Biochimica et Biophysics Acta, 1023 (1990) 133-139
Liposomes with entrapped doxorubicin exhibit extended blood
Marcel B. Bally 1,2, Rajiv Nayar ‘,*, Dana Masin ‘, Michael J. Hope I,*, Pieter R.
Cnllis I,* and Lawrence D. Mayer I,*
’ The Canadian Liposome Co. Ltd, North Vancouver, and ’ University of British Columbia, Department of Biochemistry
(Received 20 July 1989)
(Revised manuscript received 19 October 1989)
Key words: Liposome stability; Doxorubicin; Blood residence time
The blood residence time of liposomes with entrapped doxorubicin is shown to be significantly longer than for
identically prepared empty liposomes. Liposomal doxorubicin systems with a drug-to-lipid ratio of 0.2 (w/w) were
administered at a dose of 100 mg lipid/kg. Both doxorubicin and liposomal lipid were quantified in order to assess in
vivo stability and blood residence times. For empty vesicles composed of phosphatidylcholine (PC) / cholesterol (55 : 45,
mole ratio) and sized through filters of 100 nm pore size, 15-25% of the administered lipid dose was recovered in the
blood 24 h after i.v. injection. The percentage of the dose retained in the circulation at 24 h increased 2-3-fold when the
liposomes contain entrapped doxorubicin. For 100 nm distearoyl PC/chol liposomal doxorubicin systems, as much as
80% of the injected dose of lipid and drug remain within the blood compartment 24 h after i.v. administration.
Applications for liposomes as drug carriers are be-
coming apparent. In particular, acute and chronic toxic-
ities associated with certain drugs can be reduced if the
agent is presented in association with liposomes. This
reduced toxicity is accompanied by maintained or en-
hanced efficacy. Therefore, the liposomal carrier can
provide a significant improvement in the therapeutic
index of the entrapped drug. Two such formulations, an
amphotericin B-lipid complex and liposomal doxorubi-
cin, are currently being evaluated in human clinical
Many investigations have documented the potential
therapeutic benefit of liposomal doxorubicin, however,
the mechanism(s) underlying the biological activity of
these preparations are not clear. We  and others 
have demonstrated in animal models that the biological
Abbreviations: MLV, multilamellar vesicle; LUV, large unilamellar
vesicle; SUV small unilamellar vesicle; egg PC, egg phosphatidyl-
choline; DPPC, dipalmitoylphosphatidylcholine; DSPC, distearoyl-
phosphatidylcholine; chol, cholesterol; RES, reticuloendothelial sys-
tem; QELS, quasielastic light scattering; i.v., intravenous.
Correspondence: M.B. Bally, The Canadian Liposome Co. Ltd, 308,
267 W. Esplanade, North Vancouver, B.C. Canada V7M lA5.
activity of liposomal doxorubicin can be modulated by
alterations in vesicle lipid composition and size. Lipo-
somal doxorubicin systems formulated with saturated
phospholipids species (such as dipalmitoyl- or dis-
tearoyl-PC) and cholesterol are the least toxic and anti-
tumour activity increases as vesicle size decreases. It is
also well established that liposome size and lipid com-
position can dramatically alter the clearance kinetics of
liposomes [6-8]. In particular, liposomes prepared with
saturated phospholipid species and cholesterol exhibit
superior drug retention in vivo than liposomes com-
posed of unsaturated phospholipids . Further, as ves-
icle size is decreased there is a concomitant increase in
blood residence times . In this study we demonstrate
that the blood residence time is influenced not only by
the physical characteristics of the vesicle but also by the
activity of the entrapped agent, doxorubicin. This effect
is important for understanding the mechanisms de-
termining the biological activity of the encapsulated
Materials and Methods
Animals. Female DBA/2J mice 6-8 weeks old were
obtained from Jackson Animal Laboratories (Califonia).
Groups of four mice per experimental point were given
the specified treatment as a single i.v. dose via the
0005-2736/90/$03.50 0 1990 Elsevier Science Publishers B.V. (Biomedical Division)
sis of indicated samples. We thank Diane Tanguay for
her assistance in the preparation of this manuscript.
1 Sells, R.A., Owen, R.R., New, R.R.C. and Gilmore, I.T. (1987)
Lancet 8559, 2, 624-625.
2 Treat, J. Roh, J.K., Woolley, P.V., Neefe, J., Schein, P.S. and
Rahman, A. (1987) Proc. Am. Cancer Ong. 6, 31.
3 Lopez-Berestein, G., Fainstein, V., Hopfer, R., Mehta, K., Sulli-
van, M.P., Keating, M., Rosenblum, M.G., Mehta, R., Luna, M.,
Hershe, E.M., Reuben, J., Juliano, R.L. and Bodez, G.P. (1985) J.
Infect. Dis. 151, 704-710.
4 Mayer, L.D., Tai, L.C., Ko, D.S.C., Masin, D., Ginsberg, R.S.,
Cullis, P.R. and Bally, M.B. (1989) Cancer Res. 49, 5922-5930.
5 Gabizon, A., Dagan, A., Goren, D., Branholz, Y. and Fuks, Z.
(1982) Cancer Res. 42, 4734-4739.
6 Sota, Y., Kiwada, H. and Kato, Y. (1986) Chem. Pharm. Bull. 34,
7 Hunt, A.C. (1982) Biochim. Biophys. Acta 719, 450-463.
8 Huang, K.J. (1987) in Liposomes: From Biophysics to Ther-
apeutics (Ostro, M.J., ed.), pp. 1099156, Marcel Dekker, New
9 Mayer, L.D., Hope, M.J., Cullis, P.R. and Janoff, A.S. (1986)
Biochim. Biophys. Acta 817, 193-196.
10 Hope, M.J., Bally, M.B., Webb, G. and Cullis, P.R. (1985) Bio-
chim. Biophys. Acta 812, 55-65.
11 Mayer, L.D., Bally, M.B. and Cullis, P.R. (1988) Biochim. Bio-
phys. Acta 852, 123-126.
12 Mayer, L.D., Tai, L.C.L., Bally, M.B., Mitilenes, G.N., Ginsberg,
R.S. and Cullis, P.R. (1990) Biochim. Biophys. Acta, submitted.
13 Huang, L. (1983) in Liposomes (Ostro, M.J., ed.), pp. 87-124,
Marcel Dekker, New York.
Scherphof, G.L., Kuipers, F., Derksen, J.T.P., Spanjer, H.H. and
Vonk, R. (1987) Biochem. Sco. Trans. 15, (Suppl.) 62S-68s.
Scherphof, G.L., Damen, J. and Wilschut, J. (1984) in Liposome
Technology, Vol. III (Gregoriadis, G., ed.), pp. 205-224, CRC
Press, Boca Raton, FL.
Senior, J., Crawley, J.C.W. and Gregoriadis, G. (1985) Biochim.
Biophys. Acta 839, 1-8.
Poste, G. (1983) Biol. Cell 47, 19-38.
Levin, V.A. (1986) Cancer Treat. Rev. 13, 61-76.
Shemozawa, S., Mimaki, Y. and Araki, Y. (1980) J. Chromatogr.
Storm, G., Roerdink, F.H., Steerenberg, P.A., De Jong, W.H. and
Crommelin, D.J.A. (1987) Cancer Res. 47, 3366-3372.
Olson, F., Mayhew, E., Maslow, D., Rustum, Y. and Szoka, F.
(1982) Eur. J. Cancer Clin. Oncol. 18, 167-176.
22 Rahman, A., Carmichael, D., Harris, M. and Roh, J.K. (1986)
Cancer Res. 46, 2295-2299.
23 Seldin, M.F. and Steinberg, A.D. (1988) in Inflammation Basic
Principles and Clinical Correlates (Gallin; J.I., Galdstein, I.M. and
Snyderman, R., eds.), pp. 911-934, Raven Press, New York.
Finkelstein, MC., Kuden, S.H., Scheinen, H., Weissman, G. and
Hoffstein, S. (1981) B&him. Biophys. Acta 673, 286-302.
Storm, G., Van Gessel, H.J.G.M., Steerenberg, P.A., Speth, P.A.J.,
Roerdisk, F.H., Regts, J., Van Veen, M. and De Jong, W.H. (1987)
Cancer Drug Del. 4, 89-104.
Storm, G., Steerenberg, P.A., Emmen, F., Van Borssum Waalkes,
M. and Crommelin, D.J.A. (1988) Biochim. Biophys. Acta 965,
Proffitt, R.T., Williams, L.E., Presant, C.A., Tin, G.W., Wiana,
J.A., Gamble, R.C. and Baldeschweiler, J.D. (1983) J. Nucl. Med.
Gabizon, A. and Papahadjopoulous D. (1988) Proc. Natl. Acad.
Sci USA 85, 6949-6953.