Liposomes containing alkylated methotrexate analogues for phospholipase A2 mediated tumor targeted drug delivery

Article abstract of DOI:10.1016/j.chemphyslip.2008.11.005

Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO III and HT-29 human colon cancer cells. Both analogues contained a C₁₆-alkyl chain attached to the γ-carboxylic acid and one of the analogue

Full text of DOI:10.1016/j.chemphyslip.2008.11.005

Chemistry and Physics of Lipids 157 (2009) 94–103
Contents lists available at ScienceDirect
Chemistry and Physics of Lipids
journal homepage: www.elsevier.com/locate/chemphyslip
Liposomes containing alkylated methotrexate analogues for phospholipase A2
mediated tumor targeted drug delivery
Thomas Kaasgaarda,b,c,∗, Thomas L. Andresena,d, Simon S. Jensene, René O. Holtea,
Lotte T. Jensena, Kent Jørgensena
a LiPlasome Pharma A/S, Department of Chemistry, Technical University of Denmark, DK-2800 Lyngby, Denmark
b MEMPHYS – Center for Biomembrane Physics, Physics Department, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark
c Danish Technological Institute, Holbergsvej 10, DK-6000 Kolding, Denmark
d DTU Nanotech, Technical University of Denmark, 4000 Roskilde, Denmark
e Bioneer A/S, Kogle Allé 2, DK-2970 Hørsholm, Denmark
a r t i c l e i n f o
a b s t r a c t
Article history:
Two lipophilic methotrexate analogues have been synthesized and evaluated for cytotoxicity against KATO
III and HT-29 human colon cancer cells. Both analogues contained a C16 -alkyl chain attached to the ␥-
carboxylic acid and one of the analogues had an additional benzyl group attached to the ␣-carboxylic acid.
The cytotoxicity of the ␥-alkylated compound towards KATO III (IC50 = 55 nM) and HT-29 (IC50 = 400 nM)
cell lines, was unaffected by the alkylation, whereas the additional benzyl group on the ␣-carboxyl group
made the compound nontoxic. The ␥-derivative with promising cytotoxicity was incorporated into lipo-
somes that were designed to be particularly susceptible to a liposome degrading enzyme, secretory
phospholipase A2 (sPLA2), which is found in high concentrations in tumors of several different cancer
types. Liposome incorporation was investigated by differential scanning calorimetry (DSC), and sPLA2
hydrolysis was examined by fluorescence spectroscopy and high performance liquid chromatography
(HPLC). The results showed that the methotrexate (MTX)-analogue could be incorporated into liposomes
that were degradable by sPLA2. However, the in vitro cytotoxicity of the MTX-liposomes against KATO III
and HT-29 cancer cells was found to be independent of sPLA2 hydrolysis, indicating that the alkylated
MTX-analogue was available for cancer cell uptake even in the absence of liposome hydrolysis. Using
a DSC based method for assessing the anchoring stability of alkylated compounds in liposomes, it was
demonstrated that the MTX-analogue partitioned into the water phase and thereby became available for
cell uptake. It was concluded that liposomes containing alkylated MTX-analogues show promise as a drug
delivery system, although the MTX-analogue needs to be more tightly anchored to the liposomal carrier.
Also, the developed DSC-assay for studying the anchoring stability of alkylated drugs will be a useful tool
in the development of liposomal drug delivery systems.
Received 27 May 2008
Received in revised form
12 September 2008
Accepted 17 November 2008
Available online 27 November 2008
Keywords:
Methotrexate analogue
Phospholipase A2
Drug delivery
Liposome
Tumor targeting
Synthesis
© 2008 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
(Frei et al., 1984; Bertino et al., 1996; Mauritz et al., 2002; Serra et
al., 2004).
Methotrexate (MTX) is an anticancer drug that has been used in
the treatment of various cancer types for decades (Acute Leukemia
Group B, 1965). It is an antimetabolite that inhibits dihydrofolate
reductase (DHFR), which is an enzyme involved in the biosynthetic
pathway of nucleotides and MTX is therefore highly toxic to rapidly
dividing cancer cells (Subramanian and Kaufman, 1978; Bertino et
al., 1996). However, MTX has a narrow therapeutic index, and its
clinical use is hampered by severe dose-limiting side effects, as well
as intrinsic and acquired drug resistance mechanisms of cancer cells
Various approaches have been attempted to overcome drug
resistance and improve the therapeutic index of MTX. Synthesis
of MTX-analogues having a molecular structure that is slightly
different from that of the parent molecule is one approach to over-
come drug resistance that has been employed for several years
(Montgomery et al., 1979). The main strategy is to make alter-
ations to the MTX-molecule that renders the analogue insensitive
to, or able to bypass drug resistance mechanisms, and yet retain the
cytotoxic activity towards the DHFR target enzyme. An exhaustive
number of publications report on the effects that different alter-
ations to the MTX-molecule have on DHFR inhibition and cytotoxic
potency of the synthesized analogues (Piper et al., 1982a; Rosowsky
et al., 1983, 1988a,b). Such studies have shown that particularly
the ␥-carboxyl group (Rosowsky et al., 1981, 1986), and to a lesser
∗
Corresponding author at: Danish Technological Institute, Holbergsvej 10,
DK-6000 Kolding, Denmark. Fax: +45 72201919.
E-mail address: thomas.kaasgaard@teknologisk.dk (T. Kaasgaard).
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2008.11.005

T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
95
extent the ␣-carboxyl (Piper et al., 1982b; Rosowsky et al., 1984),
are amenable to derivatization without the loss of cytotoxicity.
A different approach to improve the therapeutic index of MTX
is the use of an effective drug delivery system that is capable of
tumor specific targeting. In a study by Pignatello et al. (2003),
lipo-amino acids were conjugated to both of the carboxyl groups
of the glutamate moiety, and the resulting lipophilic conjugates
were incorporated into liposomes. Apart from enabling liposomal
drug delivery, the increased lipophilicity of the synthesized MTX-
conjugates also aimed to overcome impaired active transport drug
resistance, by increasing the passive diffusion of drugs through the
plasma membrane of cancer cells. In another study, MTX as well as
another anticancer agent, docosahexanoic acid (DHA), were conju-
gated to the same phosphatidylcholine headgroup (Zerouga et al.,
2002). When incorporated into liposomes, the synthesized MTX-
DHA construct was reported to be hydrolyzed by snake venom
phospholipase A2 (snake-PLA2). Snake-PLA2 is a small water soluble
lipid degrading enzyme that cleaves phospholipids at the lipo-
some interface resulting in the formation of fatty acid and lysolipid
hydrolysis products. It is structurally and functionally similar to
human secretory phospholipase A2 (sPLA2), which is of particu-
lar interest in the context of liposomal drug delivery, as human
secretory phospholipases are overexpressed in many types of solid
tumors (Abe et al., 1997; Graff et al., 2001; Jiang et al., 2002).
The aim of the present study was to synthesize lipophilic deriva-
tives of MTX for incorporation into liposomal drug carriers, which
are made particularly susceptible to hydrolysis by human sPLA2,
whereby a tumor specific release of the MTX-analogue is expected.
We have synthesized two lipophilic MTX-ester derivatives that
each contain a C16 -alkane chain attached to the ␥-carboxylic acid,
and in one case also a benzyl group attached to the ␣-carboxylic
acid. The cytotoxicity of both MTX-analogues was evaluated against
KATO III and HT-29 cancer cell lines. These cell lines were cho-
sen because KATO III cells secrete sPLA2, whereas HT-29 cells do
not. They were therefore suitable for testing the objective of sPLA2
dependent release of the alkylated MTX-analogues from a liposo-
mal formulation. The MTX-ester derivatives were incorporated into
DPPC/DPPG/DPPE-PEG2000 liposomes and the MTX liposomal for-
mulations were characterized by differential scanning calorimetry
(DSC). The liposomal carrier was chosen as a composition expected
to be particularly susceptible to sPLA2 hydrolysis, as the phase tran-
sition temperature is close to physiological temperature, and it is
known that sPLA2 activity is high close to the phase transition,
while the incorporation of DPPG introduces a negative charge on
the liposomes, which is required for sPLA2 to be active (Buckland
and Wilton, 2000; Leidy et al., 2006). In contrast, more commonly
employed liposomal carriers, have been shown not to be susceptible
to sPLA2 hydrolysis (Andresen et al., 2005). It was decided to attach
a C16 -alkyl chain to MTX in order for the alkyl chain length to match
the chain length of the liposomal carrier lipids. The susceptibility of
the liposomal formulations towards PLA2-mediated hydrolysis was
studied by fluorescence spectroscopy and HPLC, using both human
type and snake venom PLA2 isoforms. Subsequently, the liposomal
MTX-formulations were tested for cytotoxicity in sPLA2-expressing
KATO III cells as well as non-expressing HT-29 cancer cell lines.
Finally, the anchoring stability of the MTX-ester in the drug deliv-
ery liposomes was characterized by means of a DSC method where
liposomes are used as sensors for detecting MTX-ester dissociation
from the drug delivery liposomes.
2. Materials and methods
The lipids 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine
(DMPC),
(DPPC),
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine
1,2-dipalmitoyl-sn-glycero-3-phosphatidylglycerol
(DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphatidylethanolamine-
N-[poly(ethylene glycol)-2000] (DPPE-PEG2000), were obtained
from Avanti Polar Lipids, Inc. (Alabaster, AL, USA) and used without
further purification. Boc-l-glutamic acid ␣-benzyl ester, 4-[N-
(2,4-diamino-6-pteridinylmethyl)-N-methylamino] benzoic acid
(PMAB), and all other chemicals used in the synthesis of the
MTX-esters were purchased from Sigma–Aldrich and used without
purification. Methotrexate-␥-hexadecyl ester (C16 -MTX-ester) and
methotrexate-␣-benzyl-␥-hexadecyl ester (C16 -MTX-benzyl-ester)
was synthesized in our own laboratory as described below.
2.1. Synthesis of MTX-esters
The MTX-esters were synthesized from ␣-benzyl-BOC-
protected glutamic acid as described in Scheme 1. Briefly,
N-t-BOC-l-glutamic acid ␣-benzyl ester was coupled with hexade-
cyl alcohol using standard DCC, DMAP reaction conditions. The BOC
protection group was hereafter removed by TFA and the resulting
intermediate was used without purification in the coupling to
PMAB. The desired C16 -MTX-benzyl-ester was hereby obtained in
61% overall yield. It was furthermore possible to remove the benzyl
group using boron trichloride without destroying the pteridine,
resulting in C16 -MTX-ester in 48% overall yield from the protected
glutamic acid. Experimental is provided below.
Synthesis of N-t-BOC-˛-benzyl-l-glutamic acid ꢀ-hexadecyl ester.
DCC 1 M in DCM (5.34 ml) was added to a solution of N-t-BOC-
␣-benzyl-l-Glutamic acid (1.80 g, 5.34 mmol), n-hexadecyl alcohol
(863 mg, 3.56 mmol) and DMAP (87 mg, 0.712 mmol) in dry DCM
(30 ml) under N2. The reaction mixture was stirred at room
Scheme 1. Synthesis of C16 -MTX-ester and C16 -MTX-benzyl-ester. (a) C16 H33OH, DMAP, DCC, DCM, rt, 16 h. (b) TFA, DCM, 0 ◦C, 30 min. (c) PMAB, EDCl, pyridine, rt, 12 h. (d)
BCl3, CHCl3, from −20 ◦C to rt, 30 min.

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T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
temperature overnight, after which it was filtered and concen-
trated. The crude product was purified by flash chromatography
(elution with hexane/DCM/diethyl ether 30:12:3 followed by
20:12:3) to give 1.98 g (99%) of the desired product as a white
solid. RF = 0.13 hexane/DCM/diethyl ether 30:12:3, RF = 0.33 hex-
ane/DCM/diethyl ether 20:12:3. 1H-NMR (500 MHz, CDCl3) ı = 7.36
(m, 5H), 5.18 (AB, J = 12.3 Hz, 2H), 5.11 (m, 0.5H), 4.38 (m, 0.5H), 4.05
(t, J = 6.8 Hz, 2H), 2.37 (m, 2H), 2.18 (m, 1H), 1.97 (m, 1H), 1.61 (k,
J = 6.7 Hz, 2H), 1.44 (s, 9H), 1.27 (br. s, 26H), 0.89 (t, J = 7.0, 3H). 13C-
NMR (125 MHz, CDCl3): ı = 172.7, 172.0, 155.3, 135.3, 128.6, 128.4,
128.2, 80.0, 67.1, 64.9, 53.0, 31.9, 30.3, 29.8, 29.6, 29.6, 29.5, 29.3,
29.2, 28.6, 28.3, 27.7, 25.9, 22.7, 14.1.
liposomes, DMPC was hydrated directly in PBS without prior evap-
oration from CHCl3:CH3OH. During the 60 min hydration period
at 51 ◦C the lipid suspension was vigorously shaken every 15 min,
producing multilamellar vesicles (MLVs). Subsequently, to create
large unilamellar vesicles (LUVs), the MLV lipid suspensions were
extruded (Avanti mini-extruder, Avanti Polar Lipids, Alabama, USA)
ten times through a 0.1 ␮m polycarbonate filter (Nayar et al., 1989).
MLV’s were used in all DSC measurements because of the sharper
phase transition produced by MLV’s, while LUV’s were used in all
other measurements.
2.3. DSC-measurements
Synthesis of MTX-˛-benzyl-ꢀ-hexadecyl ester. N-t-BOC-␣-benzyl-
l-glutamic acid ␥-hexadecyl ester (540 mg, 0.961 mmol) in dry DCM
(6 ml) was placed in an ice bath for 10 min after which TFA (6 ml)
was added. The solution was stirred 30 min and the solvent was
removed under reduced pressure using toluene as co-evaporant.
The crude product was redissolved in toluene (100 ml) and washed
with 1 M HCl (2 × 10 ml), brine (10 ml), sat. NaHCO3 (2 × 10 ml)
and H2O (2 × 10 ml). The organic layer was concentrated to give
450 mg of crude product. The crude product in pyridine (10 ml)
was added to a solution of PMAP (200 mg, 0.481 mmol) in dry
pyridine (20 ml) under N2, followed by addition of EDCl (461 mg,
2.40 mmol). The yellow reaction mixture was stirred at room tem-
perature in the dark for 12 h. The mixture was concentrated and
redissolved in toluene (200 ml) and washed with brine (2 × 20 ml)
and H2O (2 × 20 ml). The solution was concentrated and purified by
flash chromatography (5% MeOH in DCM) to give 232 mg (62%) of
the desired product as a yellow solid. RF = 0.30 10% MeOH in DCM.
Anal.Calcd for C43H60N8O5·H2O: C 65.62, H 7.94, N 14.24; Found:
C 65.81, H 7.85, N 14.18. 1H-NMR (500 MHz, CDCl3) ı = 8.60 (s, 1H),
7.70 (d, J = 8.5 Hz, 2H), 7.33 (m, 5H), 7.01 (d, NH), 6.71 (d, J = 8.6 Hz,
2H), 5.18 (AB, J = 12.2 Hz, 2H), 4.84 (m, 1H), 4.69 (s, 2H), 3.99 (m, 2H),
3.13 (s, 3H), 2.42 (m, 2H), 2.29 (m, 1H), 2.14 (m, 1H), 1.54 (k, J = 6.6 Hz,
2H), 1.24 (br. s, 26H), 0.87 (t, J = 7.0, 3H). 13C-NMR (125 MHz, CDCl3):
ı = 173.3, 172.3, 166.9, 162.9, 162.4, 155.2, 151.2, 149.6, 147.0, 135.3,
128.9, 128.6, 128.4, 128.2, 121.9, 121.6, 111.5, 67.2, 65.0, 55.9, 52.3,
39.1, 31.9, 30.5, 29.7, 29.6, 29.6, 29.5, 29.3, 29.2, 28.5, 27.3, 25.8, 22.6,
14.1.
Synthesis of MTX-ꢀ-hexadecyl ester. A solution of MTX-␣-benzyl-
␥-hexadecyl ester (62 mg, 0.081 mmol) in CHCl3 (5 ml) under N2,
was cooled to −20 ◦C in an acetone bath. BCl3 1 M in DCM (1.61 ml)
was added dropwise and the flask was removed from the cooling
bath. The mixture was stirred 30 min at room temperature after
which it was concentrated. Purification by flash chromatography
(first, 10% MeOH in CHCl3 then CHCl3/MeOH/H2O 80:20:1) to give
43 mg (79%) of the desired product. RF = 0.20 CHCl3/MeOH/H2O
80:20:1. 1H-NMR (500 MHz, D5-pyridine) ı = 8.93 (d, NH), 8.71 (s,
1H), 8.25 (d, J = 8.8 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 5.39 (m, 1H), 4.67
(s, 2H), 4.09 (t, J = 6.7 Hz, 2H), 2.98 (s, 3H), 2.82 (m, 3H), 2.56 (m, 1H),
1.53 (k, J = 7.0 Hz, 2H), 1.24 (br. s, 26H), 0.84 (t, J = 6.7, 3H). 13C-NMR
(125 MHz, CDCl3): ı = 173.4, 167.9, 164.4, 164.3, 156.0, 151.8, 150.2,
149.6, 146.8, 129.8, 123.3, 122.9, 111.9, 64.8, 56.0, 53.5, 39.0, 32.2,
31.6, 30.0, 30.0, 29.0, 28.3, 26.2, 23.0, 14.3.
Differential scanning calorimetry (DSC) measurements were
made using a MicroCal MC-2 calorimeter (Microcal Inc., Mas-
sachusetts, USA) at a scan rate of 30 ◦C/h. The concentrations of the
MTX-ester containing liposomes were 3 mM, which was the total
concentration of DPPC, DPPE-PEG2000, C16 -MTX-ester and free MTX.
For the measurements on anchoring stability (Figs. 9 and 10), 6 mM
MLV suspension of the MTX formulation was mixed with a 6 mM
MLV suspension of pure DMPC vesicles at a 1:1 ratio, giving 3 mM
concentrations of each type of liposomes. The mixture was trans-
ferred to the calorimeter and a DSC scan recorded from 5 to 37 ◦C
(scan 1). The sample was then incubated at 37 ◦C for 6 h and a sec-
ond scan recorded (scan 2). Appropriate baselines were subtracted
from the DSC curves.
2.4. PLA2-hydrolysis measurements
The PLA2 hydrolysis of unilamellar DPPC/DPPG/DPPE-
PEG2000/C16 -MTX (45:45:5:5) lipid vesicle suspensions (LUV’s) was
monitored by following the intrinsic tryptophan fluorescence emit-
ted at 340 nm upon excitation at 285 nm. In addition, the 90◦ light
scattering at 285 nm was measured, which is sensitive to changes
in the lipid vesicles that results from the hydrolysis (Hønger et al.,
1996). Fluorescence measurements were recorded using an SLM
DMX-1100 spectrofluorometer (SLM-Aminco, Rochester, NY). The
lipid concentration was 150 ␮M in PBS and CaCl2 was added to
give a CaCl2 concentration of 1 mM. The temperature was 37 ◦C.
In the case of snake venom PLA2 hydrolysis, PLA2 purified from
Agkistrodon piscivorus piscivorus (Maraganore et al., 1984) was
used at a concentration of 150 nM. In the case of human sPLA2
hydrolysis, 10 ␮L of human tear fluid, which contains high amounts
of sPLA2 (Qu and Lehrer, 1998), was added to 2.5 mL of the lipid
vesicle suspension. Tear fluid was collected by exposing healthy
human adult volunteers to freshly minced onions.
2.5. HPLC-measurements
HPLC measurements were performed using a Waters Mille-
nium 2010 (Milford, MA) HPLC equipped with a waters 510 pump,
a Waters 717 Plus Autosampler, and a PL-EMD evaporative light
scattering mass detector from Polymer Laboratories (Cheshire,
UK). A 5 ␮m Phenomenex (Torrance, CA) diol spherical column
and a mixture of chloroform/methanol/water (73:23:3) as iso-
cratic mobile phase was used. Concentrations that were three
times higher than in the fluorescence measurements were used
for the snake venom PLA2 experiment (i.e. 450 ␮M lipid; 450 nM
snake venom PLA2). From this reaction mixture, 100 ␮L samples
were retrieved at different times of the reaction and added to
1 mL of a chloroform/methanol/acetic acid/water (2:4:1:1) mix-
ture to stop the enzyme hydrolysis. Salts were then extracted
from the sample by shaking thoroughly with 1 mL water. From the
organic phase, 50 ␮L was used for HPLC analysis. For the tear fluid
PLA2-experiment, the lipid and enzyme concentration were the
same as in the fluorescence measurements. A 300 ␮L sample was
2.2. Preparation of liposomes
Stock solutions of DPPC and DPPE-PEG2000 were made in
CHCl3:CH3OH 1:1 (v/v), whereas DPPG was in pure CHCl3, and
C16 -MTX was made in EtOH. Appropriate amounts of the stock solu-
tions were mixed in a test tube, and the solvent evaporated using
a gentle stream of nitrogen, followed by drying at low pressure
overnight. The dry lipid mixtures were suspended in phosphate
buffered saline buffer (PBS, without calcium, magnesium, and sodi-
umbicarbonate, GIBCO, Invitrogen, United Kingdom) and placed in
a 51 ◦C water bath for at least 60 min. In the case of pure DMPC

T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
97
retrieved from the reaction mixture and added to 1 mL of a chloro-
form/methanol/acetic acid/water (2:4:1:1) mixture, and salts were
extracted by shaking with 800 ␮L of water. The degree of hydroly-
sis was calculated from the reduction in unhydrolyzed DPPC lipids,
based on the integrated signal from the light scattering mass detec-
tor.
2.6. Cytotoxic activity assays
The KATO III human gastric carcinoma cell line was purchased
from the Japan Health Sciences Foundation (Tokyo, Japan). The
HT-29 and COLO 205 human colon carcinoma cell lines were
purchased from ATCC (Manassas, VA). KATO III were grown in
RPMI 1640 medium supplemented with 10% heat-inactivated fetal
calf serum (FCS), 2 mM l-glutamine and 1 mM sodium pyruvate
in a humidified 5% CO2 atmosphere at 37 ◦C. HT-29 were grown in
McCOY’s 5A and COLO 205 in RPMI medium supplemented with
10% FCS, 2 mM l-glutamine (all from Invitrogen, Carlsbad, CA).
Cells were plated in 96-well plates at a density of 1 × 104 cells per
well, 24 h prior to addition of test compounds. MTX was solubilized
in 0.1 M NaOH, MTX-ester in EtOH, MTX-benzyl-ester in CHCl3.
MTX-liposomes were added to the cells in phosphate buffered
saline (PBS). Test compounds or formulations were added at the
indicated concentrations and incubated for 72 h (Fig. 2) or 26 h
(Fig. 7). MTX-ester release from liposomes was evaluated without
sPLA2 by addition of fresh media, and with sPLA2 by addition of
conditioned media from COLO 205 cells grown for 24 h containing
50–60 ng/mL sPLA2. KATO III cells secreted approximately 3 ng/ml
sPLA2 over a 72 h incubation period (Jensen et al., 2004). The
specific sPLA2 inhibitor LY311727 was added in order to evaluate
the specificity of sPLA2-dependent release in sPLA2-expressing
cells. The LY311727 inhibitor (kindly provided by Eli Lilly & Co.,
Indianapolis, IN) was dissolved in ethanol and added to a final
concentration of 25 ␮M 15 min before the addition of liposomes
in order to allow for optimal inhibition of the lipase. Cytotoxic
activity was assessed using a standard 3-(4,5-dimethylthiazolyl)-
2,5-diphenyltetrazolium bromide (MTT) assay (Carmichael et al.,
1987) and expressed as cell survival compared to vehicle treated
control cells. All studies were performed in triplicate.
Fig. 1. Molecular structure of C16 -MTX-ester and C16 -MTX-benzyl-ester.
MTX-ester is incorporated, the main phase transition broadens and
moves to lower temperatures, while the pretransition disappears
as highlighted in Fig. 3b. At 25% C16 -MTX-ester, an additional broad
peak appears in the 46–53 ◦C temperature range as shown in Fig. 3c.
No additional peaks were observed at the lower concentrations of
the MTX-analogue. As a control experiment, DSC scans of DPPC
3. Results
3.1. Cytotoxicity of the synthesized MTX-analogues
The molecular structures of the two synthesized MTX-analogues
are shown in Fig. 1.
Both analogues as well as the parent MTX-compound were
tested for cytotoxic activity against KATO III and HT-29 cancer cell
lines, and expressed as cell survival as presented in Fig. 2. The results
reveal that the cytotoxic activity of C16 -MTX-ester is comparable to
MTX in both cell lines. In contrast, the benzyl-MTX-ester turned out
to be non-toxic in the tested concentration range. The IC50-values
based on the survival curves are approximately 55 nM for free MTX
and the C16 -MTX-ester in KATO III cells, and 400 nM in HT-29 cells.
3.2. Incorporation into liposomes
Because the benzyl-MTX-ester was found to be non-toxic, this
analogue was not investigated any further. The C16 -MTX-ester on
the other hand, proved to be a promising candidate for liposome
based drug delivery. For this reason, the C16 -MTX-ester was incor-
porated into liposomes and analyzed by DSC. Fig. 3a shows DSC
heating curves of multilamellar DPPC liposomes containing differ-
ent amounts of C16 -MTX-ester. The DSC curve of pure DPPC displays
a main phase transition at 41.6 ◦C and a pretransition at 35.6 ◦C,
Fig. 2. Cytotoxicity of free MTX and MTX-ester and benzyl-MTX-ester after 72 h
incubation in KATO III (a) and HT-29 (b) cancer cell lines. The cytotoxicity of the
MTX-ester is comparable to that of free MTX. In contrast, benzyl-MTX-ester was
nontoxic in both cell lines.
as expected for this lipid. When increasing concentrations of C16
-

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T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
Fig. 3. DSC heating curves of DPPC multilamellar vesicles containing different concentrations of C16 -MTX-ester. (a) Main phase transition. (b) Magnification of the pretransition
region, showing that the pretransition disappears when C16 -MTX-ester is incorporated. (c) Magnification of the DSC curve of the formulation containing 25% C16 -MTX-ester.
lipid vesicles in the presence of different concentrations of free
MTX were recorded. In this experiment, the main phase transition
temperature as well as the pretransition were virtually unaffected
(data not shown) indicating a low partitioning of free MTX into the
liposomal membrane.
The effects of free MTX and C16 -MTX-ester on the main phase
transition temperature, Tm, and enthalpy, ꢁH, are plotted in Fig. 4.
The phase transition temperature decreases monotonously with
increasing C16 -MTX-ester concentrations and is almost unchanged
by free MTX (Fig. 4a). More intriguing are the enthalpy graphs
shown in Fig. 4b, which show enthalpies that have been concen-
tration normalized with respect to the DPPC lipid concentration.
The molar enthalpy increases when C16 -MTX-ester is incorporated,
while it decreases slightly with increasing concentrations of free
MTX. Interestingly, the enthalpy increase caused by the C16 -MTX-
ester is particularly pronounced when going from 0% to 2% of the
MTX-analogue.
hydrolysis in cancer tissue. Therefore, the susceptibility to PLA2-
mediated hydrolysis of the C16 -MTX-ester formulations was
examined. Fig. 5a shows the hydrolysis of a DPPC/DPPG/DPPE-
PEG2000/C16 -MTX-ester (45:45:5:5) formulation by snake venom
PLA2. The lower curve is the fluorescence intensity of intrinsic tryp-
tophan residues of PLA2, and the upper curve is the light scattering,
which is related to the morphology of the lipid vesicles. An increase
in both the fluorescence and the light scattering intensity occurs
at approximately 800 s, which is a clear indication that the lipid
vesicles are being hydrolyzed by the PLA2 enzyme, as has been
demonstrated by a number of studies (Hønger et al., 1996; Høyrup
et al., 2004). The same C16 -MTX-ester formulation was also sub-
jected to hydrolysis by tear fluid sPLA2, as shown in Fig. 5b. A steep
increase in the light scattering occurs after a characteristic lag time
of approximately 5000 s, indicating that the lipid vesicle formula-
tion is also susceptible to hydrolysis by human sPLA2 from tear fluid.
To further demonstrate that hydrolysis takes place, a parallel
experiment was carried out, in which the lipid hydrolysis was moni-
tored by HPLC. In order to get strong signals in the HPLC instrument,
the snake-PLA2 experiment shown in Fig. 6a was performed with
three times higher lipid and enzyme concentrations than the con-
centrations used in the fluorescence/light scattering experiment.
3.3. Hydrolysis by PLA2
The aim of the present study was to make liposomal formula-
tions that release the MTX-analogue as a result of sPLA2-mediated
Fig. 4. The main phase transition temperature (a) and enthalpy (b) as a function of C16 -MTX-ester or free MTX content.

T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
99
Fig. 5. Fluorescence spectroscopy measurements showing the hydrolysis of liposomes composed of DPPC/DPPG/PEG2000/C16 -MTX-ester (45:45:5:5) by snake venom PLA2
(a) and tear fluid PLA2 (b).
In addition, blank liposomes without C16 -MTX-ester, but otherwise
identical to the MTX-formulation were included in the measure-
ments to examine the specific effects that MTX-ester might have
on PLA2 hydrolysis. The two peaks in the chromatogram corre-
spond to unhydrolyzed DPPC that has a retention time between 6.5
and 7 min and palmitic acid that has a retention time of approx-
imately 3.25 min and is one of the reaction products. The data
demonstrate that DPPC is gradually degraded while palmitic acid
is concomitantly being formed, and thus confirm the fluorescence
data that the presence of MTX-ester does not inhibit PLA2 medi-
ated hydrolysis. In fact, it even appears that hydrolysis is slightly
enhanced when MTX-ester is present in the liposomes. Based on
the HPLC measurements, the extent of hydrolysis of the MTX-
ester formulation was calculated at three different time points.
The calculated hydrolysis percentages are plotted as open squares
in Fig. 5a, and are in good accordance with the fluorescence and
light scattering measurements. A quantitative HPLC analysis of tear
fluid sPLA2 catalyzed hydrolysis was not carried out because the
higher lipid concentrations required for accurate HPLC detection
would require correspondingly large amounts of tear fluid. How-
ever, Fig. 6b presents a HPLC chromatogram of a sample retrieved at
the end of the fluorescence experiment in Fig. 5b. A distinct palmitic
acid peak is seen at 3.25 min, providing qualitative confirmation
that the C16 -MTX-ester liposomes are also susceptible to hydrolysis
by human type sPLA2.
Fig. 7. Cytotoxic activity measured after 26 h in KATO III (a) and HT-29 (b) cancer cells
of free C16 -MTX-ester (MTX-ester), DPPC/DPPG/PEG2000/C16 -MTX-ester (45:45:5:5)
liposomes (MTX-L), and MTX-L in the presence of the sPLA2 inhibitor LY311727
inhibitor (MTX-L + Ly). For the non-sPLA2-secreting HT-29 cell line, sPLA2 was added
from conditioned COLO 205 media (MTX-L + sPLA2).
Fig. 6. HPLC chromatograms of DPPC/DPPG/PEG2000/C16 -MTX-ester (45:45:5:5) at
different times after the addition of snake venom PLA2 (a) and tear fluid PLA2 (b).

100
T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
Fig. 8. Schematic illustration of a DSC-method for evaluating the anchoring stability of the MTX-ester in the liposomal formulation. The DPPC/DPPG/PEG2000/C16 -MTX-ester
liposomes are shown to the left and the C16 -MTX-ester is depicted in red. The extent of migration of MTX-ester from the liposomal formulation and into the sensor-liposomes
is a measure of the anchoring stability of the MTX-ester, and can be evaluated from a DSC scan of the phase transition of the sensor liposomes. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of the article.)
3.4. PLA2-dependent cytotoxicity
that the presence of the MTX-ester formulation causes a signifi-
cant and time dependent perturbation of the main phase transition
of the DMPC liposomes. To ensure that the observed perturba-
tions of the DMPC phase transition was due to the C16 -MTX-ester
and not any of the lipid components, the lipid exchange between
DMPC and DPPC/DPPG/PEG2000 (45:50:5) without the MTX-ester
was examined (Fig. 9b). It is observed that the lipid components
have a negligible effect on the DMPC phase transition, and it can
therefore be concluded that the perturbation seen in Fig. 9a is due
to C16 -MTX-ester.
Fig. 10 shows the effect of the negatively charged DPPG lipids on
the anchoring stability of the MTX-ester in the liposome formula-
tion. The top curve shows pure DMPC liposomes, and the following
curves show DMPC liposomes after a 6-h co-incubation with MTX-
ester formulations containing increasing amounts of DPPG. The
results show that the DMPC phase transition becomes progressively
more perturbed as the DPPG content is increased, indicating that
the negatively charged DPPG decreases the anchoring stability of
MTX-ester in the liposomal formulation.
The abovementioned results indicate that it is possible to make
a liposomal formulation of the highly toxic C16 -MTX-ester, and that
such a formulation is degradable by sPLA2. For this reason, a for-
mulation consisting of DPPC/DPPG/DPPE-PEG2000/C16 -MTX-ester
(45:45:5:5) was prepared, and tested for sPLA2 dependent cytotox-
icity against KATO III and HT-29 cells (Fig. 7). KATO III cells secrete
sPLA2 whereas HT-29 cells do not. In the KATO III experiment, the
cytotoxic effect of the liposome formulation was measured after
26 h of incubation and compared to the effect in the presence of
the sPLA2-inhibitor, LY311727, as well as to the free C16 -MTX-ester.
No significant difference in cytotoxic activity was seen for these
three treatment regimens (Fig. 7a). In the HT-29 experiment, sPLA2
was added in the form of conditioned media from sPLA2-secreting
COLO 205 cells containing 50–60 ng/mL of sPLA2. The results of the
HT29 experiment show that MTX-liposomes and free MTX-ester
are equally toxic in the absence of sPLA2, whereas MTX-liposomes
become the most toxic when sPLA2 is added (Fig. 7b).
3.5. Anchoring stability
4. Discussion
To test the stability of the MTX-ester in lipid vesicles of dif-
ferent compositions, a simple DSC method was developed. The
method, which is depicted in Fig. 8, is based on co-incubating the
MTX-ester liposomal formulation with pure DMPC liposomes and
subsequently recording a DSC scan of the DMPC main phase tran-
sition. If MTX-ester is only loosely anchored in the formulation, it
will dissociate from the liposomes and, in turn, partition into the
DMPC vesicles. Upon migration of MTX-ester to the DMPC vesi-
cles, the main phase transition peak will become progressively
more perturbed by the presence of the MTX-ester, and the main
phase transition of the DMPC liposomes can thus be used as a
measure of the amount of MTX-ester which has migrated from
the MTX-formulation to the DMPC vesicles. It is noted, that this
experimental setup mimics the cytotoxicity measurements where
MTX-ester containing liposomes are incubated with cancer cells.
Fig. 9a shows DSC scans of pure DMPC lipid vesicles co-incubated
with a DPPC/DPPG/DPPE-PEG2000/C16 -MTX-ester formulation. The
4.1. Cytotoxicity of the synthesized MTX-analogues
In this study, two MTX-ester analogues were synthesized and
tested for cytotoxicity against two different cell lines. The deriva-
tive containing a C16 alkyl chain attached to the ␥-carboxylic
acid (Fig. 1a) was shown to be equally toxic as the parent MTX-
molecule, whereas the attachment of an additional benzyl group
to the ␣-carboxyl group (Fig. 1b) eliminated the toxicity of this
MTX-analogue. This lends support to previous findings that the ␣-
carboxylic acid is sensitive to derivatization (Piper et al., 1982b).
However, some studies have shown that ␣-carboxyl ester deriva-
tives retain activity (Rosowsky et al., 1984), and it was therefore
conceivable that the benzyl-ester analogue tested in the present
study would remain active as well. The main incentive for making
alkylated MTX-derivatives was to incorporate the derivatives into
sPLA2-degradable liposomes that specifically release the drug at the
tumor target site. It should be noted, however, that the increased
hydrophobicity of the alkylated MTX-analogues may also increase
the passive diffusion into cancer cells and thereby enhance the
activity of the analogues towards resistant cell lines with lost or
impaired active transport mechanisms (Frei et al., 1984; Pignatello
et al., 2004). Although the cytotoxic activity graphs in Fig. 1 do not
show indications of enhanced cytotoxicity, it is quite possible that
uppermost DSC curve shows
a scan of pure DMPC in the
absence of the C16 -MTX-formulation, and the two other curves
show DMPC vesicles at different times after mixing with the
DPPC/DPPG/PEG2000/C16 -MTX (45:45:5:5) formulation. The middle
curveisthefirstscanobtainedafterthe vesiclesweremixed, andthe
lower curve was recorded after a 6-h incubation at 37 ◦C. It is evident

T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
101
Fig. 10. Effect of DPPG content on the stability of C16 -MTX-ester in DPPC liposomes.
The upper heat capacity curve shows a scan of pure DMPC in the absence of the C16
-
MTX-formulation. The other three curves show DMPC liposomes co-incubated with
DPPC/DPPG/PEG2000/C16 -MTX formulations containing different amounts of DPPG,
as indicated above each curve. All curves correspond to the second scan that was
recorded after a 6-h incubation at 37 ◦C. The results indicate that the anchoring
stability decreases as the DPPG content is increased.
reflecting phase coexistence and would not be as sensitive to the
incorporation of small amounts of C16 -MTX-ester.
The DSC data presented in Figs. 3 and 4 strongly suggest that
C16 -MTX-ester partitions into the DPPC liposomes. First of all, the
concentration dependent and monotonous decrease in the main
phase transition, as well as the disappearance of the pretransition
indicate that C16 -MTX-ester is present in the liposomes and causes
a slight perturbation of the lipid packing. Secondly, in contrast to
free MTX, the molar enthalpy increases when the C16 -MTX-ester
is incorporated (Fig. 4b). It is pointed out that the molar enthalpy
presented in Fig. 4b is based on the DPPC concentration. Thus, the
enthalpy increase that occurs when the C16 -MTX-ester is added
implies that the C16 -MTX-ester alkyl chain is present in the apolar
part of the liposomes and contributes to the chain melting enthalpy.
The reason for the particularly large initial increase in the enthalpy
that occurs from 0 to 2 mol% C16 -MTX-ester is unknown. However,
the disappearance of the pretransition (Fig. 3b) may provide a plau-
sible explanation why the enthalpy increases more dramatically at
low C16 -MTX-ester contents. The pretransition is a low enthalpy
transition that signifies the change from a gel phase to a ripple
phase lipid bilayer, while the main phase transition signifies the
change from a ripple phase to a fluid phase lipid bilayer (Tenchov,
Fig. 9. (a) Stability of C16 -MTX-ester in the liposome formulation. The upper heat
capacity curve shows a scan of pure DMPC liposomes in the absence of the C16
-
MTX-formulation. The two other curves show DMPC liposomes co-incubated with a
DPPC/DPPG/PEG2000/C16 -MTX (45:45:5:5) formulation. The middle curve is the first
scan after the vesicles were mixed, and the lower curve is the second scan, which
was recorded after a 6-h incubation at 37 ◦C. Clearly, the DMPC main phase transition
becomes perturbed as MTX-ester partitions into the DMPC liposomes. (b) To ensure
that the observed perturbations of the DMPC phase transition was due to the C16
-
MTX-ester and not any of the lipid components, the lipid exchange between DMPC
and DPPC/DPPG/PEG2000/C16 -MTX (45:50:5:0) was examined (i.e. without C16 -MTX-
ester). The upper heat capacity curve shows a scan of pure DMPC liposomes. The two
other curves show DMPC liposomes co-incubated with DPPC/DPPG/PEG2000/C16
-
MTX (45:50:5:0) liposomes. The middle curve is the first scan after the liposomes
were mixed, and the lower curve is the second scan, which was recorded after a 6-h
incubation at 37 ◦C. It is observed that the lipids have negligible effect on the DMPC
phase transition.
enhanced activity would be observed in vivo or if the MTX-analogue
was evaluated against resistant cell lines in vitro.
1991; Biltonen and Lichtenberg, 1993). In the event that the C16
-
MTX-ester prevents ripple phase formation, as Fig. 3b indicates,
the lipid bilayer undergoes a gel to fluid phase transition without
forming the intermediate ripple phase. In this case the new main
phase transition enthalpy will be the sum of the pretransition and
the main phase transition enthalpy, except for the perturbing effects
of the MTX-analogue. The pretransition enthalpy calculated from
the pure DPPC heat capacity curve in Fig. 3b is 1.55 kcal/mol. This
value is similar in magnitude to the enthalpy jump observed in
Fig. 4, and therefore lends weight to our hypothesis.
4.2. Liposome incorporation
The
C16 -MTX-ester analogue that was equally active as
the parent MTX-compound was next investigated for liposome
incorporation by recording DSC scans of DPPC multilamellar
vesicles (MLVs) containing different amounts of the MTX-ester
(Figs. 3 and 4). The choice of pure DPPC MLVs for this experiment
might seem surprising, considering that unilamellar vesicles con-
sisting of a mixture of DPPC, DPPG, and DPPE-PEG2000 were used
for the C16 -MTX-ester formulation in the PLA2 hydrolysis and cyto-
toxic activity assays. However, pure DPPC MLVs are good probes for
investigating the incorporation into liposomes, because they give
rise to a sharp main phase transition peak that is very sensitive
to changes in the lipid vesicle structure. In contrast, unilamellar
DPPC/DPPG/DPPE-PEG2000 liposomes result in a fairly broad peak
4.3. PLA2-hydrolysis
Based on the DSC results, it was concluded that at least 10%
of the C16 -MTX-ester can be incorporated into DPPC liposomes.
Although we expect the lipid bilayer partitioning to be governed
predominantly by the hydrophobic effect, the amount that can be
incorporated into DPPC:DPPG:DPPE-PEG2000 liposomes is likely to

102
T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
be different however. To be on the safe side, only 5% C16 -MTX-ester
was therefore incorporated in the liposomal formulation, which
apart from the MTX-ester was composed of 45 mol% DPPC, 45 mol%
DPPG, and 5 mol% DPPE-PEG2000. The high amount of DPPG was
included in order to make the liposomes degradable to human
sPLA2, which is particularly active towards negatively charged lipo-
somes (Buckland and Wilton, 2000; Buckland et al., 2000; Leidy et
al., 2006).
et al., 2004; Kim et al., 2007). In addition, the hydrolysis products
may act as permeability enhancers that increase the toxicity of C16
-
MTX-ester (Noseda et al., 1989; Davidsen et al., 2002), meaning that
the increased cytotoxicity may well be unrelated to the release of
C16 -MTX-ester. For these reasons, a more likely interpretation of the
data would be that the C16 -MTX-ester is not adequately anchored
in the liposomes, resulting in the dynamic partitioning of C16 -MTX-
ester in and out of the liposomes. In this case, C16 -MTX-ester is
available for cell uptake during the time it is present as a monomer
in solution, and the cytotoxicity would resemble that of free MTX
in accordance with our observations.
The formulation was investigated for PLA2-catalyzed hydrolysis
by snake-PLA2 as well as human sPLA2, which tear fluid contains in
high amounts (Qu and Lehrer, 1998). Snake-PLA2 is structurally and
functionally similar to human sPLA2, and has the obvious advan-
tage of being available in large quantities in a purified form. It does
not possess the same preference for negatively charged lipids, but
was nevertheless considered a good model enzyme. In particu-
lar, the fluorescence and HPLC experimental data obtained using
snake-PLA2, was a valuable reference for interpretation of the data
obtained with human sPLA2. The fluorescence and HPLC measure-
ments shown in Figs. 5 and 6, strongly suggest that the liposomal
formulation is hydrolyzable by both snake venom PLA2 and human
sPLA2. The hydrolysis is particularly evident in the snake-PLA2
experiments, which showed a dramatic change in the fluorescence
intensity and light scattering in response to the PLA2 hydrolysis
(Fig. 5a), as well as a time dependent conversion of DPPC to its
hydrolysis products (Fig. 6a). We were concerned that the pres-
ence of the C16 -MTX-ester could possibly impede the hydrolysis by
perturbing the lipid bilayer structure or by inhibiting the enzyme.
It was however interesting and promising to note that the oppo-
site appeared to be the case and hydrolysis was enhanced when
the C16 -MTX-ester was present in the liposomes. The human sPLA2
experiments gave similar results, although hydrolysis was slower
and the observed changes were less pronounced due to lower
enzyme concentrations. Comparing the results to the snake venom
data, however, leaves little doubt that the liposomal C16 -MTX-ester
formulation is also being degraded by human sPLA2.
4.5. Anchoring stability
In order to test the hypothesis that the C16-MTX-ester was not
adequately anchored in the liposomes, a simple DSC method was
employed. The method is based on the addition of multilamellar
liposomes as internal sensors that mimic the cancer cells in the
cytotoxicity experiments (Fig. 8). After incubation for a specified
time period, the extent of migration of MTX-ester from the liposo-
mal formulation and into the multilamellar sensor liposomes can be
assessed by recording a DSC scan and evaluating the perturbation
of the main phase transition created by the MTX-ester. The results
demonstrated that the DSC method was highly useful in assess-
ing the anchoring stability of alkylated compounds, and showed
that the MTX-ester was indeed only loosely anchored in the tested
DPPC:DPPG:DPPE-PEG2000 (45:45:5:5) liposomes, as this formula-
tion resulted in a significantly perturbed main phase transition peak
of the DMPC sensor liposomes after incubation at 37 ◦C (see Fig. 9a).
Using the DSC method it was also shown that the MTX-ester is par-
ticularly prone to dissociating from the liposomal formulation as
the content of the negatively charged DPPG lipid increases (Fig. 10).
Unfortunately, negatively charged lipids are necessary in order for
the liposomes to be hydrolysable by sPLA2, and removing the nega-
tively charged lipids from the formulation is therefore not an option
when designing liposomes for tumor specific release by sPLA2. In
future studies it would be logical to attempt to increase the anchor-
ing stability by attaching either a longer alkyl chain or a double alkyl
chain to MTX.
4.4. PLA2-dependent cytotoxicity
As the above findings have demonstrated that the C16 -MTX-ester
is both equally toxic as free MTX, and amenable to incorpora-
tion into sPLA2-degradable liposomes, the lipsomal formulation
seemed a promising candidate for tumor targeted delivery of
MTX-analogues to cancer tissue expressing sPLA2. In Fig. 7a this
hypothesis was tested using KATO III cells, which is one example
of a sPLA2-expressing cancer cell line. In this cell line, the cytotoxic
activity of the liposomal formulation was similar to free C16 -MTX-
ester, which is expected if the C16 -MTX-ester is being released
into the aqueous media through sPLA2-catalyzed liposome degra-
dation. What was unexpected, however, was that the inhibition
of sPLA2 did not abolish the cytotoxic activity. This implies that
5. Conclusion
In this study, the synthesis of two lipophilic MTX-analogues
has been described and their potential for liposomal tumor spe-
cific drug delivery has been evaluated. One analogue was found to
be nontoxic, while the other analogue retained the full toxicity of
the parent drug. This analogue was incorporated into liposomes
that were designed to be particularly degradable by human sPLA2.
The potential benefits of this design are twofold. First of all, the
liposomal carrier lipids are specifically hydrolyzed in cancer tis-
sue that overexpress sPLA2, resulting in a tumor specific release
of the MTX-derivative. Secondly, the lysolipids and fatty acids
that are the reaction products of the hydrolysis can act as per-
meability enhancers on cell membranes, which may increase the
passive diffusion of the MTX-ester into cancer cells. The initial
studies concerning the cytotoxicity, liposome incorporation, and
PLA2-mediated liposome degradation were highly promising, and
C16 -MTX-ester is available for uptake into the KATO III cells even
without the liposomes being degraded by sPLA2. To further examine
for sPLA2 dependent cytotoxicity, the liposomal formulation was
also tested in HT-29 cells, which do not secrete sPLA2 (Fig. 7b).
In this case, the liposomal formulation behaved similarly to the
free C16 -MTX-ester, supporting our interpretation that the lipo-
some formulated C16 -MTX-ester is available for cell uptake even
without sPLA2 hydrolysis, as the formulation would otherwise be
nontoxic to the cells. Noteworthy though, the liposomal formula-
tion proved more toxic than pure C16 -MTX-ester when sPLA2 was
added externally in the form of COLO 205 conditioned media. At
first glance, this observation may seem contradictory and suggest
a sPLA2 dependent release of the MTX-derivative. However, the
enhanced cytotoxicity at higher concentrations can be explained
by the formation of lysolipid and fatty acid hydrolysis products,
which are known to be cytotoxic in this concentration range (Jensen
demonstrated that it was possible to incorporate a cytotoxic C16
-
MTX-ester analogue into sPLA2-degradable liposomes. However,
the cytotoxic activity results as well as a useful DSC method for eval-
uating the liposomal anchoring stability of alkylated compounds
indicated that the MTX-derivative leaves the liposomes even in the
absence of sPLA2 hydrolysis. Future work is therefore needed to
optimize the anchoring of the MTX-derivative in a liposomal for-
mulation. However, the possibility of using fatty acid derivatized
MTX-esters have been proven viable and it should be possible to

T. Kaasgaard et al. / Chemistry and Physics of Lipids 157 (2009) 94–103
103
anchor the MTX-esters more tightly to the liposomal membrane by
increasing the length of the hydrophobic chain.
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Acknowledgment
MEMPHYS Center for Biomembrane Physics is supported by a
grant from the Danish National Research Foundation.
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