Division of Biopharmaceutics, Leiden/Amsterdam Center for Drug
Research, University of Leiden, Leiden, the Netherlands
Many tumors express elevated levels of low-density lipoprotein (LDL)
receptors. Therefore, native LDL and synthetic LDL-like particles have
been proposed as carriers for antineoplastic drugs. We demonstrated
earlier that small apolipoprotein E (apoE)-exposing liposomes were
specifically recognized by the LDL receptor. In this study, we
incorporated a lipophilic derivative of daunorubicin (LAD) into the
apoE liposomes. Up to 11 molecules of LAD could be incorporated per
particle without significantly changing the size, lipid composition,
and ability to bind apoE of the liposomes. The biological fate of the
prodrug was largely determined by its carrier (70% of the initially
incorporated LAD was still associated to the liposomes after 4 h
of circulation in mice). Compared with free daunorubicin, the
circulation half-life of the liposome-associated prodrug was
substantially prolonged and undesired tissue disposition was reduced.
The role of the LDL receptor in the metabolism of LAD-loaded apoE
liposomes was demonstrated in rats with up-regulated hepatic LDL
receptors. In these rats, the liver uptake of the prodrug and carrier
was increased 5-fold. The addition of apoE was essential for LDL
receptor-mediated uptake of the drug-carrier complex. In LDL
receptor-deficient mice, the circulation time of both the prodrug and
the carrier increased approximately 2-fold compared with wild-type
mice. We conclude that LAD-loaded apoE liposomes constitute a stable
drug-carrier complex that is well suited for LDL receptor-mediated
selective drug delivery to tumors.
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Introduction |
In
the chemotherapy of cancer, the disposition of antineoplastic drugs in
nonmalignant tissues often causes severe side effects. The narrow
therapeutic window of these drugs hampers the administration of fully
effective doses. A therapeutic strategy in which antineoplastic drugs
are associated with carriers that are selectively taken up by tumor
cells may diminish side effects and allow the administration of more
effective doses (Tomlinson, 1987
).
The receptor for low-density lipoprotein (LDL) is an attractive target
for the selective delivery of antineoplastic drugs to tumors because it
has been found that many tumors of different origin express elevated
levels of this receptor (Catapano, 1987; Vitols, 1991). Especially
tumors of gynecological origin and myeloid leukemic cells, but also
colon, kidney, lung, and brain tumors, were found to express
exceptionally high amounts of LDL receptors (Firestone, 1994). The
elevated expression of LDL receptors on tumor cells is probably a
result of their rapid proliferation. The cells use the cholesterol
present in LDL for the synthesis of new membranes. LDL is the
predominant cholesterol-transporting lipoprotein in humans. It is a
spherical particle of about 23 nm that consists of a polar shell of
phospholipids and cholesterol, which surrounds an apolar core of mainly
cholesterol esters. A large part of the particle surface is covered
with the apolipoprotein B (apoB), which is recognized by the LDL
receptor. After internalization of LDL via its receptor, the particle
is degraded in the lysosomes (Brown and Goldstein, 1975, 1986).
However, a possible drawback for the use of endogenous LDL for tumor
therapy may be its limited availability. Furthermore, it has been found
that the incorporation of cytotoxic drugs into native LDL often induces
altered physiological behavior of the particles (Masquelier et al.,
1986; De Smidt and Van Berkel, 1990). A wide variety of methods to
incorporate antitumor drugs into LDL has been explored, but apparently,
the incorporation of drugs into LDL often causes subtle changes in the
structure of apoB, which provoke in vivo uptake by mechanisms other
than the LDL receptor (De Smidt and Van Berkel, 1990).
Because of the problems associated with the use of native LDL,
synthetic LDL-like particles constitute an attractive alternative (Ginsburg et al., 1982; Maranhão et al., 1993, 1994; Gerke et al., 1996). We recently developed small (29 nm) liposomes (Rensen et
al., 1997a). The particles are composed of natural lipids and are
completely biodegradable. The liposomes were provided with apolipoprotein E (apoE), produced by recombinant DNA technology. The
apoE (34 kDa), when associated with small lipid particles, is also
recognized by LDL receptors. In fact, the affinity of the LDL receptor
for apoE-exposing particles is even 15 to 25 times higher than for LDL
(Innerarity et al., 1979; Pitas et al., 1979, 1980; Rensen et al.,
1997a). After i.v. injection into rats, the circulating apoE liposomes
maintained their structural integrity. The biological behavior of the
particles was very similar to that of native LDL. The serum half-life
was longer than 5 h, and the particles were not recognized by the
reticuloendothelial system (Rensen et al., 1997a). Pretreatment of rats
with 17
-ethinyl estradiol (17EE, a compound that increases the
expression of LDL receptors in the liver) led to accelerated serum
clearance of the apoE liposomes and increased uptake by the liver.
These findings indicate that in vivo the LDL receptor is responsible for the clearance of apoE liposomes (Rensen et al., 1997a).
In this study, we incorporated a lipophilic prodrug of daunorubicin
(LAD) into the apoE-enriched liposomes. LAD
(3-O-oleoyl-5
-cholanic acid coupled to
alanyl-leucyl-alanyl-leucyl-daunorubicin) consists of daunorubicin that
is linked to a cholesteryl-oleate analog via a tetrapeptide spacer
(Versluis et al., 1998). The tetrapeptide spacer is susceptible to
degradation by lysosomal enzymes, which ensures the intracellular
release of free daunorubicin after LDL receptor-mediated uptake.
Daunorubicin is a very potent antileukemic agent, but its
cardiotoxicity is dose limiting (Weiss, 1992). Association of
daunorubicin with the liposomes is expected to reduce the disposition
of the drug in the heart, and thus the cardiotoxicity. We studied the
incorporation of LAD into the apoE liposomes and the stability of
LAD-loaded apoE liposomes. We further investigated the biological fate
of the particles and the incorporated prodrug, with an emphasis on the
role of the LDL receptor in the clearance.
| |
Experimental Procedures |
Materials.
Human recombinant apoE3,
isolated from Escherichia coli, was a generous gift from Dr.
T. Vogel (Biotechnology General Ltd., Rehovot, Israel) (Vogel et al.,
1985). The apoE was dissolved in PBS (10 mM sodium phosphate buffer, pH
7.4, containing 0.15 M NaCl) at a concentration of 2 mg/ml and was
stored under argon at 
80°C.
[1,2(N)-3H]Cholesteryl oleate
([3H]CO), [1-14C]CO,
and 125I-labeled sodium salt were obtained from
Amersham International (Amersham, Buckinghamshire, UK).
[3H(G)]daunorubicin (DNR, daunomycin) was
purchased from New England Nuclear Research Products (Boston, MA).
Unlabeled and radiolabeled [3H]3-O-(oleoyl)-5-cholanic
acid coupled to Ala-Leu-Ala-Leu-daunorubicin (further referred to as
LAD; see chemical structure in Fig. 1) were synthesized as described previously (Versluis et al., 1998; purity
>95%). The purity of the compounds was regularly checked by TLC on
silica gel 60 F254 (solvent:
NH4OH/CH3OH/1,2
dichloromethane, 1:10:89). Egg yolk phosphatidylcholine (EYPC, 98%)
was obtained from Fluka (Buchs, Switzerland). CO (97%) was from
Janssen Chimica (Beerse, Belgium). Cholesterol oxidase, cholesterol
esterase, peroxidase type II (200 U/mg), and PrecipathL were obtained
from Boehringer Mannheim (Mannheim, Germany). 17EE was purchased
from Sigma Chemical Co. (St. Louis, MO). All other reagents were of analytical grade.
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Fig. 1.
LAD, a conjugate of
3-O-(oleoyl)-5-cholanic acid and
alanine-leucine-alanine-leucine-daunorubicin.
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Preparation of LAD-Containing (apoE) Liposomes.
LAD-containing liposomes were prepared by sonication using a procedure
described by Rensen et al. (1997a). EYPC (50 mg) and 1.9 mg CO were
mixed in a 20-ml glass vial with 0 to 200 µg of LAD containing 0.2 µCi of [3H]LAD. For the in vivo experiments,
200 µg of LAD containing 1 µCi of [3H]LAD
and 1.9 mg of CO containing 0.3 µCi of
[14C]CO were added to 50 mg of EYPC. The
solvent was evaporated under N2 (1 h, room
temperature), followed by vacuum desiccation overnight at 4°C. The
lipids and prodrug were hydrated in 11.4 ml of 10 mM Tris·HCl buffer,
pH 8.0, containing 0.1 M KCl. The mixture was subsequently sonicated
(18 µm output) for 1 h under argon using a Soniprep 150 (MSE
Scientific Instruments, Crawley, West Sussex, UK) equipped with a water
bath to maintain the temperature at 54°C. After sonication, the
liposomes were purified and concentrated by density-gradient
ultracentrifugation at 285,000g for 18 h at 4°C,
according to Redgrave et al. (1975). After ultracentrifugation, the
liposomes, visible as a narrow opalescent layer at a density of 1.016 to 1.040 g/ml, were isolated by aspiration with glass capillary
pipettes or by fractionation of the gradients. The isolated liposomes
were stored under argon at room temperature. Liposomes prepared for the
in vivo experiments were dialyzed against PBS and were used within 2 weeks after preparation. When indicated, the liposomes were provided
with apoE by incubating the particles just before use for 30 min at
37°C with apoE at an apoE/phospholipid ratio of 0.1:1 (w/w).
Characterization of LAD-Containing (apoE) Liposomes.
The amounts of [3H]LAD and
[14C]CO in the preparations were determined by
measuring the radioactivity. The amounts of EYPC and unlabeled CO were
determined using enzymatic kits for phospholipids and esterified
cholesterol, respectively (Boehringer Mannheim, Mannheim, Germany).
PrecipathL was used as standard in both assays. The size of the
particles and the homogeneity of the population were determined by
photon correlation spectroscopy at 27°C (90° angle) using a Malvern
4700c submicron particle analyzer (Malvern Instruments, Malvern, Worcs,
UK). The net negative charge was determined by subjecting the
(LAD-loaded) (apoE) liposomes to electrophoresis in a 0.75% (w/v)
agarose gel at pH 8.8 (75 mM Tris·HCl-hippuric acid buffer,
containing 0.65 mM EDTA). After electrophoresis, the gel was cut into
small segments, which were dissolved in 0.75 ml of methyl-pyrrolidinone
and subsequently counted for 3H and
14C radioactivity.
Rf values were determined relative to
the front marker bromphenol blue. It was calculated that 1 mg of
phospholipids represents 7.6 × 1013
liposomes. For these calculations, it was assumed that the phospholipid bilayer length was 39 × 10
10 m and
that the polar head of a EYPC occupies a surface of 4.2 × 1019 m2 (New, 1990).
Radiolabeling of apoE.
The apoE was radioiodinated at
pH 10.0 with carrier-free 125I as described
previously (Van Tol et al., 1978). Unbound 125I
was removed by gel filtration followed by extensive dialysis against
PBS containing 1 mM EDTA. More than 98% of the radiolabel in the
preparations was trichloroacetic acid precipitable. The specific
activity of 125I-labeled apoE was approximately
450 dpm/ng.
Determination of Plasma Clearance and Tissue Uptake of
[3H]DNR- and [3H]LAD-Loaded (apoE)
Liposomes in Rats.
Male Wistar rats (150-200 g) were maintained
on normal chow and had free access to water. The animals were
anesthetized by i.p. injection of sodium pentobarbital (60 mg/kg
b.wt.), and the abdomen was opened. Subsequently, the rats were
injected in the vena cava inferior with 0.1 nmol of
[3H]DNR or with 5 nmol of
[3H]LAD incorporated in
[14C]CO-labeled (apoE) liposomes (4 mg of
phospholipid). At the indicated times, blood samples of 0.3 ml were
taken from the vena cava inferior, and 0.1-ml serum samples were
assayed for radioactivity. The total amount of radioactivity in serum
was calculated using the equation: plasma volume (ml) = [0.0219 × body weight (g)] + 2.66 (Bijsterbosch et al., 1989). At the
indicated times, liver lobules were tied off and excised, and at the
end of the experiment, the remainder of the liver was removed. The
amount of liver tissue tied off successively did not exceed 15% of the
total liver mass. The amount of radioactivity in the liver at each time
point was calculated from the radioactivities and weights of the liver
samples. Uptake by extrahepatic tissues was determined by removing the
tissues at the end of the experiment and counting the radioactivity.
Tissue radioactivity was corrected for radioactivity in plasma present in the tissue at the time of sampling (Bijsterbosch et al., 1989). When
indicated, rats were injected s.c. for 3 consecutive days with 17-EE
(1 mg/ml in 1,2-propylene glycol) at a dose of 5 mg/kg b.wt. The
effectiveness of the treatment was monitored by measuring loss of body
weight and reduction of total serum cholesterol levels (Chao et al.,
1979). On the fourth day, the animals were injected i.v. with
[3H]LAD-containing
[14C]CO-labeled (apoE) liposomes as described above.
Determination of Association of LAD to apoE Liposomes in
Circulation.
Male Wistar rats (150-200 g) were injected with
[3H]LAD-loaded
[14C]CO-labeled apoE liposomes (5 nmol of
[3H]LAD and 4 mg of phospholipid). After 30 min, the rats were sacrificed, and blood samples were collected. Then,
1-ml serum samples were subjected to density ultracentrifugation at
285,000g at 4°C for 18 h, according to Redgrave et
al. (1975). After centrifugation, the gradients were fractionated into
0.5-ml aliquots, starting at the top of the centrifugation tube.
3H and 14C radioactivities
were measured, and the fractions were assayed for total cholesterol.
Serum Decay of LAD-Loaded apoE Liposomes in Wild-Type and LDL
Receptor-Deficient Mice.
Male C57Bl/6J-129Sv LDL receptor (/)
mice (Ishibashi et al., 1993; 22-26 g) and male wild-type C57Bl/6J
mice (22-26 g) were maintained on normal chow and had free access to
water. The mice were injected i.v. in the tail vein with
[3H]LAD-containing
[14C]CO-labeled apoE liposomes (0.5 nmol of
[3H]-labeled LAD and 0.5 mg of phospholipid).
At the indicated time points, blood samples were taken from the tail,
and serum samples were assayed for radioactivity.
Determination of Radioactivity
3H and
14C radioactivities were determined in a Packard
1500 TriCarb liquid scintillation analyzer. Radioactivity
(3H and 14C) in serum,
liver, and other tissue samples was counted after combustion (recovery
>97%) in a Packard Tri-Carb 306 Sample Oxidizer. 14C radioactivity in the combusted samples was
measured in a Carbosorb E/Permafluor mixture (2:3 v/v), and
3H radioactivity was counted in Monophase S. Other 3H- and
14C-containing samples were counted in Emulsifier
Safe (aqueous solutions), Ultima Gold, or HIONIC Fluor (organic
solutions) scintillation cocktails. All instruments and scintillation
cocktails were from Packard Instrument Company Inc. (Downers Grove, IL).
| |
Results |
Preparation and Physicochemical Characterization of LAD-Loaded
(apoE) Liposomes.
The incorporation of LAD into small liposomes
was investigated by cosonication of EYPC and CO with a varying amount
of [3H]LAD. The resulting particles were
purified by density-gradient ultracentrifugation, and the amount of
incorporated radiolabeled prodrug and the lipid composition were
determined. Table 1 shows that LAD could
be efficiently incorporated into the small liposomes. At the lower LAD
concentrations (up to 15 µg of LAD/mg of CO), virtually all added LAD
became incorporated. At higher concentrations, the efficiency was
lower: 40 to 50%. Size, homogeneity, and lipid composition of the
liposomes were not significantly changed by the incorporation of LAD,
even when higher concentrations of LAD were used. The liposomes
prepared with 100 µg of LAD/mg of CO formed a homogeneous
(polydispersity <0.2) population of particles with a size of 29.3 ± 1.1 nm. It was calculated that these liposomes contained 11 ± 3 molecules of LAD per liposomal particle. All further experiments were
performed with these liposomes.
To induce LDL receptor-mediated uptake, it is necessary to provide the
liposomes with a specific LDL receptor-recognizing ligand. Therefore,
LAD liposomes were incubated with recombinant apoE. Figure
2 shows the association of apoE to the
liposomes at different apoE/phospholipid ratios. The association of
apoE to the liposomes was maximal at a ratio of 40 µg of apoE/mg of phospholipid. It can be calculated that at this ratio, approximately 9 molecules of apoE are associated per liposome. For all in vivo experiments, liposomes were incubated with 100 µg of apoE/mg of phospholipid to ensure that the liposomes were maximally provided with
apoE. The excess of unbound apoE present in the preparations does not
interfere with the LDL receptor recognition of the apoE liposomes
because free apoE is not able to bind to the LDL receptor (Innerarity
et al., 1979).
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Fig. 2.
Association of apoE to LAD-loaded liposomes. Density
gradient-purified LAD-loaded liposomes were prepared by adding 100 µg
of LAD/mg of CO. Aliquots of LAD liposomes were incubated with
125I-labeled apoE (5 dpm/ng of apoE) at the indicated
apoE/phospholipid ratios. Subsequently, incubation mixtures were
subjected to density gradient ultracentrifugation at
285,000g for 18 h. Gradients were fractionated, and
liposome-containing fractions were assayed for 125I
radioactivity and phospholipid content.
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To investigate the effects of apoE association and LAD incorporation on
the electric charge of the liposomes,
[3H]LAD-loaded and/or
[14C]CO-labeled liposomes were subjected to gel
electrophoresis at pH 8.8. Table 2 shows
that the association of apoE (Rf free
apoE, 0.45) and/or the incorporation of LAD do not cause major changes in the electrophoretic mobility (i.e., the electric charge) of the
particles. LAD-loaded apoE liposomes migrated in a slightly broadened
band. However, the median Rf of
the particles (0.14) was very similar to those of the free liposomes,
the LAD-loaded particles, and apoE liposomes
(Rf values 0.14, 0.09, and 0.14, respectively). Both labels of the LAD-loaded apoE liposomes migrated similarly, indicating that under these conditions the particles remain
intact. The mobility of the liposomes was slightly lower than that of
native LDL (Rf: 0.21 ± 0.01;
mean ± S.D., n = 3). The combination of a low
negative surface charge and the small size of the drug-carrier complex
is beneficial with respect to avoiding non-LDL receptor-mediated uptake
mechanisms by phagocytes of the reticuloendothelial system (Gabizon and
Papahadjopoulos, 1988).
Serum Decay and Tissue Distribution of DNR and apoE
Liposome-Associated LAD in Rats.
To investigate the effect of the
lipophilic derivatization of daunorubicin and the subsequent
incorporation of the prodrug into apoE liposomes on the biological fate
of the drug, the serum decay and tissue distribution of both the
prodrug LAD and free daunorubicin were determined (Figs.
3 and 4).
Incorporation into apoE liposomes considerably enhanced the half-life
of the derivatized daunorubicin in the circulation. After 30 min,
50.3 ± 4.7% of the injected LAD was still circulating, whereas
at that time, only 2.0 ± 0.9% of the injected amount of
daunorubicin was recovered in the circulation. Daunorubicin was
recovered in a wide variety of tissues, whereas the uptake of LAD in
these tissues was much lower. The low disposition of LAD in the
extrahepatic tissues, in particular in the heart, may reduce or even
eliminate the side effects of free daunorubicin in these tissues.
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Fig. 3.
Serum decay of free daunorubicin and LAD incorporated
in apoE liposomes. [3H]LAD-loaded apoE liposomes ( ; 5 nmol of LAD and 4 mg of phospholipid) or [3H]DNR ( ;
0.1 nmol) were injected i.v. into rats. At indicated times, blood
samples were taken, and serum was assayed for radioactivity. Results
are expressed as percentage of injected dose, and are mean ± S.E.M. of three experiments.
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Fig. 4.
Tissue distribution of free daunorubicin and LAD
incorporated in apoE liposomes. [3H]LAD-loaded apoE
liposomes ( ; 5 nmol of LAD and 4 mg of phospholipid) or
[3H]DNR ( ; 0.1 nmol) were injected i.v. into rats.
After 30 min, animals were sacrificed, and radioactivity was measured
in indicated tissues. For determination of tissue uptake, measured
values were corrected for residual serum present in collected tissue
samples. Results are expressed as percentage of injected dose per gram
tissue. Values represent mean ± S.E.M. of three experiments.
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To verify the stable association of LAD with the carrier during
circulation, rats were injected with double-labeled
([3H]LAD and [14C]CO)
LAD-loaded apoE liposomes, and a blood sample was collected at 30 min
after injection. The serum was subjected to density ultracentrifugation, and the gradients were fractionated. The fractions
were assayed for 3H and 14C
radioactivity (LAD and liposomes, respectively) and for cholesterol (rat lipoproteins). Figure 5 shows that
the distribution of liposomes and LAD over the fractions were
identical, reaching a peak at a density of 1.085 g/ml, just between the
rat LDL and high-density lipoprotein. The results suggest that after 30 min of circulation, LAD is still completely associated with the
liposomes and that no redistribution of the prodrug to the rat
lipoproteins had occurred.
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Fig. 5.
Association of LAD to apoE liposomes in the
circulation of rat. [3H]LAD-loaded ( )
[14C]CO-labeled () apoE liposomes were injected i.v.
into rats. Thirty minutes after injection, a blood sample was
collected. Serum was subjected to density ultracentrifugation
(285,000g for 18 h), and gradients were divided
into 0.5-ml fractions from top (fraction 1, lowest density) to bottom
(fraction 24, highest density). Fractions were assayed for
radioactivity and cholesterol. Dashed line, results are expressed as a
percentage of recovered activity (recoveries >97%). Arrows indicate
fractions in which serum components LDL, high-density lipoprotein, and
lipoprotein-deficient serum were recovered. Graph is representative of
two separate experiments.
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LDL Receptor-Mediated Uptake of (apoE) LAD Liposomes in Control and
17EE-Treated rats.
To allow evaluation of the role of LDL
receptor-mediated uptake in the clearance of LAD-loaded apoE liposomes,
rats were pretreated with 17EE. This steroid induces in rats an
enhanced expression of LDL receptors in the liver and therefore is
widely used as a model to study the role of the LDL receptor in the
clearance of circulating ligands (Chao et al., 1979; Harkes and Van
Berkel, 1983). Both the behavior of the liposomes (labeled with
[14C]CO) and [3H]LAD
were studied. To examine the role of apoE, both apoE-containing liposomes and liposomes devoid of apoE were used. The apoE liposomes were cleared faster from the circulation than liposomes lacking apoE
(Fig. 6, A and B). Thirty minutes after
injection, 59 ± 5% of the injected
[14C]CO label of the apoE liposomes was
recovered in the serum. After injection of liposomes lacking apoE,
90 ± 3% of the injected liposomal label was still present in the
serum at that time. The LAD present in the liposomes was also cleared
faster when the liposomes were provided with apoE. However, at all time
points, approximately 15% less [3H]LAD was
recovered in the serum than in liposomal label. The ratios of the
recovered labels (3H/14C;
ratio at time of injection set at 1) did not change in time (0.86 ± 0.02 and 0.85 ± 0.02 for apoE liposomes and liposomes lacking
apoE, respectively). Apparently, after an initial loss of some LAD from
the liposomes, the LAD is cleared simultaneously with the liposomes. A
similar phenomenon was also observed after the injection of LAD-loaded
(apoE) liposomes in control rats (results not shown).
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Fig. 6.
Clearance of LAD-loaded liposomes
17EE-pretreated rats showing effects of apoE. LAD liposomes (A) or
apoE-exposing LAD liposomes (B) were injected i.v. into
17EE-pretreated rats at a dose of approximately 5 nmol of LAD and 4 mg of phospholipid. Liposomes were labeled with [3H]LAD
() and [14C]CO (). At indicated times, blood
samples were taken, and radioactivity in serum was determined. Results
are expressed as percentage of injected dose and are mean ± S.E.M. of four experiments.
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The liver uptake of LAD-loaded (apoE) liposomes, measured at 10 min after injection into 17EE-treated rats or untreated control rats, is presented in Fig. 7. In the
control rats, the uptake of both the liposomal
[14C]CO label and
[3H]LAD was low, and the presence of apoE did
not significantly affect liver uptake. In the 17EE-pretreated rats,
the uptake of both labels was very similar to the uptake in control
rats when liposomes lacking apoE were injected (approximately 6% of the dose). However, the hepatic uptake of apoE-enriched LAD-loaded liposomes in these rats was much higher: 30 ± 4% of the injected [14C]CO label and 18 ± 1.4% of the
injected [3H]LAD were recovered in the liver
(mean ± S.E.M., n = 4). Thus, a substantial
amount of LAD was taken up by the liver together with its liposomal
carrier. These results further indicate that the addition of apoE is
crucial for the recognition of the drug-carrier complex by the LDL
receptor.
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Fig. 7.
Liver uptake of LAD and apoE liposomes in control and
17EE-pretreated rats 10 min after injection. LAD-loaded apoE
liposomes and LAD-loaded liposomes lacking apoE were injected i.v. in
17EE-pretreated rats and untreated control rats (dose, 5 nmol of LAD
and 4 mg of phospholipid). Liposomes were labeled with
[3H]LAD () and [14C]CO ().
Radioactivity in liver was determined at 10 min after injection.
Results are expressed as percentage of injected dose and are mean ± S.E.M. of four experiments. Differences with respect to control rats
injected with apoE-containing LAD liposomes were tested for
significance by ANOVA followed by Dunnett's multiple comparisons test:
*P < .01). Data represent mean ± S.E.M. of
three or four experiments for control and 17EE-pretreated rats,
respectively.
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Serum Decay of LAD-Loaded apoE Liposomes in Wild-Type Mice and Mice
Lacking LDL Receptor.
The role of the LDL receptor in the
clearance of LAD-loaded apoE liposomes was also studied by comparing
the serum decay of ([14C]CO/[3H]LAD-labeled)
LAD-loaded apoE liposomes in homozygote LDL receptor-deficient mice and
in wild-type mice (Fig. 8). A remarkable
difference in the clearance of the drug-carrier complex in wild-type
and LDL receptor deficient-mice was observed. After 4 h, 22 ± 5% and 45 ± 6% of the injected amounts of liposomal label
were recovered in the serum of wild-type and LDL receptor-deficient
mice, respectively. At this time, the recoveries of
[3H]LAD in the serum were 16 ± 3% and
31 ± 5%, respectively. Like in the rats, an instantaneous loss
of LAD of approximately 15% from the liposomes was observed in both
strains of mice. After 30 min, the
3H/14C ratios in the serum
of wild-type and LDL receptor-deficient mice were 0.80 ± 0.02 and
0.84 ± 0.05 (mean ± S.D., n = 4; ratios at
time of injection set at 1), respectively. After 4 h of
circulation, the ratios decreased to 0.73 ± 0.09 and 0.68 ± 0.07, respectively. This indicates that at 4 h after injection of
the LAD-loaded apoE liposomes, the circulating liposomes still carry
70% of the initially incorporated drug. The very similar rate of
clearance of LAD and apoE liposomes that was found after the rapid
initial loss of LAD from the liposomes suggests that LAD and the apoE
liposomes are simultaneously cleared by the same mechanism. The
difference in the clearance of the LAD-carrier complex in the two mice
strains indicates that the LDL receptor is involved in the clearance of LAD-loaded apoE liposomes.
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Fig. 8.
Clearance of LAD-loaded apoE liposomes in LDL
receptor-deficient and wild-type mice. LDL receptor-deficient mice (A)
and wild-type mice (B) were injected with LAD-loaded apoE liposomes
(1.5 mg of phospholipid) labeled with [3H]LAD () and
[14C]CO (). At indicated times, blood samples were
collected, and radioactivity in serum was determined. Results are
expressed as percentage of injected dose. Values are mean ± S.E.M. of four different experiments.
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Discussion |
The feasibility of chemotherapy in which native LDL loaded
with cytotoxic drugs is used to eradicate tumors expressing the LDL
receptor is strongly hampered by the limited availability of native LDL
and artifacts induced by incorporation of drugs. The apoE-enriched
liposomes that we recently described (Rensen et al., 1997a) and mimic
LDL in physiological behavior form a novel system that allows on a
pharmaceutical scale preparation of drug-carrier complexes for LDL
receptor-mediated targeting of antineoplastic drugs to tumors. In this
study, we incorporated a lipophilic derivative of daunorubicin (LAD)
into these liposomes and demonstrate that the prodrug remains in vivo
associated with the liposomes and that the drug-carrier complex is
cleared by LDL receptors.
Unmodified daunorubicin and doxorubicin can be incapsulated into
liposomes. The rationale for using a lipophilic prodrug in the present
study is that the small (29 nm) liposomal particles have a phospholipid
bilayer that composes 60% of their total volume. Because of the large
volume of liposomal phospholipid, a high drug load may be expected with
lipophilic drugs that are incorporated into the phospholipid bilayer.
The small size of our liposomes is dictated by the use of apoE as a
specific LDL receptor-homing marker. Larger (>50 nm) apoE-exposing
lipid particles lose their LDL receptor specificity and are mainly
cleared by the apoE remnant receptor (Rensen et al. 1997b).
The incorporation of the lipophilic daunorubicin derivative into the
liposomes was easily achieved by cosonication of the drug with the
lipid components. Up to 11 molecules of drug could be incorporated per
particle without changing the size and lipid composition of the
liposomes. The stoichiometry of the incorporation suggests that
incorporation of higher amounts of LAD may be feasible. The
incorporation efficiency (44% at the highest drug load) is not
optimal. However, the suboptimal incorporation efficiency does not
necessarily constitute a problem for pharmaceutical application because
nonincorporated LAD can easily be regained. Nevertheless, in future
experiments we will develop procedures to optimize the incorporation
efficiency of LAD. The presence of LAD in the liposomes (up to 44 µg
of LAD/mg of CO) did not affect the ability of the liposomes to acquire
apoE. The liposomes acquired maximally 9 molecules of apoE per
liposome, which should be sufficient to accomplish optimal binding to
the LDL receptor. Pitas et al. (1980) demonstrated that
dimyristoyl-phosphatidylcholine vesicles exposing minimally 4 apoE
molecules per vesicle displayed a maximal binding affinity for the LDL
receptor. Agarose gel electrophoresis of the drug-carrier complex
showed that LAD-loaded apoE liposomes have a low negative surface
charge. This is important to circumvent in vivo uptake by other uptake
mechanisms than the LDL receptor-mediated pathway, such as uptake by
the reticuloendothelial system (Gabizon and Papahadjopoulos, 1988;
Patel, 1992).
The lipophilic derivatization of daunorubicin and the subsequent
incorporation of the prodrug into apoE liposomes dramatically altered
the biological fate of the drug. Free daunorubicin was cleared from the
circulation with a half-life of less than 1 min, whereas the
liposome-associated prodrug displayed a much longer half-life.
Furthermore, the disposition of free daunorubicin in the heart (related
to the dose-limiting cardiotoxicity) and other organs was not seen with
the lipophilic daunorubicin derivative. The substantially reduced
disposition of LAD in the heart and other extrahepatic organs probably
allows the administration of higher doses of LAD than daunorubicin,
which may lead to an increased therapeutic effect.
The firm association of a drug with its carrier is a prerequisite for
the successful targeting of drugs by a drug-carrier system in vivo.
Data from our recent study suggest that LAD can form a stable
drug-carrier complex (Versluis et al., 1998). In this study, LAD and
liposomes were recovered in the same fraction after density-gradient
centrifugation of a serum sample taken 30 min after injection of
LAD-loaded liposomes into a rat. This indicates that LAD does not
significantly redistribute to serum (lipo)proteins. However, we also
observed in the present study in both mice and rats a loss of
approximately 15% of LAD from the liposomes shortly after injection.
This probably represents a nonsufficiently tight associated prodrug.
The remaining part was cleared at a rate similar to that of the
liposomes, which points to a stable anchoring of LAD into the liposomes.
The role of the LDL receptor in the clearance of LAD-loaded apoE
liposomes was clearly demonstrated in 17EE-pretreated rats. These
rats have an elevated expression of LDL receptor in the liver and are
widely used as a model to investigate LDL receptor-mediated uptake of
circulating ligands (Chao et al., 1979; Harkes and van Berkel, 1983).
The LDL receptor expression in control rats is very low (Nagelkerke et
al., 1986). In the pretreated rats, the LAD-loaded apoE liposomes were
more rapidly cleared from the circulation than in control rats. The
hepatic uptake of the liposomal label was enhanced 5-fold, and 30 min
after injection, the liver contained approximately 30% of the injected
dose. The liver uptake of these apoE liposomes therefore was comparable
to that reported for [3H]CO-labeled LDL in
17EE-pretreated rats (approximately 25% of the dose at 30 min after
injection; Pieters et al., 1991). These results clearly indicate the
involvement of the LDL receptor. The uptake of the liposomes lacking
apoE in the 17EE-pretreated rats was very similar to the uptake of
(apoE) liposomes in untreated rats. This finding indicates that the
presence of apoE on the liposomes is essential for the LDL receptor
recognition. Furthermore, the relatively rapid tissue uptake of the
apoE liposomes indicates that the 29-nm-diameter particles can readily
extravasate and become available for receptor-mediated uptake. The
hepatic uptake of apo E liposomes in the control rats was low, which
indicates that the presence of apoE on the particles does not induce
liver uptake by other mechanisms (e.g., the remnant receptor).
Further evidence for the involvement of the LDL receptor in the
clearance of LAD-loaded apoE liposomes was obtained by comparing the
clearance of the particles in LDL receptor-deficient mice with that in
wild-type mice. In LDL receptor-deficient mice, the half-lives of both
the prodrug and the liposomal label were approximately 2 times higher
than that in wild-type mice. This finding agrees well with the earlier
findings of Ishibashi et al. (1993), who reported that compared with
wild-type mice, the half-life of LDL in LDL receptor-deficient mice was
increased 2.5-fold. The difference in clearance between the two mice
strains is specifically LDL receptor mediated. An initial rapid loss of
approximately 15% of the liposome-associated LAD was also observed in
mice injected with the LAD-loaded apoE liposomes. Subsequently, the
drug and the carrier were cleared very similarly. At 4 h after
injection of the LAD-loaded apoE liposomes, the circulating particles
still carried 70% of the initially incorporated drug.
In conclusion, in the present study, we show that the lipophilic
daunorubicin prodrug LAD can be stably incorporated into apoE-enriched
liposomes. Compared with free daunorubicin, the liposome-associated LAD
showed a remarkably increased circulation half-life and a substantially
reduced tissue disposition, which may reduce the dose-limiting side
effects of daunorubicin. LAD was firmly anchored into the liposomes,
and the biological fate of the drug was largely determined by its
carrier. The LDL receptor plays an important role in the clearance of
the LAD-loaded apoE liposomes, as was clearly demonstrated in
17EE-pretreated rats and in LDL receptor-deficient mice. The
presence of apoE appeared to be essential for the LDL receptor-mediated
processing of the drug-carrier complex. Because the affinity of apoE
liposomes for the LDL receptor is 15-fold higher than that of LDL
(Rensen et al., 1997a), the drug-loaded apoE liposomes probably have a
competitive advantage over circulating endogenous LDL for binding the
LDL receptor. The present encouraging data indicate that a novel
therapy based on LDL receptor-mediated selective delivery of
antineoplastic drugs to tumor cells is feasible.
apoB, apolipoprotein B;
apoE, apolipoprotein E;
CO, cholesterol oleate;
DNR, daunorubicin;
17EE, 17-ethinyl
estradiol;
EYPC, egg yolk phosphatidylcholine;
LAD, conjugate of
3-O-oleoyl-5-cholanic acid and
alanine-leucine-alanine-leucine-daunorubicin;
LDL, low-density
lipoprotein;
PBS, 10 mM sodium phosphate buffer (pH 7.4), containing
0.15 M NaCl.
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Abstract
Introduction
