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)
lateral tail vein. The dose, administered in a volume of
200 ~1, was based on mean body weight. Blood was
collected from the carotid artery or through heart punc-
ture and was placed in EDTA-treated microtainers
(Becton-Dickinson, Canada). Plasma was prepared by
centrifuging (200 x g) blood samples for 10 min in a
clinical centrifuge. Total plasma volume per animal was
taken to be 4.55% of mean body weight. Control blood
samples containing known amounts of liposomes showed
that negligible amounts of the liposomal lipid was asso-
ciated with the pelleted blood cells. The recovery of
liposomes was similar if determined from whole blood
or from plasma.
Liposome preparation. Egg phosphatidylcholine (egg
PC), dipalmitoylphosphatidylcholine (DPPC) and di-
stearoylphosphatidylcholine (DSPC) were purchased
from Avanti Polar Lipids (Birmingham, AL). Cholesterol
was obtained from Sigma Chemicals (St. Louis. MO),
and all other chemicals were of reagent grade. Lipid
mixtures were prepared in a chloroform solution and
subsequently were dried under a stream of nitrogen gas.
The resulting lipid film was placed under high vacuum
for a minimum of 2 h. Multilamellar vesicles (100 mg
lipid/ml) were formed by hydrating the dried lipid with
300 mM citric acid (pH 4.0). The resulting preparation
was frozen and thawed five times and subsequently
extruded employing an extrusion device (Lipex Bio-
membranes, Vancouver, Canada) ten times through two
stacked polycarbonate filters (Nuclepore, Canada) of
the indicated pore size according to standard proce-
dures [9,10]. When DSPC was employed the sample was
extruded at 65OC employing a thermobarrel extrusion
device. Liposome particle size was determined by quasi-
elastic light scattering measurements (employing a
Nicomp 370 particle sizer, operating at a wavelength of
Doxorubicin encapsulation. Doxorubicin, obtained
from Adria Laboratories (Mississauga, Canada), was
encapsulated in liposomes in response to transmem-
brane pH gradients as described previously [11,12].
Briefly, the pH of the liposome suspension initially at
pH 4.0, was raised to pH 8.0-8.5 with 0.5 M Na,CO,.
The liposome preparation was subsequently heated to
60 o C for 5 min. These liposomes were then transferred
to a preheated (60” C) vial of doxorubicin, typically
adding enough lipid to achieve a final drug-to-lipid
ratio of 0.2 (w/w). This mixture was incubated with
periodic mixing for 10 min at 60 o C. Doxorubicin con-
centration, lipid concentration and encapsulation ef-
ficiency were determined as described previously .
The procedure resulted in entrapment efficiencies in
excess of 95%. Solutions for injection were prepared
with sterile physiological saline such that the specified
dose could be delivered in 200 ,ul.
Quantitation of liposomal lipid and doxorubicin. Lipo-
somal lipid was quantified employing the lipid marker
[ 3H]cholesterol hexadecyl ether (NEN, Canada). This
lipid marker does not readily exchange with lipopro-
teins, is not subject to esterase activity, and remains
associated with cells once internalized [13,14]. Due to
the long circulation times observed for the liposomes
employed in this report, the extent of [3H]cholesterol
hexadecyl ether exchange into lipoproteins was ex-
amined for the egg PC/chol and DSPC/chol liposomes
to ensure that exchange effects did not bias the results.
Vesicles were isolated from lipoproteins according to
the procedure described by Scherphof et al. . Ap-
proximately 1 ml of mouse plasma containing vesicles
was applied to a Bio-Gel A-15m gel filtration column
(30 X 1.5 cm) which was eluted at 4°C and a flow rate
of 6 ml/mm using Tris-buffered saline (pH 7.4). 80
fractions were collected and analyzed for radioactivity.
Vesicles elute in the void volume and are well separated
from HDL particles, which are the lipoprotein fraction
known to interact with lipid vesicles . Separation of
vesicles from HDL was demonstrated by calibrating the
gel filtration column by eluting various lipoprotein frac-
tions isolated using standard ultracentrifugation proce-
dures. [3H]Cholesterol labelled HDL was shown to be
included in the gel. Moreover, fasted mouse plasma
prelabelled with [ 3H]cholesterol gave an elution profile
in which the vesicle peak was clearly resolved from the
HDL peak. Plasma samples collected at 4 h and 24 h
following injection of [ 3H]cholesterol hexadecyl ether
labelled liposomes into mice showed no incorporation
of radioactivity into the HDL peak.
For scintillation counting, 50-100 ~1 plasma was
added to 400 ~1 of methanol followed by 100 ~1 of
hydrogen peroxide. Hydrogen peroxide was added to all
plasma samples due to the varying degree of hemolysis
obtained during collection and preparation of the blood
samples. Subsequently, 5 ml Pica-Fluor 40 (Packard,
Canada) scintillation cocktail was added. Samples were
left for at least 24 h prior to couting in a Beckman
model LS 3801 scintillation counter.
Doxorubicin was determined employing a fluorescent
assay procedure. Plasma (So-100 ~1) was diluted to 1
ml in physiological saline (adjusted to pH 4.0 with
HCl). Subsequently, the sample was extracted with 2 ml
of chloroform/isopropyl alcohol (1: 1, v/v). Following
vigorous mixing and a brief centrifugation, the organic
phase was collected. The fluorescence of this phase was
determined (excitation wavelength of 500 nm and emis-
sion wavelength 550 nm) employing a Shimadzu RF-540
spectrofluorometer. A standard doxorubicin curve was
preparedemploying a similar extraction procedure.
Where necessary, plasma samples were diluted such that
the doxorubicin level within the 1 ml acidic saline
solution fell within the range of the standard curve. Ail
values are represented as pg doxorubicin fluorescent
equivalents per 100 ~1 plasma. HPLC analysis of plasma
extracts obtained from the 24 h time point of animals
receiving liposomal doxorubicin systems indicated that
more than 98% of the fluorescence detected was due to
The antitumour activity of liposomal doxorubicin in
the L1210 lymphocytic leukemia model is sensitive to
vesicle size  where greater efficacy is observed for
smaller systems. Vesicle size also influences dramati-
cally the blood clearance kinetics of empty liposomes
[8,16]. This effect is illustrated in Fig. 1 for liposomes
composed of DPPC/chol (55 : 45, mol/mol). Vesicles
sized through filters with 1.0 pm pore size (Fig. lA,
open symbols) have a mean diameter of approx. ~ 1.2
pm as determined by QELS. Within one hour following
i.v. injection more than 90% of these large vesicles have
been removed from the circulation. In contrast, vesicles
sized through filters with 0.1 pm pore size (Fig. lB,
open symbols), exhibiting a mean diameter of 0.12 pm,
are retained in the circulation for extended periods.
These vesicles have a circulation half-life of approx. 8 h
15 2 0
T i m e (h)
Fig. 1. Liposomes (open symbols) and liposomes loaded with
doxorubicin (solid symbols) employing apH driven uptake were in-
jected into DBA/2J mice at a doxorubicin dose of 20 mg/kg or a
lipid dose of 100 mg/kg. Vesicles (DPPC/chol, 55:45) prepared as
indicated in Methods exhibited mean diameter of 1.2 pm (Fig. 1A) or
0.12 nrn (Fig. 1B). Blood was collected at the indicated time points in
microtainers containing EDTA. The blood samples were centrifuged
for 10 min at 500X g, subsequently the plasma was removed and
prepared for assays. Lipid was quantified employing the radiolabel
trace lipid [ 3H]cholesterol hexadecyl ether as indicated in the Meth-
ods. Each point represents an average of four animals and the error
bars reflect the standard deviation.
10 15 20
Fig. 2. The level of doxorubicin recovered in the plasma of mice
treated with either free doxorubicin (open circles), 1.2 gm DPPC/chol
liposomal doxorubicin (solid circles) or 0.12 pm DPPC/chol lipo-
somal doxorubicin (solid squares). Doxorubicin was determined em-
ploying the fluorescent assay procedure described in the Methods.
Samples diluted to 1 ml in acidic saline were extracted with 2 ml of
chloroform isopropylalcohol (1:l). The organic phase was isolated
and fluorescence was determined. Each point represents an average of
four animals and the error bars indicate the standard deviation.
and almost 20% of the injected lipid dose is still cir-
culating at 24 h.
It is generally assumed that liposome elimination
from the circulation occurs as a result of vesicle uptake
by phagocytic cells of the reticuloendothelial system
(RES) . Since doxorubicin is a potent cytotoxic
agent, it would be expected that cells which accumulate
liposomal doxorubicin may be affected by the presence
of the entrapped drug. It is therefore important to
determine whether encapsulated doxorubicin can alter
the clearance behaviour of the liposomal carrier. The
data is Figs. 1A and 1B (see closed symbols) clearly
demonstrate that entrapped doxorubicin extends the
circulation times of the associated vesicles. Regardless
of vesicle size, this results in a 2-10-fold increase in the
amount of liposomal lipid within the circulation at time
points beyond 8 h. It is interesting that the clearance
rates of 0.12 pm empty vesicles and 0.12 pm liposomal
doxorubicin systems are similar for the first 4 h follow-
ing i.v. administration.
Circulating doxorubicin concentrations in animals
injected with free drug are 500- and 25-fold lower (at
time points beyond 4 h) than obtained when the drug is
encapsulated in 0.12 pm and 1.2 pm vesicles, respec-
tively (Fig. 2). It should be emphasized that no attempt
has been made to differentiate between the level of free
drug and liposome associated drug in animals given
liposomal doxorubicin. However, since. the free drug is
cleared very rapidly from the plasma, with an initial
circulating half-life of less than 5 min , it is reasona-
ble to assume that the large majority (> 95%) of
doxorubicin measured in the plasma of animals receiv-
ing liposomally entrapped doxorubicin is associated with
the liposomal carrier. The drug-to-lipid ratio obtained
Fig. 3. Drug-to-lipid ratio determined in the plasma of mice treated
with DPPC/chol liposomal doxorubicin sized through 0.1 nm (solid
squares) and 1.0 pm (solid circles) filters. Lipid levels were de-
termined as indicated in Fig. 1. Doxorubicin was determined as
indicated in Fig. 2.
in plasma samples of animals treated with DPPC/ chol
liposomal doxorubicin (Fig. 3) supports this assumption
and indicates that this liposomal formulation is ex-
tremely stable. For the 0.12 pm diameter vesicle system
the drug-to-lipid ratio (w/ w) prior to injection was
determined to be 0.22. The plasma samples obtained 1 h
after administration indicated a drug-to-lipid ratio
(w/ w) of 0.22 and this dropped linearly to a ratio of
approximately 0.16 at 24 h. The drug-to-lipid ratio
profile obtained from animals given 1.2 pm diameter
DPPC/ chol liposomal doxorubicin indicates an ap-
parent increase in drug-to-lipid ratio for several hours
after injection. This apparent increase in drug-to-lipid
ratio may reflect sample heterogeneity with respect to
size and drug-to-lipid ratio.
The data presented thus far were obtained for vesicles
composed of DPPC/ chol (55 : 45). However, as indi-
cated in Fig. 4, the increased circulation time exhibited
by these liposomal doxorubicin systems was observed
FREE EPC DPPC
Fig. 4. The percentage of injected dose recovered in plasma 24 h after
i.v. injection of doxorubicin (hatched bars) and liposomes (solid bar:
liposomal doxorubicin and open bar: empty liposomes). The vesicles
were prepared with the indicated phosphatidylcholine species and
cholesterol (55 : 45) and sized through filters with 0.1 pm pore size.
The recovery of free drug in plasma at 24 h was less than 0.1% but
well within the detection limits of doxorubicin assay employed (see
T i m e (h)
Fig. 5. Liposomal lipid (Fig. 5A) and doxorubicin (Fig. 5B) levels
obtained in plasma of mice injected with 0.12 pm DPPC/chol
liposomal doxorubicin (solid circles) or 0.12 pm egg PC/chol lipo-
somal doxorubicin (solid squares). Doxorubicin and lipid were quanti-
fied as indicated in Figs. 1, 2 and Methods. Each point represents the
average of four animals and the error bars indicate the standard
for a number of lipid compositions. For example, 80%
of the injected lipid dose and injected doxorubicin dose
remain in the circulation 24 h after injection of lipo-
somal doxorubicin systems prepared with vesicles com-
posed of DSPC/ chol(55 : 45) and sized through the 0.1
pm pore size filters. As much as 10% of the injected
doxorubicin was still circulating three days after admin-
istration (results not shown). The data in Fig. 4 also
indicates the influence of lipid composition on
doxorubicin release. Liposomal doxorubicin systems
prepared with 0.12 ,um vesicles composed of DSPC/ chol
showed no decrease in the initial drug-to-lipid ratio 24 h
after i.v. administration. In contrast, DPPC/ chol and
egg PC/ chol vesicles display a decrease in drug-to-lipid
ratio at 24 h of 27% and 80%, respectively. This effect is
illustrated in greater detail in Figs. 5 and 6 for egg
PC/ chol and DPPC/ chol liposomal doxorubicin sys-
tems. Quantitation of liposomal lipid indicate that the
initial rates (for the first 4 h) of elimination for egg
PC/ chol and DPPC/ chol 0.l2 pm liposome are similar
(Fig. 5A). However, at times beyond 8 h the level of
lipid found in the plasma is 2-fold less for animals given
egg PC/ chol liposomes. Although the initial rates of
liposome clearance are comparable, 3-fold less
doxorubicin is found in the plasma of animals given egg
PC/ chol liposomal doxorubicin (Fig. 5B). The dif-
ferences in plasma doxorubicin concentrations become
more pronounced at time points in excess of 8 h. As
5 10 15 2 0
Fig. 6. Drug-to-lipid ratio determined in the plasma of mice treated
with 0.12 am DPPC/chol (solid squares) or 0.12 pm egg PC/chol
(solid circles) liposomal doxorubicin.
indicated by the drug-to-lipid radio profiles (Fig. 6),
these results show that almost 50% of the entrapped
doxorubicin is released from the egg PC/chol liposomes
within 1 h following i.v. administration. It is interesting
to note that the rate of drug dissociation obtained after
one hour (2.08 pg doxorubicin per mg lipid per h) is
similar to that obtained for doxorubicin encapsulated in
0.12 pm diameter DPPC/chol liposomes (2.91 pg
doxorubicin per mg lipid per h).
The data presented to this point clearly indicate that
the presence of entrapped doxorubicin decreases the
elimination of the liposomal carrier system from the
circulation. This effect cannot be induced by pre-treat-
ing animals with free doxorubicin. Administration of
free drug (20 mg/kg) 4 or 24 h prior to injection of
empty liposomes did not alter the extent of liposome
clearance (data not shown). These data support the
contention that the decreased elimination of liposomes
results specifically from the presence of entrapped drug.
It is therefore of interest to determine whether pretreat-
ing animals with a lower dose of liposomal doxorubicin
can alter the clearance rate of subsequently adminis-
tered empty liposomes. As illustrated in Table I, 24 h
after administration of lipid doses of 10 mg/kg and 100
mg/kg; the percentage of the injected liposome dose in
Influence of lipid dose on the recovery of liposomes and liposomal
doxorubicin in the plasma of mice 24 h after injection
Liposomes were composed of DPPC/chol and sized through the 0.1
nrn filters. Assays for lipid and doxorubicin are as described in the
legends of Figs. 1 and 2.
$ Recovery at 24 h
Fig. 7. Influence of liposomal doxorubicin pretreatment on the
clearance of empty 0.12 pm DPPC/chol liposomes administered at a
lipid dose of 100 mg/kg. One day prior to injection of DPPC/chol
empty liposomes the mice were given (i.v.) empty liposomes (10 mg
lipid/kg) or liposomal doxorubicin (2 mg doxorubicin/kg; 10 mg
lipid/kg). Subsequently the 100 mg/kg dose of liposomes was admin-
istered, and at the indicated time points blood was collected. Plasma
lipid levels were determined as indicated in Fig. 1. Animals pretreated
with liposomal doxorubicin are indicated by the solid circles. Control
animals are represented by the open circles. Each point represents the
mean of four animals and the error bars indicate the standard
the circulation is 0.32 and 21.67, respectively. For both
lipid doses, there is a 2-3-fold increase in the level of
liposomal lipid recovered in the plasma when the vesicles
contain entrapped doxorubicin. More importantly, pre-
treating animals with the lower dose of liposomal
doxorubicin (2 mg/kg doxorubicin; 10 mg/kg lipid)
one day prior to injection of labelled empty liposomes
(100 mg/kg) results in a dramatic reduction in plasma
clearance (Fig. 7). In particular, the animals which were
pretreated with liposomal doxorubicin show no change
in the circulating level of liposomal lipid (96% of the
injected dose) at 1 h and 4 h after i.v. administration,
and at 24 h, 62% of the injected lipid dose remains in
circulation of these pretreated animals.
This report shows that small (100 nm) liposomes
composed of phosphatidylcholine and cholesterol dis-
play prolonged circulation times when administered at
appropriate dose levels, and that entrapped doxorubicin
markedly reduces the clearance rates of the liposomal
carrier system. Here we discuss the considerable bio-
logical significance for liposomal delivery systems that
are implicit in these results.
The toxicity and therapeutic activity of drugs are
known to be dependent on the concentration and/or
the lifetime of the agent at the site of potential toxicity
or the target site. In this regard many investigators have
examined the pharmacokinetic behaviour of doxorubi-
cin [18,19] and of liposomal systems  to obtain insight
on mechanism of action. However, previous studies on
the elimination of liposomal doxorubicin from the blood
[4,5,20,21,22] have focused primarily on the circulating
level of doxorubicin and not on the liposome dose
required to achieve these circulating drug levels. For
example, liposomal doxorubicin (drug-to-lipid ratio of
0.2, w/w) displays antitumour activity equivalent to the
free drug when administered at a dose of 20 mg/kg
doxorubicin . This doxorubicin dose corresponds to a
lipid dose of 100 mg/kg which is high enough to
saturate the RES and reduce liposomal clearance rates
. This effect would be expected to be even more
marked for other liposomal doxorubicin preparations
which exhibit drug-to-lipid ratios 3-10-fold lower . A
comparable dose of doxorubicin (20 mg/kg) in other
formulations would be delivered with lipid doses rang-
ing from 300 mg lipid/kg to well over 1000 mg lipid/kg.
Since lipid dose dramatically influences the blood
clearance kinetics of liposomes, therapeutic and toxico-
logical studies of liposomal drug formulations must
consider the amount of lipid required to achieve a
specific drug dose.
The effect of encapsulated drug on the circulation
lifetime of liposomal delivery systems has not been
observed previously. Doxorubicin is a potent cytotoxic
agent  and administration of this drug encapsulated
in liposomes could result in a direct toxicity to the cells
responsible for the clearance of the liposome carrier.
Cells of the reticuloendothelial system as well as other
phagocytic cell types accumulate liposomal systems
[24-261 and subsequent release of the entrapped drug
within the cell could result in impaired phagocytic abil-
ity or cell death. The data presented in Fig. 7 is con-
sistant with this model of RES toxicity. Pre-treating
animals with a low dose of liposomal doxorubicin,
which is cleared rapidly from the circulation, effectively
blocked the clearance of subsequently administered
liposomes. We are currently determining the nature and
extent of doxorubicin mediated cytotoxicity on phago-
cytic cell populations which may accumulate the lipo-
somally entrapped drug.
With regards to the mechanism(s) of biological activ-
ity of liposomal doxorubicin, these results do provide
new insight. The acute toxicity of these formulations
may be governed by the extent of drug dissociation
from the liposomal carrier in the circulation. For egg
PC/chol liposomal doxorubicin the drug-to-lipid ratio
profiles (see Fig. 6) indicate that 50% of the drug
dissociates from the carrier within 1 h following i.v.
administration. As would be predicted from these re-
sults, the LDsO dose of doxorubicin entrapped in these
liposomes is approximately 2-fold higher than the LDsO
dose of free drug . The drug-to-lipid ratio profiles
also indicate that saturated liposomes (DSPC/chol or
DPPC/chol) with entrapped doxorubicin display the
greatest in vivo stability. These observations are con-
sistant with previous investigations  and with the
dramatic reduction in acute toxicity of doxorubicin
entrapped in DSPC/chol liposomes .
What is remarkable about the liposomal doxorubicin
preparations formulated with the saturated lipids con-
cerns the maintenance of biological activity. As indi-
cated elsewhere [4,20], DSPC/chol liposomes with
encapsulated doxorubicin display antitumour activity
similar to that of the free drug. Further, the increased
circulation half-life exhibited for liposome systems with
entrapped doxorubicin was most pronounced for vesicles
which displayed the greatest in vivo stability. Regard-
less of the specific nature of the cytotoxic effects which
result in this increase in circulation time, these drug
mediated changes are detected rapidly (within 4 h)
following i.v. administration (see Fig l), and occur with
little or no measurable release of doxorubicin from
vesicles within the blood compartment. It is not unrea-
sonable, therefore, to suggest that this biological activity
is due to intracellular processing of the saturated lipo-
somes containing doxorubicin. Storm et al. , utiliz-
ing a liver tumour model, had postulated a similar
mechanism for antitumour activity of saturated lipo-
somal doxorubicin systems.
With reference to other tumour models, the increased
circulation time exhibited by liposomal doxorubicin sys-
tems may result in increased accumulation of the lipo-
somal carrier and associated drug at the sites of tumour
growth. It has been demonstrated previously that small
vesicles composed of cholesterol and saturated
phospholipid species can localize at sites of tumour
growth . More recently, Gabizon and Papa-
hadjopoulous  have developed ‘and characterized
liposomal systems which displays extended circulation
times. These liposomal systems also appear to accu-
mulate at sites of tumour growth. The results presented
here and elsewhere  suggest that drug mediated bio-
logical effects can be measured following i.v. adminis-
tration of stable, long-lived liposomes with entrapped
doxorubicin. To date, however, there are no clear indi-
cations suggesting that extended circulation times will
be of any therapeutic advantage.
In summary, we have demonstrated that the clearance
of liposomal doxorubicin from the circulation is
governed by the physical characteristics of the lipo-
somes, the cytotoxic activity of the entrapped drug as
well as the dose of lipid administered. Specifically, for
liposomes composed of distearyol PC/cholesterol ad-
ministered (i.v.) at a dose of 100 mg/kg 25% of the
injected lipid dose is retained in the circulation at 24 h.
This value increases to almost 80% when the liposomes
The authors gratefully acknowledge Dr. R. Thies for
his helpful discussion and for performing HPLC analy-
sis of indicated samples. We thank Diane Tanguay for
her assistance in the preparation of this manuscript.
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