, i.e., liver, spleen, lung, was significantly higher in rats receiving LIPO-HEP than in those receiving untreated heparin, e.g. 4.7 and 11.8 times higher in the liver and the spleen, respectively at 150 min after the injection. Thereby, in contrast to the untreated heparin, LIPO-HEP was selectively incorporated into the reticuloendothelial system (RES) and it may be suggested that prolonged biological activity in LIPO-HEP is due to a gradual release of heparin from the liposomes entrapped in RES, and that it is not due to prolonged circulation in blood.
INTRODUCTION Although it has been well known that liposome encapsulation of a certain drug or agent results in the prolongation of the drug effect and reduction of side effect due to selective uptake by specific tissue or organ, the exact metabolism of the liposome encapsulated agent has not been fully elucidated yet. We have reported in the foregoing paper that encapsulation of heparin in multilamellar liposomes yields the prolongation of in vivo anticoagulant
Key words:
liposome, heparin, metabolism, rats
369
370
LIPOSOME-HEPARIN
Vol. 56, No. 3
effect (1). In order to study the mechanism of the prolongation, we prepared liposome-encapsulated heparin (LIPO-HEP) containing 3H-labelled heparin (3H-HEP) and/or '\C-labelled phosphatidyl-choline (14C-PC). Upon intravenous administration of the radiolabelled liposome or heparin into rats, the radioactivity as well as the biological activity in blood and various organs was serially determined.
MATERIAL AND METHODS Preparation of liposomes: In order to radio-label liposome, L-a'4C-Phosphatidylcholine (PC) (0.02 mCi/0.0132 mg, Amersham Japan, Tokyo) was mixed with 1 ml chloroform solution containing 50 mg yolk phospholipid (The Green Cross Co. Ltd., Osaka), which was composed of PC (79%), phosphatidylethanolamine (13%), sphingomyeline - lyso PC (4%) and alpha-tocopherol (1%). The mixture was placed in a round bottomed flask and it was evaporated for about 2 hours in nitrogen gas until a thin lipid film was formed. Then the mixture of heparin calcium (3,000 u, Mitsui Pharm. Co.Ltd., Tokyo) and 3H-heparin (0.33 mCi, 506 u/3.3 mg, Amersham Japan, Tokyo) was placed over the lipid film and encapsulation was completed by vigorous shaking of the flask for one hour at room temperature. All the lipids collected from the wall of flask were subjected to the repeated washing and centrifugation at 10,OOOxg for 10 min unti thrombin clotting time of the resultant supernatant returned to normal. Then an aliquot was taken for scintillation counting and assay of anticoagulant activity and the remainder was kept at 4°C under nitrogen in a sealed polyethylene tube until use. Animal experiments: Male Wistar rats weighing 180-230 g were obtained from Nihon Animals Co. Ltd., Osaka and were fed on standard diet for at least two weeks before the experiments. Rats were anesthetized by intramuscular injection of ketamine hydrochloride and jugular vein was cannulated using Silastic@ tubing (Dow Corning, MI). Through the catheter, LIPO-HEP (40 u/kg) was administered and titrated blood was drawn in plastic syringe at 5, 10, 30, 60, 90, 120, 150 min after the administration. After the completion of the last blood collection, the animal was sacrificed by injecting an excessive dose of pentobarbiturate intravenously and the liver, spleen, lung, kidney were harvested. Measurement of anticoagulant activity: To determine the anticoagulant activity of the prepared LIPO-HEP, one ml of chloroform was added to 0.1 ml of LIPO-HEP and the mixture was vortexed vigorously for about 2 min to disrupt liposomes. After 1 ml of saline was added further, the mixture was thoroughly mixed and centrifuged at 17OOxg for 10 min at 4"C, yielding three (aqueous, interface, and chloroform) phases. Twenty microliter of the aqueous phase was incubated with 100 pl of human pooled titrated plasma for 2 min at 37°C. Then, 50 1.11 of 0.05 M CaClz solution was added to the mixture and the clotting time was recorded in a Clotem system. Samples were usually diluted with saline so that thrombin clotting time (TCT) became in the range between 10 and 27 set, in which clotting time was well correlate with heparin concentration. The amount of heparin was then calculated, using a standard curve of TCT obtained in the presence of the known amount of heparin. Anticoagulant activity in vivo was determined by the plasma recalcification clotting time (RCT) as described before (1). Measurement of radioactivity: In order to measure radioactivity of 3H and/or 14C of liposome encapsulated heparin, 0.05 ml of sample was solubilized in
Vol. 56, No. 3
LIPOSOME-HEPARIN
371
Univer-Gel@ (10 ml) and the radioactivity was measured in a liquid scintillation counter (Packard Tri-CAEB, IL). As for the preparation of blood sample, 0.1 ml of plasma prepared from titrated whole blood was likewise solubilized in Univer-Gel@. The freshly harvested organs were treated as follows for radioactivity determination. After each organ was weighted, 1 ml of saline was added per 1 g of tissue. The mixture was thoroughly homogenized and 0.1 ml 50% KOH and 0.1 ml Hz02 were added to the homogenate. The mixture was heated for 30 min at 70°C for digestion and it was neutralized by adding 0.1 ml 2 N HCl. Finally 0.05 ml of the resultant mixture was solubilized in 10 ml Univer-Gel@. Analysis of the degradation of 14C-PC by thin layer chromatography (TLC): At designated times after intravenous administration of LIPO-HEP containing 14C-PC, the blood was collected and plasma was prepared as described earlier. The plasma was subjected to lipid extraction by the method of Bligh & Dyer (2) and the lipid was separated on TLC (solvent system, as described previously (3)) and was visualized by autoradiography.
FRONT _.
i
I
i
i
,\
/
,::j / , . I
PC-
“*
LYSO-PC-
ORIGIN-
0
2
5 TIME
10 60 150 (min>
TLC autoradiogram of 14C-PC labelled LIPO-HEP in blood after its FIG. 1 intravenous injection into rats.
372
Vol. 56, No. 3
LIPOSOME-HEPARIN
RESULTS Properties of the prepared liposomes: The final preparation of LIPO-HEP contained 35-50 U heparin/ml. The radioactivity of 3H or 14C ranged from 2 to 8 million dpm/ml. The amount of phospholipid in the final preparation was estimated to be 15-25 mg/ml. Other characteristic as stability, size distribution were essentially identical with those reported previously (1). Possible degradation of PC of LIPO-HEP: 14C-PC labelled heparin (40 u/kg) was intravenously administrated into rats and 1 ml of titrated blood was collected at the designated times. Then plasma obtained from each sample was subjected to lipid extraction and to lipid analysis by TLC. The chromatography was finally analyzed by autoradiography. As shown in FIG. 1, there was no significant radiolabelled metabolites except a trace amount of lyso-PC, indicating no significant hydrolysis of PC at least in plasma. Interrelationship between anticoagulant activity in blood of rats to which either untreated 3H-HEP or LIPO-HEP was intravenously administered. When untreated heparin (40 u/kg) was injected intravenously, RCT was significantly prolonged until 30 min after the injection and it returned to normal 60 min after. Upon injection of the same unit of LIPO-HEP, the anticoagulant effect judged by RCT was significantly prolonged as shown in FIG, 2. The amount of 3H-radioactivity in blood was significantly higher in rats receiving LIPO-HEP than those receiving untreated 3H-HEP. There was a rapid decline in the 14C-radioactivity until 30 min after the injection, and thereafter the activity remained almost unchanged at 5% of the total injected dose. This elimination pattern was significantly different from that of 3H-HEP, indicating an apparent dissociation of heparin and liposome in blood.
1
‘c. .N-L_____I~H-HE
118 510
I
I
30
60
TIME AFTER
90
120
150 (ml”)
INJECTION
FIG. 2 Recalcification clotting time and the radioactivity of LIPO-HEP (solid lines) and untreated 3H-HEP (broken lines), which were intravenously administered into rats (40 u/kg). The results were expressed as mean & SD (n-5). *p
Vol. 56, No. 3
LIPOSOME-HEPARIN
373
The amount of 14C-radioactivity in liver, spleen, lung and kidney FIG. 3 after intravenous injection of radiolabelled LIPO-HEP (40 u/kg). The results were expressed as mean ? SD (n=5).
LPO-HEPARIN 3 z
The amount of 3H-radioactivity in various organs after an intravenous FIG. 4 injection of untreated heparin (a) and of LIPO-HEP (b) into rats (40 u/kg). The results were expressed as mean 2 SD (n=3).
374
LIPOSOME-HEPARIN
Vol. 56, No. 3
14C-radioactivity of in organs: 14C-labelled liposome solution (2 ml/kg) was into 15 rats and each 5 rats were sacrificed at 10, 60, 150 min after the administration of the liposome. The radioactivity in liver, spleen, lung, and kidney was determined as described earlier. The results were summarized as % injected dose per gram tissue in FIG. 3. There was a significant accumulation of radioactivity in spleen, liver and lung, while almost no radioactivity was detected in kidneys. In both liver and spleen, the value at 150 min was significantly lower than that at 60 min, indicating liberation of liposome from those organs.
injected
3H-radioactivity in organs: Untreated 3H-heparin (40 u/kg) or the same unit of liposome encapsulated 3H-heparin was injected intravenously into 9 rats and each 3 rats were sacrificed at 10, 60, 150 min after the injection and the radioactivity in the four organs was determined. In rats receiving untreated 3H-heparin, there was some accumulation up to 5% of the total amount per gram tissue, as shown in FIG. 4-a. In cases when lipo-3H-heparin was injected, a significantly higher amount of 3H-radioactivity was detected especially in liver and spleen as shown in FIG. 4-b. Distribution of 3H- & r4C-radioactivity in various organs and plasma of rats receiving doubly radio-labelled Lipo-HEP: Doubly radio-labelled Lipo-HEP (40 u/kg) was injected intravenously in 9 rats and 3 each was sacrificed at 10, 60, 150 min after the injection and each radioactivity in organs and plasma was examined as described earlier. As shown in FIG. 5, the 3H-radioactivity in plasma was significantly higher than "C-radioactivity, which was compatible with the earlier results (FIG. 2). However in organs, almost an equal amount of the radioactivity was detected.
The amount of radioactivity [3H (a) & 14C (b)] invarious organs FIG. 5 and plasma after the administration of LIPO-HEP (40 u/kg). The results were expressed as % injected dose per whole tissue (mean f SD, n=5).
Vol. 56, No. 3
LIPOSOME-HEPARIN
375
DISCUSSION The metabolism of heparin administered intravenously to rats has been well elucidated by the use of 3H-labelled heparin (4) and the elimination rate of the untreated heparin in the present study was in accordance with that reported previously (4). When LIPO-HEP was injected, the amount of 3H-heparin in blood remained significantly higher between 30 to 150 min after the injection, which may account for the prolonged biological activity of LIPO-HEP. However, as far as the early phase (up to 10 min after administration) elimination rate is concerned, there was no significant difference between the two. Also, there was a dissociation between the radioactivity in blood of 14C-PC and 3H-HEP in rats receiving LIPO-HEP. This might indicate liberation of 3H-HEP from LIPO-HEP deposited in tissues. However, the ratio of 3H-HEP and 14C-PC was reversed at 90 min after the administration, the reason of which has yet to be elucidated. The liberation of heparin from liposome in circulating blood is unlikely, as there was no significant hydrolysis of lipids constituting the liposome used. The rapid elimination of LIPO-HEP from circulating blood may be partly due to the size and charge of the liposomes we used, as large multilamellar vesicles are cleared off much rapidly than small unilamellar vesicles (5,6). The determination of radioactivities in various organs in the present study confirmed highly specific uptake of liposomes into reticuloendothelial system (RES) (7-9), as a significantly smaller amount of untreated heparin was deposited in these organs. It has been reported that larger liposomes were selectively incorporated into RES (9,lO) and therefore marked uptake into RES was seen in our study as large multilamellar liposomes were used. However, no time dependent changes in the deposition of 3H-HEP in RES of rats receiving LIPO-HEP, which may not be compatible with the gradual liberation of heparin from the liposomes trapped in RES into circulating blood (11). The amount of liberated heparin might be too small to affect the total amount of deposition. These observations suggest that the prolonged biological activity of liposome-encapsulated heparin is not due to retarded elimination of the liposome from the circulating blood but to gradual liberation of heparin from liposome entrapped in RES.
REFERENCES 1.
KIM, T.D., SAKON, M., KAWASAKI, T., KAMBAYASHI, J., OHSHIRO, T., and MORI, T. Studies on liposome-encapsulated heparin. Thrombos. Res. 43, 603-612, 1986.
2.
BLIGH, E.G. and DYER, W.J. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911-917, 1959.
3.
KAWASAKI, T., KAMBAYASHI, J., MORI, T. and KOSAKI, G. Analysis of platelet phospholipids by high performance liquid chromatography. Thrombos. Res. 36, 335-344, 1984.
4.
KAMBAYASHI, J. Fate and anticoagulant activity of tritium labelled heparin in rats. Acta Haematol. Jpn. 43, 131-144, 1980.
5.
JDLIANO, R.L. and STAMP, D. The effect of particle size and charge on the clearance rates of liposomes and liposome encapsulated drugs. Biochem. Biophys. Res. Commun. 63, 651-658, 1975.
Vol. 56, No. 3
376
LIPOSOME-HEPARIN
6.
ABRA, R.M. and HUNT, C.A. Liposome disposition in vivo - III. dose and vesicle-size effects. Biochim. Biophys. Acta 666, 493-503, 1981.
7.
ALLEN, T.M., MURRAY, L., MacKEIGAN and SHAH, H. Chronic liposome administration in mice: effects on reticuloendothelial function and tissue distribution. J. Pharmacol. Exp. Ther. 229, 267-275, 1984.
8.
SPANJER, H.H., MORSELT, H. and SCHERPHOF, G.L. Lactosylceramide-induced stimulation of liposome uptake by Kupffer cells in vivo. Biochim. Biophys. Acta 774, 49-55, 1984.
9.
RAHMAN, Y.E., CERNY, E.A., PATEL, K.R., LAU, E.H. and WRIGHT, B.J. Differential uptake of liposomes varying in size and lipid composition by parenchymal and Kupffer cells or mouse liver. Life Sci. 2, 2061-2071, 1982.
10.
HWANG, K.J., PADKI, M.M., CHOW, D.D., ESSIEN, H.E., LAI, J.Y. and BEAUMIER, P.L. Uptake of small liposomes by non-reticuloendothelial tissues. Biochim. Biophys. Acta 901, 88-96, 1987.
11.
GABIZON, A., DAGAN, A., GOREN, D., BARENHOLZ, Y. and FUKS, Z. Liposomes as in vitro carriers of Adriamycin: Reduce cardiac uptake and preserved antitumor activity in mice. Cancer Res. 42, 4734-4739, 1982.