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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:[email protected] (A. Gabizon).

www.elsevier.com/locate/addr

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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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