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Tumor cell targeting of liposome-entrapped drugs with
phospholipid-anchored folic acidPEG conjugates
Alberto Gabizon*, Hilary Shmeeda, Aviva T. Horowitz, Samuel Zalipsky
Oncology Department, Shaare Zedek Medical Center, Hebrew University School of Medicine, Jerusalem, Israel
ALZA Corporation, Mountain View, CA, USA
Received 6 October 2003; accepted 5 January 2004
Abstract
Targeting of liposomes with phospholipid-anchored folate conjugates is an attractive approach to deliver chemotherapeutic
agents to folate receptor (FR) expressing tumors. The use of polyethylene glycol (PEG)-coated liposomes with folate attached to
the outer end of a small fraction of phospholipid-anchored PEG molecules appears to be the most appropriate way to combine
long-circulating properties critical for liposome deposition in tumors and binding of liposomes to FR on tumor cells. Although a
number of important formulation parameters remain to be optimized, there are indications, at least in one ascitic tumor model,
that folate targeting shifts intra-tumor distribution of liposomes to the cellular compartment. In vitro, folate targeting enhances
the cytotoxicity of liposomal drugs against FR-expressing tumor cells. In vivo, the therapeutic data are still fragmentary and
appear to be formulation- and tumor model-dependent. Further studies are required to determine whether folate targeting canconfer a clear advantage in efficacy and/or toxicity to liposomal drugs.
D 2004 Elsevier B.V. All rights reserved.
Keywords: Folate; Liposome; Targeting; Chemotherapy; Murine tumor model; PEGylation
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1178
1.1. Folate-targeted liposomes (FTL) versus nontargeted liposomes (NTL) . . . . . . . . . . . . . . . . . . . . . . . 1178
1.2. FTL versus nonliposome-based folate-targeted systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11792. FR expression and tumor models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1180
3. Formulation issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
3.1. Achieving prolonged circulation time . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
3.2. Optimization of the PEG-folate conjugate concentration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1181
3.3. PEG steric interference with binding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1182
3.4. Insertion versus conventional incorporation of folatePEGlipid into liposomes . . . . . . . . . . . . . . . . . . 1183
0169-409X/$ - see front matterD 2004 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2004.01.011
* Corresponding author. Oncology Department, Shaare Zedek Medical Center, POB 3235, Jerusalem 91031, Israel. Tel.: +972-2-655-5036;
fax: +972-2-652-1431.
E-mail address:alberto@md.huji.ac.il (A. Gabizon).
www.elsevier.com/locate/addr
Advanced Drug Delivery Reviews 56 (2004) 11771192
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4. In vitro studies with FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183
4.1. Kinetics of liposome binding to tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1183
4.2. Delivery of doxorubicin encapsulated in FTL to tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 1184
4.3. In vitro cytotoxicity of doxorubicin encapsulated in FTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1185
5. Pharmacokinetics and tissue distribution studies with FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . . . . . 11866. Therapeutic effects of FTL-encapsulated drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1188
6.1. Folate-targeted PEGylated (STEALTHR) liposomal doxorubicin (FTL-Dox) . . . . . . . . . . . . . . . . . . . 1188
6.2. Folate-targeted PEGylated (STEALTHR) liposomal cisplatin (FTL-cisplatin) . . . . . . . . . . . . . . . . . . . 1189
7. Toxicity of cytotoxic drugs encapsulated in FTL . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
8. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1190
1. Introduction
The rationale for cancer targeting with folate
ligands attached to the liposome surface is based on
two layers. First, there is a common layer to all folate-
targeted systems which relates to the choice of the
tumor cell folate receptor (FR) as the target. FR
upregulation or over-expression is commonly associ-
ated with a broad variety of tumor types including
solid and hematological malignancies, and it appears
to be morefrequently observed in advanced stages of
cancer [1]. How specific and frequent is FR over-
expression in cancer cells to justify its choice as target
is discussed in other articles of this issue ofAdv. DrugDeliv. Rev.and will not be addressed here. The second
layer contains elements unique to liposomal and
perhaps other nanoparticulate drug carrier systems
and will be addressed here. The strength of the
folate-targeted liposome approach stems from concep-
tual advantages over two alternative approaches: non-
targeted liposomal systems and nonliposome-based
folate-targeted systems.
1.1. Folate-targeted liposomes (FTL) versus non-
targeted liposomes (NTL)
A schematic illustration of the folate liposome
targeting concept is presented in Fig. 1. Long-circu-
lating liposomes, such as polyethylene glycol (PEG)
coated liposomes (also known as STEALTHR lip-
osomes)[2], tend to accumulate in tumors as a result
of increased microvascular permeability and defective
lymphatic drainage, a process also referred to as the
enhanced permeability and retention (EPR) effect[3].
This is a passive and nonspecific process of liposome
extravasation that is statistically improved by the
prolonged residence time of liposomes in circulation
and repeated passages through the tumor microvascu-
lar bed[4].However, except for rare instances, tumor
cells are not directly exposed to the blood stream.
Therefore, for an intra-vascular targeting device to
access the tumor cell FR, it must first cross the
vascular endothelium and diffuse into the interstitial
fluid. Experimental data with antibody-targeted lip-
osomes and FTL indicate that liposome deposition in
tumors is similar for both targeted and nontargeted
systems[57],supporting the hypothesis that extrav-
asation is indeed the rate-limiting step of liposome
accumulation in tumors. However, once liposomeshave penetrated the tumor interstitial fluid, binding
of targeted liposomes to FR may occur thus shifting
the intra-tumor distribution from the extracellular
compartment to the tumor cell compartment, as shown
recently for a mouse ascitic tumor [7]. Binding to
tumor cells may be followed by internalization of
liposome contents via folate-receptor mediated endo-
cytosis (Fig. 1). Retrograde movement of liposomes
into the blood stream, if any, will be reduced for
liposomes with binding affinity to a tumor cell recep-
tor. Obviously, when the parameter of drug delivery isconsidered, there will always be a combination of in
situ release from an extracellular liposome depot and
intra-cellular release from internalized liposomes.
Therefore, the theoretical advantages of FTL over
NTL are related to a shift of liposome distribution to
the tumor cell compartment, delivery of liposomal
contents to an intra-cellular compartment in liposome-
associated form, and, possibly, prolonged liposome
retention in the tumor. On the negative side, the main
disadvantage of a targeted system to a cancer cell
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receptor such as FR is the difficulty of a large nano-
size assembly, such as FTL, to penetrate a solid tumor
mass, specially considering the high interstitial fluid
pressure that is often present in tumor masses of
clinically detectable size[8].
1.2. FTL versus nonliposome-based folate-targeted
systems
Liposomal systems offer an elegant drug deliveryamplification system. Each liposome vesicle carries a
drug cargo usually in the order of 103 104 molecules.
For instance, in the case of a STEALTHR liposome
formulation known as Doxil, there are between
10,000 and 15,000 doxorubicin molecules per vesicle
[9], and these may be targeted with the help of as few
ligands as 10 per vesicle, i.e. a 1001000-fold deliv-
ery amplification factor when the drug:ligand ratio is
considered. Another theoretical advantage of liposo-
mal systems is that their size far exceeds the critical
glomerular filtration threshold. Therefore, unlike low
molecular weight folate-targeted complexes, FTL do
not have access to the luminal side of kidney tubular
cells where FR is expressed, thereby sparing kidneys
of massive FR-mediated liposomal drug delivery and
subsequent toxicity[10].One of the disadvantages of
FTL vis-a-vis small drug folate conjugates is that
liposomes are bulky structures that are difficult to
internalize by nonphagocytic cells. The best charac-
terized pathway of liposome internalization, mediatedby clathrin-coated pits, often leads to sequestration of
liposome contents within the lysosome compartment.
An alternative pathway of endocytosis, known as
potocytosis, may operate for receptors associated with
cell caveolae or lipid rafts, such as FR [10], and
facilitate access to the cytosol via acidic endosomes
bypassing lysosomes. It is well established that FTL
enter cells by FR-mediated endocytosis (FRME)[11].
In addition, experimental data with folate-targeted,
pH-sensitive liposomes are consistent with liposome
Fig. 1. Schematic drawing illustrating the concept of folate targeting of liposomes to tumor cells. The blue dots represent the liposomal folate
ligands. The red dots represent the drug molecules encapsulated in the liposome water phase. The various steps involved in the targeting process
are numerically designated from 1 to 6. Steps 13 are common to nontargeted and targeted liposomes. Steps 46 are specific to FTL. (1)
Liposomes with long-circulating properties increase the number of passages through the tumor microvasculature. (2) Increased vascular
permeability in tumor tissue enables properly downsized liposomes to extravasate and reach the tumor interstitial fluid. (3) Drug is gradually
released from liposomes remaining in the interstitial fluid and enters tumor cells as free drug to exert a cytotoxic effect. (4) Other liposomes bind
to the FR expressed on the tumor cell membrane via the folate ligand. Because of the limited diffusion capacity of liposomes, binding is likely to
be limited to those tumor cells in closest vicinity to blood vessels. (5) Liposomes are internalized by tumor cells via FRME. (6) Internalized
liposomes release their drug content in the cytosol enabling the drug to exert its cytotoxic effect.
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transit through an acidic vesicle compartment[12]. A
connection between post-caveolar or post-raft endo-
somes and lysosomes is possible, since markers of the
clathrin-coated pit pathway and folate conjugates havebeenshown to co-localize in the same cell organelles
[13]. Thus, an important fraction of internalized lip-
osomes may end up in lysosomes. The cell trafficking
of liposomes following FRME needs to be better
understood, specially since intra-cellular trafficking
of small molecular weight folate conjugates may be
different from that of nanoparticles with multimeric
binding such as FTL.
2. FR expression and tumor models
A prerequisite for investigation of any targeted
system is the availability of tumor models with stable
overexpression of the target receptor. Routine cell
culture conditions expose tumor cells to high folate
concentrations so that even if a fresh tumor explant
overexpresses FR, in vitro culture may gradually
cause downregulation of FR. The standard approach
we have used to generate a FR-overexpressing cell
line is to cultivate the cells in a folate-free culture
medium. FR upregulation is a common response of
cells grown in a folate-depleted environment. Manytumor cell lines respond to folate-depleted culture
conditions with upregulation of FR. This is generally
a reversible process, i.e. when folate supplies are
restored FR is downregulated [14]. Therefore, FR-
overexpressing cell lines should be maintained in
folate-free medium. The addition of 10% nondialyzed
serum to folate-free medium results in a sub-physio-
logic concentration of folic acid (3 nM) under which
these cell lines maintain high FR expression [14].
We have studied several animal tumor models
overexpressing the FR, including mouse M109 carci-
noma and itsmultidrug-resistant cells (MDR)subline,
M109R[14], the human KBcarcinoma[15], and themouse J6456 lymphoma [16]. High FR (HiFR)-
expressing cells have been selected from these tumor
cell lines as previously described for M109 and KB
tumors[14]. A high FR-expressing J6456 subline was
similarly obtained by a single in vivo passage of
tumor cells followed by repeated in vitro passage in
a folate-free culture medium.
Baseline information on the uptake of free folic
acid and on the effect of folate-depleted diet on
receptor expression in vivo is obviously of great
importance in the testing of FTL. Since we found that
folate binding by the M109 tumor was not affected by
the diet within the short time frame required for a
tissue distribution study, experiments with this tumor
model and with the KB human carcinoma (another
well-established model of inducible and stable high
FR expression[11,13,16])proceeded with animals on
normal diet. In contrast, J6456 lymphoma quickly
downregulated FR in animals with a normal, folate-
enriched diet (Table 1). Therefore, experiments with
the J6456-HiFR should be carried out preferably in
animals maintained on a folate-depleted diet. The
results of folic acid uptake and targeted versus non-targeted liposomal uptake in the J6456-HiFR tumor,
presented in Table 1, point to a 30-fold drop in
radiolabeled folate in cells from mice fed a normal,
folate-enriched, diet, and to a 312-fold increase in
liposome uptake when FTL are compared to NTL.
The importance of using a folate-depleted diet in in
vivo experiments with folate-targeted systems has
been questioned. Clearly if tumor FR expression is
quickly downregulated under a folate-rich diet, then
Table 1Folic Acid (F.A.) and liposome uptake of J6456 and J6456-HiFR tumor cell linesa
Cells/source 3H-F.A.
fmole/106 cells
3H-CHE-NTL
pmole/106 cells
3H-CHE-FTL
pmole/106 cells
J6456 (parental line) 2F 1 215F 12 199F 23
J6456-HiFR (in vitro F.A.-depleted medium) 14,675F 1403 286F 29 3719F 340
J6456-HiFR (mice on normal diet) 186F 127 Not done Not done
J6456-HiFR (mice on F.A.-depleted diet) 5660F 931 430F 8 1470F 133
a J6456 cells (parental line) obtained from in vitro passage using standard (folate-rich) culture medium. J6456-HiFR cells were obtained
from either in vitro passage in F.A.-depleted medium or in vivo passage in mice on normal diet or F.A.-depleted diet. Cells incubated at 37 jC
for 30 min in the presence of radiolabeled F.A. (0.1 pmol/ml), and for 24 h in the presence of 3H-CHE (cholesterol hexadecyl ether) labeled NTL
or FTL (300 nmol phospholipid/ml). Results are expressed as fmol F.A. per million cells, or pmol phospholipid per million cells.
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the use of a special folate-depleted diet is necessary. In
our experience, the folate-depleted diet in specific
pathogen-free (SPF) mice exposed to chemotherapy
causes serious weight loss and is problematic for long-term therapeutic experiments lasting several weeks or
months after treatment has been completed. The
approach we have generally adopted is to put mice
on a folate-depleted diet shortly before tumor inocu-
lation and put them back on a normal diet 1 week after
the last treatment is administered. FTL have been
cleared from circulation and their interactions, if
any, with the tumor FR should be over.
3. Formulation issues
3.1. Achieving prolonged circulation time
It has been well established that prolonged circu-
lation is a prerequisite for tumor accumulation of
liposomes [17,18]. PEGylated liposomes are the best
basis for a formulation that confers a long half-life in
circulation. In addition, optimal drug retention is
critical to ensure delivery of an intact drug payload
upon reaching the target cell. For drugs encapsulated
in the water phase of liposomes, stable retention canbe achieved by using high Tm (>37 jC) phospholi-
pids and cholesterol. Therefore, the formulations we
have used are STEALTHR type and consist of fully
hydrogenated soybean PC, cholesterol, and PEG
DSPE conjugate. Vesicle size is tailored to V 100
nm by sequential extrusion to ensure that there is no
physical hindrance to extravasation and internalization
[9].
3.2. Optimization of the PEG-folate conjugate
concentration
In a liposome coated with PEG polymers, a rea-
sonable strategy is to present the folate ligand at the
outer end of the PEG chain. Folate has been coupled
to amino-PEGDSPE [19] mainly through the gam-
ma-carboxyl to PEGDSPEas seen inFig. 2 and as
described before[11,14,20].The affinity of the result-
Fig. 2. Structures of various lipopolymers discussed in this review: mPEG DSPE, Folate PEG DSPE, and the disulfide-linked cleavable
lipopolymer, mPEGDTPDSPE. Note that approximately 80% of the folate moieties are linked as shown, through the gamm a carboxyl, while
the remaining 20% are alpha-carboxyl linked analogs, as determined by HPLC assay of carboxypeptidase G-treated conjugate [Ref. 14].Degree
of polymerization, n = 45 for derivatives of mPEG of molecular weight 2000 Da; n = 75 for derivatives of PEG of 3350 Da.
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ing conjugate to FR is about 10-fold lower than
unconjugated FA [14]. However, since liposome
binding is multivalent, i.e. mediated through several
ligands, its overall affinity for a target cell is theproduct of the individual affinities of the ligands
participating in binding. This is why the affinity of
FTL for FR-expressing cells is much higher, and
nearly a 1000-fold greater concentration of soluble
folic acid is needed to compete effectively with FTL
for binding to FR[14,21].
Because of the flexibility of the PEG chain, a small
number of folate residues on the liposome surface
may be sufficient to enable liposome binding to the
cell FR. In STEALTHR liposomes, the molar ratio of
PEGDSPE is approximately 5%, probably far more
than the amount of ligand we would need on the
liposome surface to secure an optimal chance of
binding to FR. Based on earl ier work from the
laboratory of Low and colleagues [11,20] and our
subsequent studies [14,21], it appears that a molar
fraction of 0.2 0.5% folate PEG DSPE is sufficient
for effective interaction with the cell membrane FR.
The rest of liposomal PEG would be in the form of the
standard methoxy(m)PEG DSPE conjugate. Howev-
er, a recent study [22] testing a wide range of PEG-
folate concentrations indicates that optimal binding is
obtained with low levels of 0.03%, about 10-fold lessthan those commonly used in previous studies. The
authors hypothesize that, at high surface density, a
folatefolateinteraction prevents folate binding to the
receptor[22].
3.3. PEG steric interference with binding
In our early studies, we observed that mPEG
(2000)DSPE significantly interferes with the bind-
ing and uptake of liposomes targeted with 0.5%
folatePEG(2000)DSPE [14]. Therefore, we in-creased the PEG length in the folate conjugate to
MW 3350, a change that resulted in a major improve-
ment of the targeting effect. Even then, interference
with binding to FR is not entirely overcome (as shown
below in Fig. 3A). One option, not yet tested, is to
extend further the PEG length of the folate conjugate.
Excluding PEGDSPE from the liposomes adversely
affects their in vivo circulation time (as shown below
in Fig. 8) and is, therefore, not an option. One
alternative strategy to avoid the interference of PEG
coating on binding and uptake of FTL while main-
taining its shielding effect on circulating liposomes is
to design a cleavable PEG lipid [23,24]. This has
been done using a conjugate with a thiolytically
cleavable disulfide linked mPEGdithiodipropionate
(DTP)DSPE. As illustrated in Fig. 2, this lipopol-
ymer contains DTP as a linking moiety between the
PEG and lipid components [23].
Ideally, cleavable PEG should be sufficiently sta-
ble in plasma. Gradual cleavage and release of PEG
Fig. 3. (A) Effect of substituting mPEG DSPE (PEG) with mPEG
DTP DSPE (thiolytically cleavable lipopolymer, PEG-SS) onliposome binding to M109R-HiFR tumor cells. In vitro incubation
of 3H-CHE labeled liposome preparations at phospholipid concen-
tration of 300 nmol/ml in the absence or presence of 1 mM cysteine
for 24 h. Test conditionswere as previously reported for liposome
cell binding assays [14]. (B) Effect of substituting mPEGDSPE
(PEG) with mPEG DTP DSPE (thiolytically cleavable lipopol-
ymer, PEG-SS) on plasma clearance of FTL. 3H-CHE labeled preps
(2 Amol phospholipid per mouse) were injected i.v. into BALB/c
mice (n = 4 8 per group), and animals were sacrificed after 24 h.
Average of two experiments. All differences are significant by one-
way Anova with Bonferroni post-test atP< 0.001 level, except for
FTL PEG-SS vs. FTL w/o PEG, which is not significant.
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will take place in the extracellular fluid or upon
contact with cell membrane components. This will
enable avid binding and internalization of FTL by
FR-expressing cells.Fig. 3A shows the results of anin vitro experiment testing binding of FTL coated
with cleavable mPEGDTPDSPE to FR-expressing
cells in the absence or presence of cysteine. There is a
clear enhancement of liposome binding to cells in the
presence of cysteine when mPEG DTP DSPE is
included in the FTL formulation. In contrast, control
FTL prepared with standard, noncleavable mPEG
DSPE show poor binding and no effect of cysteine.
However, in vivo, FTL prepared with mPEGDTP
DSPE were cleared much faster than control FTL
(Fig. 3B)indicating that this particular lipopolymer is
too labile and is not entirely stable in circulation. New
cleavable lipopolymers with increased stability were
recently prepared [25] and await testing. Although
this approach is a promising one, it still requires
optimization.
3.4. Insertion versus conventional incorporation of
folate PEG lipid into liposomes
Another important aspect of formulation is wheth-
er the folate ligand can be inserted into preformed
liposomes as opposed to conventional incorporationduring liposome preparation. The latter procedure
requires the ligand-PEGlipid to be co-mixed with
other liposome components during the initial step of
liposome preparation. Recently it was demonstrated
that incubation of pure ligand-PEGlipids with lip-
osomes results in their clean insertion into the outer
leaflet of the liposomal bilayer[26].This has several
advantages from the pharmaceutical point of view
(reviewed by Zalipsky et al.Ref. [27]): (i) FTL could
be prepared without modifying the production line of
a commercial liposomal formulation; (ii) folate target-ing can be applied to a variety of liposomal drug
formulations; (iii) folate ligand will be inserted only
in the outer leaflet of the bilayer relevant for targeting
and thus will not be buried in the liposomal interior,
and will not be able to interact with the encapsulated
drug; (iv) provided that pure ligand-PEG lipid is
used, the insertion into preformed liposomes is the
only method of preparation of ligand-bearing PEG-
liposomes that completely avoids incorporation of
any extraneous residues [27]. Considering that fo-
latePEGDSPE [14] is a well-characterized pure
conjugate, all four advantages of the insertion ap-
proach can potentially apply. Ligand insertion has
been reported for antibody targeted liposomes withsatisfactory yields [28]. We have tested the folate
PEG DSPE insertion method with two PEGylated
liposomal formulations: DOXILR (STEALTHR lipo-
somal doxorubicin) and SPI-77 (STEALTHR liposo-
mal cisplatin), and obtained a high rate of ligand
association (range 6294%) with liposomes, resulting
in a final concentration of 0.35 0.55% of folate
PEGDSPE in mol% of total phospholipid [29]. A
recent report has also demonstrated an efficient yield
for post-insertion of folatePEG lipophilic conjugates
into preformed liposomes loaded with doxorubicin
[30].
4. In vitro studies with FTL-encapsulated drugs
In vitro observations may give a false assessment
of a targeting strategy using liposomes or other
carriers due to the complexity of the in vivo setting
and the enormous drug pharmacokinetic changes
caused by the use of particulate drug carriers [9].
Nonetheless, in vitro studies are still important in
assessing the ability of targeted liposomes to interactwith FR-expressing cells and to deliver bioavailable
drug into the relevant cellular compartments.
4.1. Kinetics of liposome binding to tumor cells
Fig. 4depicts the effects of liposome concentration
and incubation time on the in vitro binding of FTL
labeled with 3H-cholesterol hexadecyl ether (3H-CHE)
to M109-HiFR cells. Although an increase in liposome
uptake with time of incubation is clearly seen for all
three lipid concentrations tested (30, 100, and 300nmol/ml), the steepest slope, a 3-fold increase within
20 h, was obtained with the lowest concentration. In
addition, at any given incubation time, the highest
relative uptake of liposomes was observed with the
lowest concentration. These results indicate that satu-
ration of liposome uptake begins at 100 nmol/ml and
possibly at lower concentrations. Furthermore, the
kinetics are consistent with recycling of receptors,
enabling a gradual, albeit slower rise of liposome
uptake with time.
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4.2. Delivery of doxorubicin encapsulated in FTL to
tumor cells
Our studies on in vitro delivery of doxorubicin byFTL are described in detail in Goren et al. [21].FTL
loaded with doxorubicin are taken up by FR-bearing
cells and the drug is swiftly transferred from the intra-
cellular compartment to the nucleus, indicating that a
significant fraction of the drug is released from lip-
osomes in the cytosol and redistributes to the nucleus
due to its known affinity for DNA. Although there is
strong evidence from confocal microscopy that a
substantial fraction of the drug enters cells in liposo-
mal form, it cannot be ruled out that part of the drug
gaining access to the nucleus may originate from
destabilized membrane-bound liposomes. For an ex-
ample of the nuclear localization of doxorubicin (Dox)
in KB tumor cells after exposure to FTL-Dox, seeFig.5. The doxorubicin transfer to the nucleus is clearly
drug-specific since, when similar FTL were loaded
with rhodamine, the fluorescent label remained in the
cytoplasm. Perhaps, the most attractive feature of
doxorubicin-loaded FTL was the ability to deliver
doxorubicin to MDR cells as effectively as to the
parental, doxorubicin-sensitive cells. As seen in Fig.
6, resistant cells accumulated much less drug than
sensitive cells when exposed to free Dox. In contrast,
a similar level of drug uptake was observed in both
types of cells when exposed to FTL-Dox. The in vitro
uptake of NTL-Dox was negligible. This and other
studies with FTL [20,31,32] support the hypothesis
that FR-mediated drug delivery is an effective ap-
proach to deliver anthracyclines to tumor cells. Fur-
thermore, the bypass of the P-glycoprotein (Pgp)
efflux pump suggests a potential role of FTL in
overcoming drug resistance[21].
Fig. 5. Confocal fluorescence microscope picture of KB-HiFR
tumor cells after 2-h in vitro exposure to 10 AM FTL-Dox.
Doxorubicin fluorescence (orange) is readily recognized in the
nucleus sparing the nucleolus. A rim of membrane-bound
fluorescent liposomes is also seen. Test conditions were as
previously reported for confocal microscope observations of
liposomal doxorubicin cell uptake[21].
Fig. 4. Kinetics of tumor cell liposome uptake in vitro: M109-HiFR
cells were incubated in the presence of 3H-CHE labeled FTL. Test
conditions were as previously reported for liposome cell binding
assays[14].(A) Effect of phospholipid concentration. (B) Effect of
incubation time.
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4.3. In vitro cytotoxicity of doxorubicin encapsulated
in FTL
As shown in a number of studies[20,31,32], drug
delivered by FTL is always more cytotoxic than drug
delivered by NTL. When FTL-Dox is compared to
free Dox, the results are variable with small differ-
ences in both directions. This is likely due to varia-
tions in drug bioavailability that may result fromliposome formulation, drug loading method, satura-
tion of liposome uptake, and cell-dependent liposome
uptake pathways. In our studies [21], we found a
slight advantage for free drug with lower IC50 than
that for FTL-Dox. This finding was difficult to rec-
oncile with the higher drug levels measured in cells
exposed to FTL-Dox as compared to cells exposed to
free Dox, suggesting that part of the drug delivered by
FTL is not internalized and/or remains sequestered on
the cell surface or within intra-cellular compartments.
To test the full cytotoxic potential of FTL-Dox in a
longer assay exposing tumor cells to the in vivo
milieu, we performed a Winn assay. In this assay,
the cells are exposed to the drug in various forms in
vitro. After a short exposure (12 h), the cells are
washed to remove any noncell-associated material,
counted, and inoculated in the animal. The number of
animals developing tumors and the timing of tumor
development provide an indication of the cytotoxic
activity exerted by the treatment upon direct in vitro
Fig. 6. Enhanced Dox delivery via FTL to M109R-HiFR (MDR+)
tumor cells. Tumor cells were exposed to free or liposomal drug as
FTL or NTL for 1 h, at a Dox concentration of 10 AM. Drug notassociated with cells was washed out by centrifugation. Cell-
associated drug was extracted and measured as previously
described[21].
Fig. 7. Winn assay to examine in vivo the cytotoxic effect of FTL-Dox after in vitro exposure of M109R-HiFR cells. M109R-HiFR cells
incubated for 2 h in the presence of free Dox, FTL-Dox (w/o mPEG), or NTL-Dox (DOXILR), and then washed to remove any nonassociated
drug/liposome. Mice were inoculated with 106 cells in the footpad. Curves show the probability of preventing tumor development. FTL-Dox
was significantly more effective than free Dox or NTL-Dox. Untreated vs. Free Dox P= 0.0075; Free Dox vs. NTL-DoxP= 0.0313; Free Dox
vs. FTL-Dox, P= 0.0250; Doxil vs. FTL-DoxP< 0.0001 (log rank test).
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exposure to tumorcells. As seen in one representative
experiment (Fig. 7), FTL-Dox was significantly more
effective than free drug, and certainly more than the
nontargeted formulation, in agreement with the invitro drug delivery data shown in Fig. 6. Thus, we
conclude that FTL is capable of delivering Dox to
tumor cells at high levels with potential biologic
activity superior to free drug and NTL.
5. Pharmacokinetics and tissue distribution studies
with FTL-encapsulated drugs
As mentioned above, PEG coating interferes with
the uptake of FTL. However, PEG coating is critical
for the long circulation time of liposomes, and this is in
turn a prerequisite for liposome accumulation in
tumors, as can be seen when the plasma clearance of
PEGylated FTL is compared to that ofnonPEGylated
FTL after i.v. injection in mice Fig. 8. Therefore, in
most of our in vivo experiments we use FTL formu-
lated with mPEG DSPE at approximately 4.7% molar
ratio and folatePEGDSPE at approximately 0.3%
molar ratio. The PEG tether used for the folate conju-
gate is longer (3.35 K) than that of mPEGDSPE (2
K) in an effort to reduce the interference of the latter
with FR-mediated cell uptake [14]. The pharmacoki-netics of FTL in rats is shown inFig. 9.In comparison
to NTL of similar composition, FTL showed a faster
clearance despite the fact that both formulations were
PEGylated. The accelerated clearance of FTL may be
the result of direct liposome uptake by the liver FR.Alternatively, binding of the plasma folate binding
protein, a soluble form of FR, to circulating liposomes
could result in opsonization and liposome tagging for
removal by the reticulo-endothelial system by nonspe-
cific mechanisms. There was a slight, additional ac-
celeration of clearance of FTL when rats were put on
folate-deficient diet, owing to higher uptake in liver
and particularly in spleen (Fig. 9 inset), suggesting
upregulation of FR. Clearly, the most relevant biodis-
tribution data are those addressing the comparative fate
of FTL and NTL in tumor-bearing mice [7]. Fig. 10
depicts the results of such a biodistribution experiment
comparing FTL with NTL in tumor (M109-HiFR)-
bearing mice. The main conclusions of our recently
published biodistribution studies[7] are:
i) FTL retain the folate ligand in vivo, even after
prolonged circulation and extravasation into
malignant ascitic fluid.
ii) Liver uptake of FTL is greater and faster in
comparison to NTL, resulting in lower plasma
levels of the former(Fig. 10).
iii) Tumor levels of FTL-injected mice in mouseM109 and human KB models are not significant-
ly different from those of NTL-injected mice
(Fig. 10),although kinetically there is a trend for
greater FTL deposition in the tumor at early time
points (6 h) and greater NTL deposition at late
time points (4872 h).
iv) Liver uptake of FTL is significantly reduced by
concomitant injection of a large dose of free folic
acid. However, tumor levels of FTL remain
unaffected by such a co-dose of folic acid
suggesting that liposome accumulation in tumorsis dictated by liposome extravasation rate rather
than by binding to FR.
v) In an ascitic tumor model that enables discrimina-
tion between the tumor cell compartment and the
extracellular fluid, tumor cell-associated liposome
levels were significantly greater for FTL-injected
mice than for NTL-injected mice, indicating that
folate targeting shifts liposome distribution from
the tumor extracellular space towards association
with FR-expressing tumor cells.
Fig. 8. Plasma and tumor levels of 3H-CHE labeled liposomes 24
h after i.v. injection of 2 Amol phospholipid per mouse in M109-FR
bearing mice. Higher plasma and tumor levels were observed after
injection of PEGylated FTL (FTL with PEG) as compared to
nonPEGylatedFTL (FTL w/oPEG).Differencesin plasmaand tumor
levels between FTL with PEG and FTL w/o PEG were statistically
significant,P= 0.0021 andP= 0.0114 (t-test), respectively.
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Fig. 10. Tissue distribution of NTL and FTL (2 Amol per mouse) 48 h after i.v. injection in M109-HiFR tumor-bearing BALB/c female mice.
There were five mice per group, and two subcutaneous tumor implants per mouse. Liposomes were radiolabeled with 3H-CHE. Plasma, spleen
and kidney levels of NTL were significantly higher than those of FTL atPlevels of 0.0001, 0.0144, and 0.0017, respectively, while liver levels
of FTL were significantly higher than those of NTL atP< 0.0001. Tumor and skin levels were not significantly different.
Fig. 9. Pharmacokinetics of FTL in rats fed normal or folate-deficient diet. Data were obtained from the mean of two experiments, with four rats
(Simonsen Albino, Gilroy, CA) per group in each experiment. Liposomes radiolabeled with a 67Ga-deferoxamine complex encapsulated in the
liposome water phase were prepared as previously described and injected into the rat tail vein [39]at a dose of 68 Amol per rat. Figure inset
shows tissue levels 24 h after injection.
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The in vivo tumor uptake of FTL loaded with
boron-containing compounds has also been investi-
gated in the M109 and KB FR-expressing tumor
models by Lee and colleagues. Their results showsimilar [6] or slightly greater [33] levels for FTL as
compared to NTL peaking at 24 h after injection.
6. Therapeutic effects of FTL-encapsulated drugs
6.1. Folate-targeted PEGylated (STEALTHR) liposo-
mal doxorubicin (FTL-Dox)
We have examined the activity of FTL-Dox in three
tumor models. Initially, we tested the M109-HiFR
tumor. NTL-Dox (DOXILR) and FTL-Dox were both
highly and equally effective against this tumor achiev-
ing a high percentage of cures and a major improve-
ment in activity over free Dox (unpublished data). The
second model tested was the M109R-HiFR tumor. This
tumor has an MDR phenotype conferring resistance to
doxorubicin. Given our in vitro data indicating that
folate-mediated drug delivery can bypass the MDR
efflux pump, and enhance drug uptake, we reasoned
that FTL-Dox will be more active than free drug and
NTL-Dox in vivo. However, here again, FTL-Dox and
NTL-Dox were equally active and both were moreactive than free drug (Fig. 11). These experiments were
done in mice under a normal, folate-enriched diet. In a
third model, J6456-HiFR cells which quickly down-
regulate the FR in animals under normal diet, were
tested with FTL-Dox in this under a folate-depleted diet
(Fig. 12). The use of folate-depleted diet was compli-
cated by the increased sensitivity of these mice to toxic
effects of chemotherapy. In fact, NTL-Dox (DOXILR)
was highly toxic in animals under a folate-depleted diet
(100% deaths). FTL-Dox was toxic to a small fraction
(30%) of the animals while retaining a strong antitumor
activity. The reason for this difference is still unclear
although it is conceivable that the small amount of
folate present in conjugate of the FTL-Dox formulation
can rescue the animals from lethal toxicity. At any rate,
the toxicity issue precludes a net assessment of a
therapeutic advantage of FTL-Dox.
A recently published study on the therapeutic
efficacy of FTL-Dox of similar composition to
Fig. 11. Therapeutic test of FTL-Dox in mice inoculated with M109R-HiFR (MDR+ tumor). 10 6 cells were implanted into the footpad of
BALB/c male mice. Formulations were injected i.v. on days 9, 17, 35, and 42 at a dose of 8 mg/kg Dox (total 32 mg/kg). There were 10 mice
per group (free Dox, NTL-Dox, FTL-Dox). Curves show the probability of tumor growth control and survival. Free Dox vs. NTL-Dox or FTL-
Dox,P< 0.0001; NTL-Dox vs. FTL-Dox, not significant (log rank test).
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STEALTHR points to significantly greater tumor
inhibition as compared to NTL-Dox in the KBcarcinoma model growing in nude mice fed a fo-
late-free diet [34]. However, there are two method-
ological issues requiring cautious interpretation: the
drugs used in that study were administered i.p.
instead of i.v., a factor that may distort the pharma-
cokinetics of liposomal vehicles, and the doxorubicin
dose (10 mg/kg 6 injections) is far above the
mouse LD50 (reviewed in [35]).
Another published study on the therapeutic effica-
cy of FTL-Dox deals with upregulation of FR-h
expression
1
in acute myelogenous leukemia with all-trans retinoic acid to render these cells more sensitive
to the targeted agent [36]. In these mouse ascites
leukemia models, FTL-Dox was more efficacious than
NTL-Dox using, as above, the i.p. treatment route.
6.2. Folate-targeted PEGylated (STEALTHR) liposo-
mal cisplatin (FTL-cisplatin)
A formulation of FTL-cisplatin was prepared in
our laboratory by post-insertion of folate PEG
DSPE into a PEGylated liposomal cisplatin formu-
lation (SPI-77, provided by ALZA) and tested in
early clinical studies [37]. SPI-77 is definitely less
toxic than free cisplatin, but, at the same time, it
appears to be significantly less active in several
tumor models [38] thus rendering its therapeutic
potential of limited value. Preliminary results have
been presented[29]indicating that the folate-targetedpreparation is more effective than the nontargeted
one, yet not more effective than free cisplatin in the
M109-HiFR tumor model. Further studies with FTL-
cisplatin are ongoing.
There are marked differences between the FTL-
cisplatin and FTL-Dox formulations: in the former,
the drug-to-lipid ratio is f 5-fold lower, and the drug
release rate is much slower [38]. From the data
gathered on these two formulations in folate-targeted
form, it is evident that properties of the basic formu-
1 FR-h, a receptor of lower affinity as compared to FR-a, is
often expressed in CD34+ bone marrow cells in inactive form and in
some leukemias in active form. In most tumors and epithelial
tissues, dealt with in this review, FR-a is expressed.
Fig. 12. Therapeutic test of FTL in mice inoculated with J6456-HiFR and fed a folate-deficient diet. BALB/c female mice were inoculated i.p.
with 106 J6456-FR lymphoma cells. Seven days later, mice were treated i.v. with 10 mg/kg, NTL-Dox (DOXIL), or FTL-Dox. Survival was
recorded and analyzed by log rank test. Mice were fed a folate-deficient diet from 1 week before tumor inoculation till 1 week after treatment.
All mice treated with NTL-Dox died of toxicity. Only 3/10 mice treated with FTL-Dox died of toxicity. The difference between NTL-Dox and
FTL-Dox is significant by log rank test atP= 0.0032.
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lation have important implications on pharmacologic
performance and that conclusions cannot be extrapo-
lated from one formulation to another.
7. Toxicity of cytotoxic drugs encapsulated in FTL
Except for strictly phase-specific drugs, liposome
encapsulation generally reduces toxicity of cytotoxic
drugs, such as doxorubicin and cisplatin[35].Limited
information is available on the toxicity of these drugs
when delivered by FTL. The presence of FR in normal
tissues such as liver and kidney may raise the concern
of toxicity to these tissues when liposomal drugs are
targeted with folate. As seen in the previous section,
our experience from therapeutic trials with Doxil
suggests that FTL-Dox is tolerated at least as well
as NTL-Dox. Similar results have been obtained when
FTL-cisplatin is compared to SPI-77, both prepara-
tions being several-fold less toxic than cisplatin [29].
Other published therapeutic studies with FTL and
doxorubicin have not reported on any unexpected
toxicities. The results of tissue distribution studies
suggest that enhanced toxicity to kidney is unlikely
since FTL show actually a slightly decreased uptake
by kidney as compared to NTL [7]. This is not a
surprising finding given the fact that the FR of kidneytubular cells is located in the luminal (urinary) side
which can only be accessed by molecules undergoing
glomerular filtration[10], meaning that this compart-
ment is clearly inaccessible to intact liposomes. Thus,
although the data are still preliminary, the concerns
that folate targeting may worsen liposomal drug
toxicity appear to be unfounded.
8. Concluding remarks
Although the use of liposomes as passive devices
for drug delivery in cancer has recently taken a firm
hold in our standard clinical armamentarium, the
concept of ligand-mediated active liposome targeting
still needs further experimental proof of validity in
animal models and surely in clinical trials. A number
of important questions remain open and need further
testing before the added value of folate targeting to
liposome delivery can be thoroughly evaluated at a
preclinical level. Formulation issues that need to be
addressed are related to the optimal density of folate
ligands on the liposome surface and to the interference
of PEG coating with FRME. They may have an impact
on in vivo liposome clearance and liposome interac-tion with tumor cells. Studies evaluating the toxicity of
various dose levels of drug-loaded FTL and comparing
it with that of their nontargeted counterparts are also
required. Finally, therapeutic studies that (i) cover a
broad range of tumor models and dose levels and (ii)
address the issue of modulation of FR expression by
dietary folate content and/or treatment need yet to
mature. In agreement with the principle that liposome
extravasation is the rate-limiting step of tumor local-
ization, the current body of data does not support the
claim that folate targeting increases to a sizable extent
the overall liposome concentration in subcutaneously-
growing tumor implants. However, folate targeting
may still lead to significant pharmacodynamic changes
with improvement of the therapeutic index by shifting
drug distribution from the extracellular to the intra-
cellular tumor compartment while systemic toxicity is
left unchanged or even reduced.
Acknowledgements
This work was supported in part by the Israel
Science Foundation, by the Israel Society against
Cancer, and by ALZA Corporation (Mountain View,
CA). We wish to thank the technical help all along
these studies of Dina Tzemach, and Lidia Mak (Shaare
Zedek Medical Center). We also wish to acknowledge
Charles Engbers and Mary Newman (ALZA Corp.,
and formerly SEQUUS Pharmaceuticals) for per-
forming the rat pharmacokinetic experiments.
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