Pharm Res (2012) 29:1435–1443

DOI 10.1007/s11095-011-0619-y

RESEARCH PAPER

Sterically Stabilized Liposomes Incorporating the Novel

Anticancer Agent Phospho-Ibuprofen (MDC-917): Preparation,
Characterization, and In Vitro/In Vivo Evaluation
George Mattheolabakis & Ting Nie & Panayiotis P. Constantinides & Basil Rigas

Received: 12 August 2011 / Accepted: 26 October 2011 / Published online: 10 November 2011
# Springer Science+Business Media, LLC 2011

ABSTRACT KEY WORDS anticancer agent . liposomes . NSAIDs .

Purpose To incorporate phospho-ibuprofen (P-I), a lipophilic, pegylated phospholipids . phospho-ibuprofen
water insoluble novel anti-cancer agent, into pegylated lip-
osomes and upon formulation optimization to evaluate its ABBREVIATIONS
antitumor activity in vitro and in vivo. EE entrapment efficiency
Methods P-I loaded liposomes were prepared using the thin- egg-PC egg-phosphatidylcholine
film hydration method, and characterized for size, zeta P-I phospho-ibuprofen
potential, drug content and drug release. We examined their RES recticuloendothelial system
physical stability by particle size changes; their lyophilization soy-PC soy-phosphatidylcholine
ability in the presence of cryoprotectants; and their antitumor soy-PE soy-phosphatidylethanolamine
activity in vitro in human cancer cell lines and in vivo in a soy-PS soy-phosphatidylserine
xenograft murine model.
Results P-I was successfully loaded into liposomes consisting
of soy-PC and PEG2000-PE. These liposomes were <150 nm INTRODUCTION
in diameter; exhibited prolonged stability in suspension and can
be lyophilized using sucrose as cryoprotectant. P-I liposomes Inflammation is a critical component of tumor progression
inhibited the growth of human cancer cell lines in vitro and in and non-steroidal anti-inflammatory drugs (NSAIDs) are
vivo of xenograft in nude mice to a greater extent than free P-I. efficacious in inhibiting early neoplastic progression and
Conclusions High levels of P-I can be incorporated into malignant conversion (1). For example, the NSAID ibu-
liposomes which can be lyophilized in the presence of sucrose profen reduces the risk of various human cancers, including
and showed good stability upon storage. Moreover, these drug- those of the colon, breast, lung and prostate (2–5). A major
incorporating liposomes were capable of inhibiting the growth of obstacle in the long-term use of NSAIDs for the control of
xenografted tumors in mice more effectively than free P-I. These cancer is their high toxicity and relatively low efficacy. To
results justify further development of the P-I liposomes. overcome these limitations, we developed novel chemical
modifications of conventional NSAIDs (6–8). One such
modified NSAID is phospho-ibuprofen (P-I), a derivative of
ibuprofen, consisting of ibuprofen covalently attached to
G. Mattheolabakis : T. Nie : B. Rigas (*) the diethylphosphate group via a spacer moiety. We have
Division of Cancer Prevention, Department of Medicine
recently reported on its metabolism, pharmacokinetics and
Stony Brook University Stony Brook
HSC, T-17 Room 080 pharmacodynamics in mice (8). P-I inhibited the growth of
Stony Brook, New York 11794-8173, USA colon cancer xenografts in nude mice, but its effect was
e-mail: basil.rigas@stonybrook.edu suboptimal (68% maximal inhibition under our experimen-
P. P. Constantinides
tal protocol). We reasoned that an improved method of
Medicon Pharmaceuticals, Inc. drug delivery could enhance its antitumor efficacy and/or
Stony Brook, New York 11790-3350, USA afford us the opportunity to use lower drug doses.
1436 Mattheolabakis, Nie, Constantinides and Rigas

Liposomes, used extensively as drug carriers, have to prepare a thin lipid film in the inner wall of a 100-ml
already found significant clinical applications due to their round bottom flask. For every liposome formulation a
versatility and biocompatibility (9). They can accommodate standard initial molar ratio of lipids and drug was used
poorly soluble lipophilic agents and increase their thera- consisting of lipids:PEG-PE:drug 4.8:0.5:4.7. The lipid film
peutic index (10,11) as it was demonstrated with pegylated was hydrated with 3 ml of phosphate buffered saline (PBS,
liposomes incorporating various anticancer agents (12). pH 7.4) and vortexed for 10 min. The resultant suspensions
Major contributors to this effect are the prolonged of multilamellar vesicles were subjected to sonication for
circulation time of these liposomes due to avoidance of 10 min in a 5510 Branson bath type sonicator (Danbury,
the recticuloendothelial system (RES), and their preferential CT) followed by extrusion 5 times through a 0.4-μm-
accumulation in solid tumors through the “enhanced polycarbonate membrane and 20 times through a 0.2-μm-
permeability and retention” (EPR) effect (13,14). polycarbonate membrane using an extruder device (Lipex
P-I has low water solubility (logP = 5.22) and the use of Northern Lipids Inc., BC, Canada). Non-entrapped drug
hydrophobic vehicles is required to dissolve it. In this study, we was removed by overnight dialysis with excess PBS.
report on the preparation of P-I loaded liposomes. We studied Entrapped P-I was quantified with high-performance-
the effect of lipid compositions and cryoprotectants on drug liquid-chromatography (HPLC) Waters Alliance 2695
entrapment efficiency, liposome size, zeta potential and long- equipped with a Waters 2998 photodiode array detector
term stability. We also assessed the effect of optimized liposome (220 nm) (Milford, MA) and a Thermo BDS Hypersil C18
formulations on the growth of several human cancer cell lines column (150×4,6 mm, particle size 3 μm) (Thermo Firsher
in vitro and on the growth of a xenograft tumor in mice. Scientific, Waltham, MA) by dissolving a small volume of
the liposome suspension into 1 ml of acetonitrile. The
mobile phase followed a gradient between buffer A (H2O,
MATERIALS AND METHODS acetonitrile, trifluoroacetic acid 94.9:5:0.1 v/v/v) and buffer
B (acetonitrile). The retention time for P-I was previously
Materials determined to be 7.42 min (8).
The size and zeta potential of the liposomes were
Soy-phosphatidylcholine (soy-PC), egg-phosphatidylcholine determined using dynamic light scattering (DLS) and
(egg-PC), soy-phosphatidylserine (soy-PS), soy- microelectrophoresis, respectively, 10 min after diluting
phosphatidylethanolamine (soy-PE) and methoxy-poly the samples in PBS and housing them at 25°C using the
(ethylene glycol) 2000-distearoylphosphatidylethanolamine Zeta-Plus Brookhaven instrument (Holtsville, NY).
(PEG2000-PE) were obtained from Avanti Polar Lipids The morphology of the liposomes was determined by
(Alabaster, AL). Human insulin, cholesterol, octadecylamine transmission electron microscopy (TEM). For negative stain-
(stearylamine), acetonitrile, chloroform, miscellaneous ing, liposomes were diluted with distilled water and dropped
reagents and solvents, all of analytical grade, were obtained on a copper grid and air-dried for 1 min at room temperature.
from Sigma (St. Louis, MO). After adhesion of liposomes, 10 μl of 2% uranyl acetate
solution, filtered through a 0.2 μm filter prior to use, were
Cell Culture dropped onto the grid as a staining solution. The excess
staining solution was removed with filter paper after 30 s and
MCF-7 and MDA-MB-231 human breast cancer and SW840 the sample was air-dried for 10 min at room temperature,
human colon cancer cell lines were purchased from American followed by observation under TEM.
Type Culture Collection (ATTC, Manassas, VA) and grown Nuclear magnetic resonance (NMR) was performed
as monolayers. Cell culture media (MEM for MCF-7 cells; L- using a Varian Instrument (Santa Clara, CA) at 500 Mhz
15 for MDA-MB-231 cells; and RPMI 1640 for SW480 cells) by dissolving lyophilized liposomes with entrapped drug in
were supplemented with 10% heat-inactivated fetal bovine deuterated chloroform to determine the PEG content of the
serum (FBS), 1% antibiotics (50 U/ml penicillin and dispersion.
0.05 mg/ml streptomycin) and 0.01 mg/ml human insulin The entrapment efficiency of the drug (EE) is defined as
(insulin used only for MCF-7). Cells were maintained at 37°C the ratio of the amount of drug entrapped in liposomes to
in a humidified incubator containing 5% CO2. the total amount of drug initially introduced in the
formulation.
Preparation and Characterization of Liposomes
Containing P-I Lipid Substitution

A chloroform solution of lipids and P-I was slowly We used various lipid mixtures to obtain liposomal
evaporated in a rotary evaporator (Buchi R11, Switzerland) dispersions of P-I and determined their EE, size and zeta
Liposome Formulation of Phospho-Ibuprofen 1437

potential. The lipid mixtures included either partial studied as cryoprotectant agents in different molecular
substitution of soy-PC with other lipids as soy-PS and soy- ratios with the lipids.
PE or complete substitution of soy-PC with egg-PC. We
also evaluated the addition of cholesterol or stearylamine to In Vitro Cytotoxicity Assay
the liposome formulations. (Table I lists all tested formula-
tions). All experiments were performed at least in duplicate. The cytotoxicity of P-I loaded liposomes against MDA-MB-
231 and MCF-7 breast and SW480 colon cancer cells was
In Vitro Release of P-I from Liposomes and Liposome determined using the MTT assay and compared to that of
Stability free P-I (15). Briefly, 104 cells/well were plated in 96-well
flat-bottom tissue-culture plates in 100 μl culture medium,
Release of P-I from P-I loaded liposomes was studied using and incubated for 24 h, when the test compounds were
the dialysis method at room temperature. Liposome added. Twenty four hours later MTT solution (5 mg/ml in
samples of 0.5 ml (7 mg/ml) were placed in dialysis bags PBS pH 7.4) was added into each well and the plates were
(MWCO 3.500, Thermo Scienctific, Rockford, IL) and incubated at 37°C for 4 h. A volume of 100 μl of 10% SDS
tightly sealed. The bag was immersed in 100 ml of PBS in 0.01 M HCl was added into each well and the plates
under mild stirring. Samples of the release medium (0.5 ml) were incubated at 37°C overnight to solubilize the
were taken in predetermined time intervals and replaced formazan crystals. Absorbance was determined at 570 nm
with the same volume of fresh medium. The concentration using a microplate reader (Molecular Devices, USA). The
of P-I was determined by HPLC. experiments were performed in hexaplicates and repeated
Liposome dispersions were kept at room temperature twice.
and at predetermined time intervals their size and zeta
potential were assessed to evaluate the stability of the Treatment of Nude Mice with Colon Cancer
liposome formulations. Xenografts

Liposome Lyophilization SW480 cells (2× 106, suspended in 100 μl Matrigel (BD
Matrigel Matrix, BD Biosciences)/PBS (1:1)) were xeno-
Aliquots of freshly prepared liposomes were lyophilized in grafted subcutaneously in both flanks of 5–6 weeks old
the presence or absence of cryoprotectant. Briefly, 0.2 ml of female SCID mice (Harlan Sprague–Dawley, Indian-
liposome suspension were placed inside Eppendorf tubes apolis, IN). When the average xenograft tumor volume
and mixed with a small volume of concentrated cryopro- reached ∼70 mm3, animals were divided into three treatment
tectant solution. The mixture was vortexed for 1 min groups: a) vehicle (n=8); b) free P-I 100 mg/kg (n=10); c)
followed by rapid freezing with liquid nitrogen and 24- liposome P-I 100 mg/kg(n=10); and d) liposome P-I
h lyophilization. The lyophilized liposome powder was re- 300 mg/kg(n=10), all administered by intraperitoneal
suspended in the original volume of water and vortexed for injection once a day 5 d/week for 17 d. Tumor volume
1 min. The size of the liposomes in the suspension was was calculated by measuring with a caliper its length (L) and
determined as above. Sucrose, mannitol and trehalose were width (W) according to the formula, L×W×(L+W/2)×0.56.

Table I Liposome Formulation Groups

Group Soy-PC Egg-PC Soy-PS Soy-PE Cholesterol Stearylamine

100% of total 10% of total 10% of total % of total l % of total
lipids mol:mol lipids mol:mol lipids mol:mol ipid mol:mol lipids mol:mol

A + − − − − −
B + − + − − −
C + − − + − −
D − + − − − −
E + − − − + 1% −
F + − − − + 5% −
G + − − − + 10% −
H + − − − − + 5%
I + − − − − + 10%
J + − − − − + 15%
1438 Mattheolabakis, Nie, Constantinides and Rigas

Xenograft growth inhibition in response to treatment was increase the drug EE without significantly altering the zeta
calculated by comparing the difference in percent increase potential of the liposome suspension. Complete substitu-
of tumor growth from their 0 time volume to their volume at tion, however, of egg-PC for soy-PC failed to meaningfully
sacrifice between drug- and vehicle-treated groups. improve these two parameters. Although the average size of
the liposomes was reduced on average by 15 nm, drug EE
was also significantly reduced by 8%. Similarly, partially
RESULTS substituting soy-PS and soy-PE for soy-PC provided no
substantial improvement.
Liposome Formulations To ensure that the ratios of the component lipids were
not significantly altered during liposome preparation, the
P-I liposomes were prepared by extrusion of multi- drug:lipid ratio was monitored throughout their prepara-
lamellar liposomes using various phospholipid mixtures tion process using 1H NMR spectroscopy. Figure 1 (lower
(Table I). To generate homogenous unilamellar vesicles, panel) shows a 1H NMR spectrum of a liposome suspension
hydration of the drug-lipid film was employed, followed at its final stage. Assignments of the 1H resonances reveal
by extrusion through polycarbonate membranes. Lip- that the molar ratio of the soy-PC to the PEG-PE is 10:1
osomes were characterized in terms of their size, zeta and the molar ratio of drug:soy-PC is 1:1. Both results are
potential, drug loading and long-term stability. Table II identical to the input ratios, indicating that the drug:lipid
summarizes these results. ratio is maintained throughout the formation of the
Liposomes composed of phosphatidylcholine (PC) and liposomes. Similar results we obtained with egg-PC
equimolar quantities of P-I showed satisfactory entrapment liposomes (data not shown).
efficiency, (around 60%), with drug content of about
15 mg/ml, which is considered sufficiently high for a Liposome Stability and Release
hydrophobic molecule like P-I (10). The average hydrody-
namic diameter of these liposomes was 140 nm and their Liposome stability is a property that can critically impact
zeta potential −28.7 mV. Images from negative-staining their intended clinical application (9). Instability of a
TEM revealed liposomes of discrete and round structure, liposome suspension leads to aggregation or fusion of
ranging in diameter from 100 to 150 nm (Fig. 1); these liposomes during storage, which in turn increases particle
results are in close agreement with their hydrodynamic size. Particles of greater size are generally taken up rapidly
diameter (Table II). by the RES resulting in their rapid clearance and shortened
Using phophatidylserine (PS) and, to a lesser extent, half-life. Additionally, the instability of the liposomes
phosphatidylethanolamine (PE) in liposome preparation has accelerates drug release during storage.
been shown to reduce their size without significantly The development of liposomes as effective drug delivery
altering their zeta-potential at physiological pH (16,17). systems was aided by the incorporation of membrane
Thus, we substituted, partially or completely, egg-PC, soy- rigidifying agents such as cholesterol. Early studies demon-
PE and soy-PS for soy-PC and determined the effect of strated that cholesterol enhanced the retention of entrapped
each substitution on drug entrapment and liposome size agents by modulating the fluidity of the liposomal mem-
and other physicochemical characteristics. The goal of brane (18,19). Indeed, cholesterol in the phospholipid
these substitutions was to reduce the liposomal size or membrane bilayer can increase the stability of liposomes

Table II Characteristics of
Liposome Formulations Group Hydrodynamic diameter, nm PDI Zeta potential EE Drug content
mV % mg/ml

A 140±5 0.122±0.03 −28.7±4 57±3 15±3

B 132±6 0.120±0.04 −31.1±1 54±6 15±4
C 133±8 0.117±0.04 −30.4±4 60±4 17±2
D 125±4 0.181±0.03 −40±4 49±2 13±1
E 150±3 0.112±0.03 −34.7±1 60±1 16±1
F 148±6 0.131±0.04 −32±2 61±1 16±1
G 170±5 0.179±0.03 −32.7±4 60±4 16±2
H 131±8 0.082±0.08 −24±4 48±6 13±2
All determinations were per- I 127±2 0.098±0.01 −9.7±2 51±4 13±3
formed at least in duplicate; values, J 123±3 0.112±0.01 10.3±4 52±7 14±2
mean±SEM
Liposome Formulation of Phospho-Ibuprofen 1439

Fig. 1 Soy-PC liposomes with

incorporated P-I. Upper panel:
TEM microphotographs of
liposomes. Bar = 100 nm. Lower
panel: 1H NMR spectrum of
lyophilized liposomes with
incorporated P-I at 500 MHz after
dissolving them in deuterated
chloroform.

(19). We therefore assessed the effect of cholesterol on Surface Charge

liposome stability. To this end, only soy-PC liposomes was
used since, as shown above, soy-PE, soy-PS and egg-PC The surface charge of nanocarriers affects the mechanism
had no favorable effects on the characteristics of the and extent of their interactions with cells and tissues and
liposomes. may accelerate their plasma clearance after systemic
As shown in Table II (groups E-G), cholesterol 1–10% administration (20). For example, the combination of
did not influence drug EE but increased the diameter of the neutral surface charge with a hydrophilic polymer like
liposomes (from 150 to 170 nm), a change that paralleled PEG significantly prolongs the circulation time of nano-
cholesterol content. Figure 2 demonstrates that stability of carriers in vivo, enhancing their therapeutic effect (13). The
the liposomes, evaluated by liposome size and the rate of use of stearylamine in liposomes has been demonstrated to
drug release, was independent of cholesterol content under alter the positive surface charge of the liposomes, facilitat-
the employed experimental conditions. Based on these ing their interaction with negatively charged targets (21).
results, cholesterol was not included in further studies. Of Consequently, we prepared liposomes with stearylamine
interest, this study also established the prolonged stability of content between 5% and 15% and determined their size,
liposomes with little or no aggregation or precipitation for zeta potential and drug EE (Table 2, groups H–J). While
at least up to 1 month of storage. size and drug EE of the liposomes were not significantly
1440 Mattheolabakis, Nie, Constantinides and Rigas

the presence of cryoprotectants. Lyophilized liposomes

exhibit prolonged shelf-life and are reconstituted with
vehicle immediately prior to their use. (20). Three potential
cryoprotectants were evaluated, sucrose, mannitol and
trehalose, by determining their effect on liposome size
upon reconstitution. Figure 4 shows the effect of various
molar ratios of total lipids:sugar (mol:mol; range 3:1–1:24)
on the hydrodynamic radius of liposomes after they were
resuspended following lyophilization. Sucrose at lipid:
sucrose ratios ≥1:9 protected the liposomes from aggregat-
ing during storage. In contrast, mannitol was unable to
protect the liposomes from aggregating even at molecular
ratios of lipid:mannitol 1:24. Trehalose, a natural α-linked
disaccharide often successful in such applications (22), also
failed to protect the liposomes in the same molecular ratios
(data not shown).

The Effect of Liposomal P-I on the Growth of Cancer

Cells In Vitro and In Vivo

Initially, we used the MTT assay to determine the

cytotoxicity of P-I incorporated in liposomes (liposomal
Fig. 2 Liposome size and drug release during storage. Changes in the P-I) in cancer cell lines in vitro. We studied two breast cancer
size of liposomes (upper) and release of PI from the liposomes (lower) cell lines, MDA-MB-231 and MCF-7, and the SW480
upon storage as a function of storage time for 0%, 1%, 5% and 10% colon cancer cell line and compared the effect of liposomal
molecular ratios of incorporated cholesterol.

altered by stearylamine, their zeta potential changed signifi-

cantly from −28 mV for liposomes without stearylamine
(controls) to +10 mV for 15% of stearylamine (Fig. 3).

Liposome Behavior upon Lyophilization

Loss of liposome stability during storage can be overcome

by lyophilization during which liposomes are freeze-dried in

Fig. 4 Changes of the hydrodynamic diameter for liposomes during

lyophilization. Liposomes with 0%, 5% and 10% of incorporated
stearylamine were studied in the presence and absence of sucrose (upper)
Fig. 3 The effect of stearylamine on liposome characteristics. Particle size, or mannitol (lower) used as cryoprotectants. *Pre: liposomal size prior to
zeta potential and drug EE as a function of the stearylamine content of the lyophilization; No sugar: liposomal size after lyophilization without
liposomes. cryoprotectants.
Liposome Formulation of Phospho-Ibuprofen 1441

to optimize the liposomal formulation of P-I using

PEGylated liposomes and have now demonstrated the
feasibility of this approach.
We used a series of lipid mixtures to prepare liposomes
and assessed their potential relevance using standard
criteria for their intended clinical application. Two of the
crucial investigated parameters were liposome size and zeta
potential, as they can affect the therapeutic efficacy of the
incorporated P-I. Size and charge are determinants of the
efficacy and biodistribution of liposomes, especially through
the EPR effect (20–22,24). Based on these considerations,
we sought to generate liposomes less than 200 nm in
Fig. 5 Effect of free P-I and P-I loaded liposomes on the growth of diameter with zeta potential close to neutrality (13), and
SW480 xenografts. Each formulation of P-I was administered daily i.p.
optimize drug loading, and stability that makes their use
Values: Mean±SEM. *, p<0.05; **, p<0.01; compared to control.
practical by balancing these parameters.
Of the evaluated lipids, soy-PC generated optimum
P-I to that of free P-I. The values of 24-h IC50 (concentra- results as judged by the above criteria. All other phospho-
tion that inhibits cell growth by 50% at 24 h) were as follows: lipids generated liposomes with unfavorable size or drug
MDA-MB-231 cells: P-I=18 μM, liposome P-I=40 μM; EE, whereas high cost was an additional factor in rejecting
MCF-7 cells: P-I=27 μM, liposome P-I=56 μM; and SW480 soy-PS and soy-PE. Despite strong evidence in the literature
cells: P-I=32 μM, liposome P-I=52 μM. Empty liposomes that cholesterol can enhance the stability of liposomes, in
had no effect on cell growth (data not shown). the present studies cholesterol made no additional contri-
We also evaluated the effect of liposomal P-I on the bution to liposome stability. Stearylamine, known to add
growth of SW480 subcutaneous xenografts in SCID mice. cationic charges to the surface of liposomes (21), was of no
As shown in Fig. 5, liposomal P-I inhibited in a dose- added benefit to liposome formulation and thus it was not
dependent manner the growth of these xenografts, reducing included in the final formulation.
at the end of the study the growth of xenografts by 42% Successful long-term storage of liposomes, as already
(p<0.05) at its lower concentration (100 mg/kg/d) and mentioned, facilitates their clinical use. Sucrose was shown
67% (p<0.01) at its higher concentration (300 mg/kg/d), to protect liposomes during lyophilization, a widely
compared to control. Free P-I (100 mg/kg/d) had only a employed method to prolong the shelf-life of liposomes. It
modest (25%) and statistically not significant inhibitory was of interest that neither mannitol nor trehalose, two
effect on tumor growth. Interestingly, the difference in commonly used cryoprotectants that have been useful in
efficacy between free P-I and liposome P-I at the same dose various systems (25), had no appreciable effect on the
(100 mg/kg/d) is statistically significant (p<0.05). Liposo-
mal P-I was well tolerated by the mice, which showed no
weight loss or other signs of toxicity.

DISCUSSION

Facilitated drug delivery emerges as a key contributor to

the efficacy of novel anticancer agents. Nanocarriers are
being increasingly appreciated as useful drug delivery
vehicles and their initial promise has now been realized in
the clinic (12). For example, liposomal formulations of
paclitaxel and doxorubicin have already been approved for
the treatment of cancer patients. We have responded to the
current paucity of anti-cancer agents by synthesizing the
novel modified NSAIDs, represented here by P-I (8). All
modified NSAIDs that we have synthesized to date,
Fig. 6 Proposed relative position of P-I in the liposomal bilayer. In this
including P-I, display significant lipophilicity and poor diagram, P-I is positioned between the lipids; two PC molecules are
aqueous solubility requiring lipid vehicles for solubilization shown. The hydrophilic part of the P-I molecule protrudes from the outer
and delivery (23). Through a systematic process we sought margin of the bilayer.
1442 Mattheolabakis, Nie, Constantinides and Rigas

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