Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells
Anti-GD2 Immunoliposomes for Targeted Delivery of the Survivin Inhibitor Sepantronium Bromide (YM155) to Neuroblastoma Tumor Cells
Shima Gholizadeh 0 1 2 3
Emmy M. Dolman 0 1 2 3
Rebecca Wieriks 0 1 2 3
Rolf W. Sparidans 0 1 2 3
Wim E. Hennink 0 1 2 3
Robbert J. Kok 0 1 2 3
Robbert J. Kok 0 1 2 3
0 Princess Maxima Center for Pediatric Oncology Utrecht , the Netherlands
1 Department of Oncogenomics, Academic Medical Center University of Amsterdam Amsterdam , the Netherlands
2 Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences Utrecht University Utrecht , the Netherlands
3 Department of Chemical Biology and Drug Discovery, Utrecht Institute for Pharmaceutical Sciences Utrecht University Utrecht , the Netherlands
Purpose Sepantronium bromide (YM155) is a hydrophilic quaternary compound that cannot be administered orally due to its low oral bioavailability; it is furthermore rapidly eliminated via the kidneys. The current study aims at improving the pharmacokinetic profile of YM155 by its formulation in immunoliposomes that can achieve its enhanced delivery into tumor tissue and facilitate uptake in neuroblastoma cancer cells. Methods PEGylated YM155 loaded liposomes composed of DPPC, cholesterol and DSPE-PEG2000 were prepared via passive film-hydration and extrusion method. Targeted (i.e. i m m u n o - ) l i p o s o m e s w e r e p r e p a r e d b y s u r f a c e functionalization with SATA modified monoclonal antidisialoganglioside (GD2) antibodies. Liposomes were characterized based on their size, charge, antibody coupling and YM155 encapsulation efficiency, and stability. Flow cytometry analysis and confocal microscopy were performed on IMR32 and KCNR neuroblastoma cell lines. The efficacy of developed formulations were assessed by in-vitro toxicity assays. A pilot pharmacokinetic analysis was performed to assess plasma circulation and tumor accumulation profiles of the developed liposomal formulations. Results YM155 loaded immunoliposomes had a size of 170 nm and zeta potential of −10 mV, with an antibody coupling efficiency of 60% andYM155 encapsulation efficiency of14%. Targeted and control liposomal formulations were found to have similar YM155 release rates in a release medium containing 50% serum. An in-vitro toxicity study on KCNR cells showed less toxicity for immunoliposomes as compared to free YM155. In-vivo pharmacokinetic evaluation of YM155 liposomes showed prolonged blood circulation and significantly increased half-lives of liposomal YM155 in tumor tissue, as compared to a bolus injection of free YM155. Conclusions YM155 loaded immunoliposomes were successfully formulated and characterized, and initial in-vivo results show their potential for improving the circulation time and tumor accumulation of YM155.
immunoliposomes; neuroblastoma cells; sepantronium bromide (YM155); targeted delivery
AUC Area under curve
BIRC5 Baculoviral inhibitor of apoptosis
BSA Bovine serum albumin
CA Compartmental analysis
CLSM Confocal laser scanning microscope
CNS Central nervous system
Neuroblastoma (NB) is an aggressive malignancy of the
sympathetic nervous system and is the most frequently occurring type
of solid extracranial tumor in children (
). Although the survival
rate in low-risk patients is currently over 90%, children with
high-risk neuroblastoma currently have a very poor prognosis.
For these patients, the 5-year disease free survival rate is
between 25%–35%, despite many patients undergoing aggressive
multi-modality therapies (i.e. combinations of chemotherapy, surgery,
stem cell rescue and radiation therapy) (
). This highlights the
urgent need for new therapeutic strategies (
The pathology of neuroblastoma (as well as most other
types of cancer) is complex and differs between individual
patients. Anti-apoptotic proteins have been shown to play a
role in tumor development, thus making such proteins
promising drug targets. One of these proteins is survivin, which is
encoded by the ‘baculoviral inhibitor of apoptosis
repeatcontaining 5’ (BIRC5) gene. Over-expression of the
survivinencoding gene has been shown to correlate strongly to a poor
patient prognosis, metastatic spread and (increased) resistance
to chemotherapy (
). Hence, survivin inhibitors could
potentially be used in neuroblastoma treatment, especially for high
risk patients with poor prognosis (
). Anti-BIRC5 antisense
based therapies such as gataparsen sodium (LY2181308) and
small molecular inhibitors of anti-apoptotic proteins are
currently being tested in phase I/II clinical trials (
One of the promising small molecular inhibitors of survivin
is sepantronium bromide (YM155) of which the structure is
shown in Fig. 1 (
). Recent phase I/II ‘single agent’ clinical
trials have shown acceptable toxicity in patients with advanced
solid malignancies (
). The physicochemical and
pharmacological properties of YM155 are listed in Table I. Due to the
hydrophilicity and permanent cationic charge on one of the
nitrogen atoms of the molecule, YM155 is rapidly eliminated
via organic cation transporter (OCT) mediated excretion in
bile and urine. Because of this, YM155 has a short plasma
half-life of approximately 1–2 h, as determined in
pharmacokinetic measurements in mice and rats (
). The rapid
elimination of YM155 dictates administration as intravenous
infusion rather than intravenous bolus injection (
Nano-encapsulation technologies and application of the
resulting so-called nanomedicines in health care have received
Fig. 1 Molecular structure of Sepantronium bromide (YM155).
much attention in recent years, in particular for application in
cancer therapy (
). Long circulating nanomedicines such as
PEGylated liposomes can improve tumor drug delivery by
virtue of the enhance permeability and retention (EPR) effect, i.e.
the accumulation and retention of nanocarriers in the tumor
site due to differences between normal vasculature and tumor
blood vessels (18). Thus, the rapid elimination of YM155 and
its poor pharmacokinetic properties can potentially be
improved by encapsulating the YM155 molecules in a
nanocarrier that can prevent renal excretion of the compound.
In recent studies, such a PEGylated liposomal dosage form of
YM155 has been introduced and studied in rodent cancer
models for tumor drug uptake and efficacy (
). We now
propose a novel immunoliposomal formulation for YM155,
which can further improve its delivery to neuroblastoma.
Targeted liposomes (immunoliposomes) have been modified
with ligands such as antibodies (Ab) that bind to cell-surface
exposed receptors on target cells. Such a binding of the
nanomedicine to receptors can be an efficient strategy to
facilitate the intracellular delivery of drugs into target cells, which
can further enhance their specificity for the targeted cell type
). Disialoganglioside GD2 is an attractive target for an
immunoliposome based strategy since GD2 is extensively
expressed by neuroblastoma tumor cells (
), while it is
virtually absent in nonmalignant tissues outside the central
nervous system (CNS) (
). GD2-positive tumor cells can be
recognized specifically by anti-GD2 antibodies (Ab) or anti-GD2
Ab functionalized nanoparticles (
). Chimeric and humanized
anti-GD2 antibodies have been investigated for
immunotherapy, tumor vaccination and as targeting ligand for drug delivery
). Although GD2 is expressed in neurons, the
human brain is protected from parenteral anti-GD2 Ab and
antiGD2 decorated nanoparticles by the blood-brain barrier.
In this study, we investigate the formulation of YM155 in
GD2-targeted immunoliposomes for the specific delivery of
YM155 to neuroblastoma tumor cells. The obtained
formulation was evaluated for its in-vitro stability and YM155 release
kinetics at different release conditions. The cell specific binding
and uptake of the liposomal formulations (i.e. targeted and
nontargeted control) were analyzed in cultured NB tumor cells,
followed by an in-vitro efficacy evaluation of the YM155
liposomes and free YM155. Finally, a pharmacokinetic pilot study
was conducted to investigate the plasma half-life and tumor
accumulation of the YM155 immunoliposomes in mice.
MATERIALS AND METHODS
The lipids 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
1,2-distearoyl-sn-glycero-3-phosphoethanolamineN-[methoxy(polyethylene glycol)-2000] (DSPE-PEG2000)
1,2-dimyristoyl-sn-glycero-3-phosphoethanolamineN-(lissamine rhodamine-B sulfonyl) (Rho-PE) were purchased
from Avanti Polar Lipids (Alabaster AL, USA). Cholesterol
(Chol) was obtained from Sigma Aldrich (St. Louis MO,
USA). Sepantronium bromide (YM155) was purchased from
B i o - c o n n e c t ( H u i s s e n , T h e N e t h e r l a n d s ) . A n t i
disialoganglioside GD2 (Anti-GD2) antibody was obtained
f r o m B D P h a r m i n g e n ( A l p h e n a a n d e n R i j n ,
The Netherlands). N-succinimidyl S-acetylthioacetate
(SATA) and Hoechst 33,342 Fluorescent Stain were obtained
f r o m T h e r m o F i s c h e r S c i e n t i f i c ( L a n d s m e e r ,
The Netherlands).4′,6-Diamidino-2-phenylindole (DAPI)
was obtained from Roche (The Netherlands). FluorSave
mounting agent was obtained from Merck Millipore (San
Diego, CA, USA). All chemicals used were analytical grade
unless otherwise stated. Cell culture media and supplements
were obtained from Gibco laboratories (Gaithersburg,
United states). Fetal calf serum (FCS) was obtained from
Preparation of Liposomes
Immunoliposomes were prepared from a mixture of DPPC,
Cholesterol, DSPE-PEG2000 and DSPE-PEG2000-Mal in
mol a r r a t i o s o f 1 . 3 6 : 1 . 3 6 : 0 . 1 4 : 0 . 1 4 , b a s e d o n p r i o r
immunoliposomal formulations developed in our department
). Control liposomes consisted of DPPC, Cholesterol and
D SP E - P E G 2 0 0 0 i n m o l a r r a t i o s o f 1 . 3 6 : 1 . 3 6 : 0 . 2 8 .
Fluorescently labeled liposomes were prepared by adding
rhodamine-PE to the lipid mixture at 0.2 mol% of total lipid
(TL). Lipids were dissolved in chloroform: methanol (9:1, v/v)
and to a final concentration of 90 mM TL. YM155 was
codissolved with the lipid mixture at a concentration of
0.25 μmol/μmol TL. Organic solvents were removed using a
rotary evaporator and subsequent drying under a nitrogen
flow. The resulting drug-lipid film was hydrated by adding
3 ml of HBS buffer pH 7.4 (10 mM Hepes containing
135 mM NaCl). Unilamellar liposomes were prepared by
multiple extrusion steps over polycarbonate membranes
(Nuclepore, Pleasanton, CA, USA) at decreasing pore size
range (from 0.4 to 0.1 μm). Next, targeted liposomes (but not
control liposomes) were reacted with Sata-anti-GD2 antibody
at a final concentration of 30 μg/μmol TL (2 mg/ml antibody)
that had been deprotected with hydroxylamine 0.5 M for
45 min prior to its addition to the liposomes. Coupling of
Sacetylthioacetyl (SATA) to anti-GD2 (8:1 SATA:Ab mol:mol
ratio) was performed as described previously (
non-reacted antibody and non-encapsulated YM155 were
removed by ultracentrifugation at 60,000 g at 4°C for 30 min and
resuspension of liposomes in 3 ml HBS This step was repeated
in three times. After the final purification step, liposomes were
stored under nitrogen atmosphere at 4°C in the dark for a
maximum of 4 weeks prior to usage.
Characterization of Liposomes
Liposome size and polydispersity was measured using
dynamic light scattering (DLS) on an ALV CGS-3 system (Malvern
Instruments, Malvern, UK). Zeta-(ζ) potential of liposomes
was measured using a Malvern Zetasizer Nano-Z (Malvern
Instruments) with universal ZEN 1002 dip cells and DTS
(Nano) software. The total lipid concentration was determined
according to Rouser (
). YM155 content of the liposomes
was determined from 100 μl aliquots which were disrupted
by diluting in 300 μl of acetonitrile (ACN). YM155 content
was measured with a UPLC Waters Acquity system (Waters
Corporation, Milford, MA, USA) equipped with Acquity
UPLC ®BEH C18, 1.7 μm column (2.1 × 50 mm)
thermostated at 50°C and a UV detector (Waters
Corporation, Milford, MA, USA) operated at 252 nm. The
gradient mobile phase was operated at a flow rate of 0.5 ml/
min and consisted of 5% (v/v) ACN in water containing in
total 0.1% (v/v) trifluoroacetic acid (solvent A) and 100%
ACN containing 0.1% trifluoroacetic acid (solvent B).
YM155 encapsulation efficiency (EE %) was calculated from
the determined YM155/TL ratio versus the initial drug/TL
ratio. Conjugation of anti-GD2 to DSPE-PEG2000-Malwas
confirmed by western blotting using anti-mouse IgG
immunodetection. In brief, reduced samples were subjected
to SDS-PAGE using 4–12% gradient NuPAGE Novex
BisTris mini-gel (Thermo Fischer Scientific). Samples were
electrotransferred onto a nitrocellulose membrane via an
iBlot Dry Blotting system (Invitrogen). Next, the membrane
was blocked with 5% (w/v) BSA in Tris-buffered saline
containing 0.1% Tween-20 (TBS-T) and incubated with
goatanti mouse-IgG secondary antibody conjugated to
horseradish peroxidase (HRP) (Thermo Fischer Scientific), diluted
1:1000 in 5% BSA in TBS-T. Proteins were visualized and
detected using SuperSignal West Femto Chemiluminescent
Substrate (ThermoFischer Scientific) and a Gel Doc imaging
system equipped with a XRS camera and Quantity One
analysis software (Bio-Rad, Hercules, CA, USA). Coupling of
antiGD2 to targeted liposomes was quantified were further
characterized by micro-BCA assay (Pierce Biotechnology,
Rockford, IL, USA) using mouse IgG (Sigma Aldrich) as
calibration standards. Antibody coupling degree
(antibody/liposome; ) was calculated using geometric arguments according
to Adrian (
) using the following formula: = π/
6 × CAb × (3dbl × R2 – 3R × dbl2 + dbl3) × Mab−1× VLs−1,
in which (CAb) is the measured concentration of coupled
antibody (g/mol TL), (MAb) is the molecular mass of the
antibody, (R) is the average diameter of liposomes (nm) of the
spherical liposomes and (VLs) and (dbl) represent the specific
lipid volume and lipid bilayer thickness, respectively.
Storage Retention of YM155 Liposomes
The storage retention of encapsulated YM155 in
liposomal formulations in HBS buffer was studied at 4°C for a
total duration of 4 weeks. At 0, 5, 10, 20 and 30 days
samples were collected in duplicate. Released
(nonencapsulated) YM155 was separated from liposomal
YM155 by ultrafiltration using Vivaspin centrifugal
concentrators with a molecular weight cut-off membrane of
10 kDa (Sartorius AG, Aubagne, France) for 5 min at
4000 g at 4°C. YM155 was determined in both the filtrate
(free YM155, released fraction) and the remaining
fraction in the upper compartment of the Vivaspin device
(total YM155, free and liposomal fraction) which
represented at least 70% of the starting volume. The collected
samples (both fractions) from each time point were stored
in the dark at 4°C for further analysis. Aliquots of 50 μl
were taken and diluted in 150 μl ACN, centrifuged at
22,000 g for 10 min at 4°C and subjected to UPLC
analysis as described above. Experiments were repeated two
times with individually prepared batches of liposomes.
Particle size and polydispersity were studied at the
beginning and end of the study by DLS as described above.
In-Vitro Release of YM155 from Liposomes
Release studies were conducted to study the influence of
serum proteins and temperature on retention of YM155
in liposomes. Aliquots of anti-GD2 targeted and control
liposomes with a final YM155 concentration of 25–30 μM
were prepared by diluting the stock solutions prepared for
each batch with incubation media consisting of either
HBS buffer, 5% bovine serum albumin (BSA) in HBS
buffer, or pooled female mouse serum containing
EDTAdisodium (Seralab Laboratories, Haywards Heath, UK)
diluted 1:1 with HBS buffer. Samples were incubated at
either 37°C or 4°C while gently shaking on a roller bench
for a total duration of 24 h. At pre-determined time
periods of 6, 12 and 24 h, samples were removed from the
incubators and concentrations of released YM155 and
total YM155 were determined as described above.
Experiments were repeated two times with individually
prepared batches of liposomes. YM155 retention in
liposomes at each time point was calculated after correction
for low levels (<5%) of free YM155 at the beginning of
the incubations. The YM155 release data were fitted with
zero-order or first order kinetic models, thereby using the
FZierrsot‐‐oorrddeerr kkiinneettiiccss :: MMtt==MM00 ¼¼ e−−kk**tt
Where M0 is the amount of encapsulated YM155 at the
start of the incubation and Mt is the amount of
encapsulated YM155 at each time point; k is the release rate
constant. The release rate constants (k) reported in this
work are calculated from first-order kinetics fits to
experimental release data. The calculated release rate constants
allow a quantitative comparison of the experimental
YM155 release profiles of the (liposomal) formulations
under the various conditions.
Culturing of Tumor Cell Lines
Neuroblastoma tumor cell lines KCNR, IMR32 and WiDR
colon adenocarcinoma cells were obtained from ATCC™. Cells
were cultured in Dulbecco’s Modified Eagle’s Medium
(DMEM) containing D-glucose (4.5 g/L) and 2 mM
glutamate, without pyruvate, and supplemented with 10% fetal calf
serum (FCS), 1% MEM Non-Essential Amino Acids (NEAA)
and 1% L-Glutamine. Cells from passage 5 to 7 were seeded
one day before the experiment in culture flasks (T-75 cm2) at
2–4 × 106 cell densities, unless stated differently.
Assessment of GD2 Expression in Neuroblastoma Cell
Neuroblastoma cell lines were studied for GD2 expression
levels by flow cytometry. Cells from each cell line were
trypsinized and resuspended at a concentration of
approximately 4 × 106 cells/ml in 10 ml of PBS supplemented 6.6%
FCS and 20 mM EDTA (flow cytometry buffer). Cells were
incubated with either anti-GD2 Ab or mouse isotype control
Ab (Thermo Fischer Scientific) as a negative control, at a 1:200
dilution for 45 min at room temperature. Next, cells were
washed three times with flow cytometry buffer by
centrifugation for 5 min at 300 g and suspended in flow cytometry buffer.
After the final washing step, cells were suspended in flow
cytometry buffer FACS containing FITC-labeled
Goat-antiMouse IgG (H + L) secondary antibody (Thermo Fischer
Scientific) at a dilution of 1:960, followed by incubation at room
temperature in the dark for 20 min. Next, the cells were washed
as explained above. Gates were set for GD2-positive and
negative fluorescence signals, based on the fluorescence intensity
obtained from cells as measured by the AccuriTM C6 Flow
Cytometer (BD Biosciences, Erembodegem, Belgium). The
analysis was based on both living and dead cells. Generally,
10,000 events were acquired per sample. Data were analyzed
with CFlow Plus software (BD Biosciences).
Binding and Uptake Studies with Rhodamine Labeled
The binding of rhodamine labeled GD2-targeted and control
liposomes to GD2 positive (i.e. IMR32) and GD2 negative (i.e.
WiDR) tumor cells was studied by flow cytometry, using an
average of 40 × 103 cells/ml in flow cytometry buffer. Cells were
incubated with liposomes for 1 h at 4°C in the dark at different
lipid concentrations ranging from 0.1 to 2 mM TL. Cells were
washed three times with FACS buffer as described in the
previous section after which mean fluorescence intensity (MFI) of the
rhodamine signal was determined using the AccuriTM C6 Flow
Cytometer. Generally, 10,000 events were acquired per sample.
Data were analyzed with CFlow Plus software.
The uptake of rhodamine-labeled liposomes was studied
using KCNR neuroblastoma cells by fluorescence microscopy
using a Leica TCS-SP confocal laser-scanning microscope
(Leica, Heidelberg, Germany). KCNR cells were seeded at a
concentration of 40 × 104 cells/ml on FluoroDish™ glass
bottom petri-dishes and allowed to adhere overnight. Cells were
incubated with rhodamine labeled liposomes at a TL
concentration of 1 mM for 4 h at 37°C, followed by washing with
cold PBS. Cells were fixed (4% (v/v) formaldehyde in PBS; 30
mi at room temperature) and nuclei were stained using
Hoechst 33,342 (5 min at room temperature). After washing
with cold PBS, the petri-dishes were dried and covered with
FluorSave mounting agent and kept at 4°C until confocal
microscopy analysis. Z-stack of optical sections (11.5 μm in total
thickness) was captured with a 20× objective, using 10 sections
with a step size of 1.15 μm. In order to illustrate cell uptake,
2D images were captured from the middle section of the
Zstack (i.e. 5th section).
In-Vitro Effects of YM155 Loaded Liposomes
Efficacy of YM155 loaded liposomes (targeted and
nontargeted control liposomes) was studied on KCNR by
alamarBlue viability assay (Thermo Fischer Scientific),
according to the supplier’s instruction. KCNR cells were seeded
at 20 × 103 cells/well in 96 well Greiner polystyrene plates
(Sigma Aldrich) in complete DMEM medium and allowed
to adhere overnight. The medium was replaced by cell culture
medium supplemented with YM155 or YM155 liposomal
formulations at concentrations ranging from 0.8 to 100 nM,
after which the cell culture were incubated for 24 h at 37°C.
Control experiments included incubations with empty
liposomes (i.e. without YM155) at equivalent TL concentrations
in the range of 0.08–11 μM TL. After 24 h of incubation,
medium was replaced with drug-free medium containing
alamarBlue reagent (10 vol%), after which cells were
incubated for an extra 3 h at 37°C. Fluorescence was
quantified with a microplate reader (Mithras LB940) and used to
calculate the relative percentage of living cells normalized
against control cells that had not been treated with YM155.
IC50 values were calculated by curve fitting using non-linear
regression in Graph Pad Prism 6.0.
Pharmacokinetic Pilot Study
Animal experiments were conducted in compliance with the
national regulations and have been approved by the local
ethical committee for animal experimentation. NMRI nu/
nu female mice (25 g, Charles River, Massachusetts, USA)
were housed in a temperature-controlled room
(approximately 22°C) with 55 to 65% relative humidity, a photoperiod of
12/12 h with free access to water and pelleted rodent food.
The mice were challenged with KCNR neuroblastoma
(passage 1–2) derived from DAG102776 by serial
xenotransplantation in both flanks. When the tumor growth reached the size
of approximately 8 × 8 mm, mice were randomly divided in
three groups that were injected intravenously with either free
YM155 dissolved in HBS (1 mg/kg, n = 11), YM155 loaded
anti-GD2-immunoliposomes (3 μmol TL/kg diluted in HBS,
equivalent to 1 mg/kg YM155, n = 11), or an equivalent dose
of YM155 loaded control liposomes (n = 11). At designated
time points (i.e. 5 min, 9 min, 15 min, 30 min, 1 h, 4 h, 8 h,
24 h, 48 h and 72 h), blood samples (approximately 0.5 ml per
sampling time point) were collected in tubes containing
EDTA as anticoagulant and mice were sacrificed. Blood
samples and excised tumors were stored at −80°C until analysis of
their YM155 concentration by liquid
chromatographytandem mass spectrometry (LC-MS/MS) (
Pharmacokinetic and Statistical Analyses
Pharmacokinetic analysis of the in-vivo data was performed
using the Pk Solver 2.0 add-in template for Microsoft Excel, as
described previously (
). Pharmacokinetic parameters were
determined by non-compartmental analysis (NCA) using
linear-logarithmic trapezoidal model fitting, or by
compartmental analysis (CA) using 1-compartment model fitting.
Statistical significance was analyzed using two-tailed
unpaired Student’s t-test. A p-value <0.05 was considered
RESULTS AND DISCUSSION
Preparation and Characterization of Liposomes
YM155 loaded liposomes were prepared by drug/lipid film
hydration and extrusion, and subsequently purified by three
cycles of ultracentrifugation and re-dispersion of the pelletized
liposomes in HBS. SATA-modified anti-GD2 was conjugated
to the maleimidyl anchor of DSPE-PEG2000-Mal before the
ultracentrifugation procedure, thus avoiding an additional
purification process to remove non-conjugated anti-GD2 from
the final immunoliposomes. The physicochemical properties
of the different types of liposomes are given in Table II. The
average liposome size determined by DLS was in the range of
140–170 nm, with a corresponding polydispersity index of
about 0.1. The prepared liposomes had a slightly negative
zeta-potential of around −10 mV. Attachment of anti-GD2
antibody to the surface of the liposomes did not significantly
alter their size or surface zeta-potential as compared to control
liposomes, and neither did the inclusion of YM155. The
average YM155 encapsulation efficiency was found to be
around 14%, which is comparable to encapsulation
efficiencies of other hydrophilic drugs, resulting in an average loading
content of approximately 0.8 mg/ml (1.8 mM encapsulated
drug, corresponding to a drug/lipid ratio of 0.035; see
Table II). The reported encapsulation efficiency of 14% is
comparable to encapsulation efficiencies by film hydration
method of other hydrophilic drugs Quantification of attached
Data are presented as mean values of 3–5 preparations ± SD
a Drug/Lipid ratio determined after liposomal disruption. The initial YM155/Lipid ratio prior to liposomal formulation was determined to be 0.25, see
paragraph 2.2 in main text
b YM155 encapsulation efficiency is defined as: [liposomal drug/lipid ratio/ [initial drug/lipid ratio]
anti-GD2 by microBCA showed an antibody coupling
efficiency of around 60% for both YM155-loaded and empty
liposomes, which corresponds to an average of approximately
33 μg Ab/μmol TL. Assuming an average liposomal size of
140 nm, this results in a calculated 16 to 19 antibody
molecules coupled per single liposomal particle (see Table III).
To confirm that anti-GD2 had indeed been coupled
covalently to the liposomal surface, (i.e. by thioether bond resulting
from the reaction of SATA-anti-GD2 with the
maleimidylPEG-DSPE lipids) immunoblotting was performed as
described in section 2–3. SATA-modified anti-GD2 Ab
(SATAGD2) migrated as two bands of approximately 25 and 55 kDa,
which correspond to the respective light and heavy chains of the
antibody (Fig. 2, left). Control (i.e. non-targeted) non-loaded
liposomes (L) and control YM155 loaded liposomes
(YM155L) did not display any bands, while both GD2-targeted empty
(non-loaded) liposomes (GD2-L) and GD2-targeted YM155
loaded liposomes (GD2-YM155-L) displayed several bands
between 25 and 80 kDa corresponding to the molecular weight of
anti-GD2 Ab heavy or light chains coupled to one or multiple
DSPE-PEG2000-Mal anchors (2.9 kDa per unit). Since not all of
the anti-GD2 subunits had been modified with SATA and/or
c o n j u g a t e d t o t h e m a l e i m i d y l - P E G a n c h o r , t h e
immunoliposomal formulations also contained bands that
migrated in parallel to the subunits of the parent anti-GD2 protein
(i.e. around 25 and 55 kDa).
Storage Retention and Release of Encapsulated YM155 from Liposomes at Different Conditions
Both liposomal formulations showed good storage stability for
at least 3 weeks at 4°C in HBS buffer, as reflected in DLS
particle size and polydispersity measurements (Supplemental
fig. S3A) and YM155 retention (Supplemental Fig. S3B).
The results of the in-vitro release tests of GD2-targeted
immunoliposomes and control liposomes loaded with
YM155 are shown in Fig. 3. The conditions of the incubation
of panel 3B were chosen to reflect cell culture or in-vivo
conditions (50% serum, 37°C), in which effects of either
temperature or serum proteins on bilayer stability can be
demonstrated. Control and anti-GD2 immunoliposomes released 30.0%
Table III Efficiency of Anti-GD2
Antibody Coupling to Liposomes
± 11.3 and 34.5% ± 5.2 of the encapsulated YM155,
respectively, over the studied 24 h time period. The observed
leakage of the liposomes could be largely attributed to
temperature-related changes in liposomal stability (Fig. 3c,
d, showing release in HBS buffer at 37°C vs. 4°C) while the
presence of either serum albumin or 50% serum only
marginally affected YM155 release (Fig. 3a, b, c showing YM155
release in presence of 5% BSA or 50% serum, versus release
in HBS buffer without serum proteins). Comparison of
firstorder release rates (Table IV) confirms a slight increase in the
YM155 release rate at 37°C in the presence of serum
albumin. The data in Fig. 3 and Table IV also show that there is
no significant difference in YM155 release rate between the
c o n t r o l l i p o s o m e s a n d t h e a n t i - G D 2 m o d i f i e d
immunoliposomes, regardless of the release medium and
temperature. Furthermore, it was shown that the YM155 release
GD2 liposomes (empty)
μg Ab/ μmol TL
Ab coupling efficiency (%)a
Ab / liposomeb
35.7 ± 9.9
60.1 ± 5.5
19 ± 5
30.9 ± 9.8
58.7 ± 7.8
16 ± 5
Data are presented as mean values of 3 preparations ± SD
a Antibody (Ab) coupling efficiency (%) = (amount of Ab coupled to liposomes/initial amount of Ab added) × 100%
b Number of Ab molecules coupled per liposome, assuming all recovered Ab is attached covalently to liposomes, and
assuming an average liposome size of 140 nm
Fig. 3 Release experiments were
performed at different conditions.
(a) at 37°C in 5% BSA in HBS
buffer, (b) at 37°C in 50% serum in
HBS buffer, (c) at 37°C in only HBS
buffer and (d) at 4°C in 5% BSA in
HBS buffer. Each value represents
the mean value (± S.D) of two
performed in duplicate.
in the presence of HBS buffer +5% BSA could be halted for at
least 24 h if the temperature was dropped to 4°C, as can be
seen in Fig. 3d.
A possible explanation for the increased YM155 release at
higher temperatures is an increase in fluidity of the lipid bilayer
at 37°C (
). Overall, it can be concluded that YM155
Table IV Comparison of the First-Order Rate Constants k (h−1), Based on
Fitting of the in-vitro YM155 Release Profiles Shown in Fig. 3, Showing the
Effects of Temperature and Medium Composition on the YM155 Release
Temp. (°C) Medium
Control liposomes Targeted liposomes
a Value of k equals the slope of the linear fit (R2 > 0.99 in all cases) of the
semi-logarithmic plot[(YM155 retained in liposomes) versus time] (not
shown), extrapolated from the experimental release profiles shown in Fig.
b YM155 release from the liposomes in HBS + 5% BSA at 4°C remained
unchanged (< 5% for all samples) during a 24 h period, indicating the
YM155-containing liposomes are stable in the medium at low temperatures
liposomes have moderate stability at body temperature, while
neither the presence of proteins (i.e. serum or BSA) in the
medium, nor the coupling of anti-GD2 to the liposomal surface
appear to greatly influence the stability of the liposomes. The
observed release of YM155 from liposomes can be important in
the interpretation of other results concerning efficacy and
pharmacokinetics, in which free YM155 and encapsulated drug may
undergo different uptake and clearance routes. For potential
invivo applications of YM155-loaded PEGylated liposomes their
instability and the related release of the encapsulated drug is not
necessarily problematic, as has been reported by Shakushiro
and coworkers (
). In their study, YM155-loaded liposomes
with various lipid compositions were extensively studied and
tested for antitumor activity. Among the investigated
sulfate (DSPC/AS) liposomes were shown to be the most stable
formulation, based on the in-vitro YM155 release study. The
YM155 release rate from DSPC/AS liposomes reported by
Shakushiro and coworkers (
) is comparable to the YM155
release rate of the formulations described here in the present
work. However, Shakushiro et al. reported that the optimal
formulation which demonstrated most potent in- vivo antitumor
activity was in fact the DSPC/phosphate buffer (BP) liposomes,
which showed a relatively higher YM155 in-vitro release rate
than the DSPC/AS formulation mentioned earlier. The
Fig. 4 Binding of liposomal
formulations to (a) IMR32 cells and
(b) WiDr cells. The data points are
aligned (fitted) by non-linear
regression (using one phase
association model). MFI = Mean
Fluorescence Intensity; TL = total
difference in YM155 release rates between the optimum
formulations found previously by Shakushiro et al. and the
formulations used in this work can be explained by the different
nanomedicine targeting approaches in both studies.
Generally, both passively and actively targeting
nanomedicine accumulate in the tumor tissue via the enhanced
permeability and retention (EPR) phenomenon. When
passively targeting nanomedicine accumulates in the target tissue, the
encapsulated drug molecules need to be released from their
delivery system so that they can be taken up by the target cells.
Thus, their release rate needs to be precisely engineered, as
described by Shakushiro et al. However, for actively targeting
nanomedicine direct endosomal uptake of the nanomedicine is
facilitated by the presence of (surface) targeting moieties (e.g.
anti-GD2 antibody), which is then followed by cytosolic release
of the encapsulated drug molecules. Since this study aims to use
actively targeting nanomedicine formulations for the delivery of
YM155, the more stable liposomal formulations are expected
to be most suitable.
GD2 Expression in Various Neuroblastoma Cell Lines
GD2 expression levels in neuroblastoma tumor cell lines were
measured by flow cytometry as explained in section 2–7.
KCNR (and IMR32) neuroblastoma showed relatively high
levels of GD2 expression (i.e. about 95% and 86%,
respectively), while WiDr colon carcinoma cells showed negligible
fluorescent signal at the pre-determined gate settings,
confirming the lack of GD2 expression on this cell line (data
not shown). WiDr cells were further used in flow cytometry
experiments as negative control cells.
Cell Binding and Uptake of Liposomes
The ability of GD2-targeted immunoliposomes to recognize
GD2 positive cells (IMR32) was demonstrated by incubating
rhodamine labeled liposomes with the cells at 4°C, after which
cell-associated liposomes were detected by flow cytometry. A
f o u r - f o l d i n c r e a s e i n b i n d i n g o f G D 2 - t a r g e t e d
immunoliposomes to IMR32 cells was observed in
comparison to non-targeted control liposomes (Fig. 4a). Moreover,
both formulations showed similar low binding to
GD2negative WiDr cells (Fig. 4b), thus confirming the
GD2dependency of liposomal binding.
The binding and internalization of GD2-targeted
immunoliposomes by KCNR cells was studied by confocal
microscopy (Fig. 5). Incubation of cells with liposomal
formulation for 4 h at 37°C allowed binding and receptor mediated
internalization of immunoliposomes, which was detected by a
distinct increase in captured fluorescent intensity at the middle
section of z-axis, which was not observed for control
liposomes. The obtained results confirm the active (cellular)
uptake of GD2-targeted immunoliposomes by KCNR cells.
In-Vitro Evaluation of GD2 Targeted Loaded Liposomes in Cell Culture
The in-vitro efficacy of YM155 loaded liposomes was studied in
KCNR cells by incubating the cells for 24 h with the developed
formulations. Inhibition of survivin by YM155 will change the
balance between cell survival and apoptosis, which can be
analyzed via analysis of mRNA or surviving protein expression or
apoptosis assays, or indirectly by a cell viability assay
(AlamarBlue assay). Dose response curves of cell viability
experiments are shown in Fig. 6a and have been analyzed by
nonlinear curve fitting to determine IC50 values of each formulation.
IC50 values of 27, 60 and 83 nM were calculated for free
YM155, anti-GD2 immunoliposomes loaded with YM155,
and control liposomes loaded with YM155, respectively.
Liposomes not containing YM155 did not induce cell toxicity
at the concentrations examined in this work (Fig. 6b). The
overall data suggest that treatment of neuroblastoma cells (KCNR)
with free YM155 is more effective compared to the treatment of
cells with liposomal formulations (targeted or control), although
YM155 loaded GD2-targeted immunoliposomes showed an
increased (around 20%) efficacy compared to the non-targeted
YM155-loaded control liposomes.
Amphiphilic small molecular inhibitors generally diffuse
rapidly through cellular membranes, which enable small
molecules to effectively inhibit intracellular molecular targets.
However, YM155 is a positively charged small molecule not
capable of passive diffusion over lipid membranes. Instead, its
intracellular accumulation is actively mediated by cell specific
influx transporter channels (
). Apparently, the active uptake
of free YM155 is very efficient, outperforming the uptake of
immunoliposomes by GD2-positve cancer cells cultured
in vitro. The major benefit of encapsulating YM155 in
liposomes is however in avoiding its rapid renal elimination,
which will be presented below. It should thus be noted that
part of the effects observed for the liposomal formulations
might be due to a fraction of YM155 that is released from
the liposomal formulations during the 24 h incubation in
protein containing cell culture media at 37°C and which is
subsequently transported over the cell membrane similar to free
YM155. However, the absence of sample agitation means that
YM155 released from the liposomes during the incubation
period is likely to be lower than the corresponding YM155
release in the stability tests shown in Fig. 3. More importantly,
the difference in efficacy observed between the anti-GD2
immunoliposomes and the non-targeted control liposomes
clearly shows that a significant part of the total YM155 is taken
up by the cells while remaining encapsulated in the liposomal
nanocarrier system. Figure 7 schematically depicts the
available uptake routes of YM155 upon its addition to the cells as
liposomal formulation or free drug.
Fig. 6 Cell toxicity was determined in neuroblastoma cells (KCNR) via MTS
assay after 24 h of total incubation period. (a) Semi-logarithmic plot of the cells
exposed for 24 h to YM155 loaded liposomal formulations and free YM155.
The IC50 values were determined with Graph Pad Prism 6.0. (non-linear
regression model was used for curve fitting). (b) Cells were exposed for
24 h to non-loaded liposomal formulations. Data are plotted as mean values
± SEM (n = 3).
Fig. 7 Uptake routes for cytosolic
delivery of YM155. Receptor
mediated uptake delivers
GD2targeted YM155 liposome into
endosomal and eventually
lysosomal vesicles. Cytosolic
delivery of liberated YM155 is
facilitated by passive diffusion
(possibly as in complex with an
organic anion or via organic cation
transporters (OCT) when they are
present in the lysosomal
membrane). Cytosolic delivery of
free YM155 is facilitated via organic
cation transport (OCT) channels at
the cell membrane. Adapted with
permission from ref. (
Copyright 2006, Springer.
The experiments show no toxicity of the anti-GD2
antibodies attached to the surface of liposomes on KCNR cells.
However, toxic effects of free anti-GD2 antibodies were
previously reported by Gottstein et al. on IMR32 cells (
cause for these alternate observations regarding anti-GD2
toxicity can be the difference in sensitivity of the
neuroblastoma cell types (i.e. IMR32 and KCNR) to anti-GD2 antibody.
Another explanation is that Gottstein et al. (
) observed toxic
effects because in their work the IMR32 cells were exposed to
concentrations of anti-GD2 antibody (IC50 1.5 μg/ml) that
were much higher than the concentrations of anti-GD2
antibody that the liposomal surfaces were exposed to in this work.
The highest concentration of anti-GD2 (coupled to a
liposomal surface) that was added to the cell culture media in our
experiments was 0.08 μg/ml, which is well below the above
mentioned IC50 value of 1.5 μg/ml.
Pharmacokinetic Profiles of Free YM155 and its
The distribution kinetics of YM155-loaded liposomes was
evaluated in nude mice with subcutaneously implanted
neuroblastoma tumors. Upon intravenous injection of either
targeted or control liposomes or free YM155, plasma and
tumor samples were collected over a time period of 5 min till
3 days and analyzed for YM155 by LC-MS/MS. Figure 8
shows the YM155 plasma concentration versus time curves of
free YM155 (Fig. 8a), YM155-loaded control liposomes (Fig.
8b), and YM155-loaded anti-GD2 immunoliposomes (Fig.
8c). The calculated pharmacokinetic (PK) parameters for free
YM155 and liposomal YM155presented in Table V have
been determined by non-compartmental analysis (NCA) using
a linear-logarithmic trapezoidal method, in which linear
interpolation is used if drug concentrations are increasing or
constant (Ci + 1 ≥ Ci), while logarithmic interpolation is used
if drug concentrations are decreasing (Ci + 1 < Ci). The slope
of the drug concentration versus time curve in the terminal
phase (λz) was determined by linear regression on a
semilogarithmic scale, and λz was then used to derive
pharmacokinetic parameters such as drug half-life (t1/2) and volume of
distribution in the terminal phase (Vz). PK parameters were
also determined by compartmental analysis (CA), in which the
YM155 plasma concentration versus time curve is fitted using a
1-compartment model. These fitted data are based on 11–13
animals per group, corresponding to 3–5 time points.
Combined with the large error margins in some of the data
points, the results from the pharmacokinetic analyses listed in
Table V should be treated as estimates when comparing them
with pharmacokinetic results from other studies. Within this
work however, the PK parameters listed in Table V
sufficiently show the differences between the three different
formulations (i.e. free YM155, Liposomal YM155, and
GD2liposomal YM155). As expected, free YM155 showed a large
initial volume of distribution and rapid elimination from the
circulation, resulting in plasma levels that were detectable only
for about 8 h post administration. In contrast, liposomal
formulations of YM155 (targeted and control) displayed much
smaller distribution volumes as compared to free YM155,
which can be expected for PEGylated nanocarriers that are
retained within the circulation initially and that only distribute
slowly over the endothelial barrier. Moreover, we observed
the expected long-circulating properties of PEGylated
liposomes, as both formulations were able to sustain elevated
plasma levels of YM155 for at least a 3 day time period (Fig. 8b, c,
see also Supplemental Fig. S4 for better comparison during
the first hours). The increased longevity of liposomal YM155
in blood plasma can be attributed to the small hydrodynamic
size of the liposomes and stealth properties provided by PEG
chains, which allow liposomal formulations to escape
recognition and subsequent clearance by the mononuclear phagocyte
system (MPS) (
). As long as the drug is still retained in the
liposomal nanocarriers, the normal rapid clearance of free
YM155 is prevented.
The difference between free YM155 and liposomal YM155
formulations is also clear when examining the pharmacokinetic
parameters derived from the NCA fitting model, as listed in
The PK values are calculated by non-compartmental analysis (NCA) using a linear-logarithmic trapezoidal method
C0, concentration at t = 0, extrapolated, t1/2 = elimination half-life; AUC0-∞, extrapolated area under the curve from
zero to infinity; CL, plasma clearance; Vz, volume of distribution during terminal phase, derived from slope (λz) in terminal
phase; Vss, volume of distribution at steady state; MRT0-∞, mean residence time
a PK analysis for free YM155 is based on data points until t = 2 h. Inclusion of the t > 2 h data points will result in an
unrealistic estimate of the terminal slope λz (see main text for details)
Half-life values determined by non-compartmental analysis (NCA) using
linear-logarithmic trapezoidal model, and compartmental analysis (CA) using
a 1-compartment model. PKSolver CA model fit parameters, correlation
coefficient (R2 ) and standard error of weighted residuals (SE): Free YM155
(2 h): R2 = 0.81, SE = 153 (ng/ml); YM155 Liposomes: R2 = 0.97, SE =
2576 (ng/ml); YM155 GD2-Liposomes: R2 = 0.99, SE = 142 (ng/ml).
a PK analyses of free YM155 are based on data points until t = 2 h. See also
Table V and main text
interesting, for instance with labeled liposomes that can be
traced immunohistochemically in liver and spleen as these are
the main organs responsible for clearance of PEGylated
To illustrate the difference in initial and terminal YM155
clearance rates in the different formulations, the
pharmacokinetic evaluation was also performed by compartmental
analysis (CA), using a 1-compartment model (data not shown).
Contrary to NCA, which determines the YM155 half-life
from the fitted terminal slope, CA (1-compartment model) fits
a first-order exponential decay function to the dataset to
determine the PK parameters. The (t1/2) values of the YM155
concentration in plasma derived from the NCA and CA
methods are listed for comparison in Table VI.
It should be noted that the CA used a 1-compartmental
model, which means that the fit results are only meaningful for
the initial period of rapid decline in YM155 plasma
concentration that occurs in the first two hours. Instead, the more
gradual decline in the terminal period that can go up to t =
72 h is accurately described by the NCA fit results. The
difference is most pronounced for the GD2-liposomes loaded
with YM155, for which the rapid decrease observed in
YM155 plasma concentration during the first 2 h results in a
Table VI Comparison of Half-Life Values for YM155 Concentration in
Blood Plasma Determined by Non-compartmental (NCA) and
Compartmental Analysis (CA)
CA half-life estimate that is even comparable to that of free
YM155 (0.23 and 0.20 h, respectively).
Tumor Accumulation of Free YM155 and its Liposomal
As can be observed in Fig. 9, intratumor levels of YM155 could
be detected up to 24 h post injection for free YM155, or up to
three days post injection for liposomal YM155. It is important
to notice that free YM155 was eliminated more slowly from
tumor tissue than from the circulation which can be attributed
to the cation-transporter mediated uptake of the drug into
(tumor) tissue and its subsequent intracellular retention.
Tumor accumulation of liposomal YM155 was delayed but
sustained as compared to free YM155, which can be attributed
to accumulation of nanocarriers via leaky tumor blood vessels,
i.e. by EPR, and the prolonged residence of the nanocarriers in
the circulation (
). Moreover, when comparing anti-GD2
immunoliposomes and non-targeted control liposomes, highest
intratumoral levels were observed for control liposomes as
compare to targeted liposomes (AUC0-∞ values of 13,194 ng/mL*h
and 3803 ng/mL*h for control liposomes and anti-GD2
immunoliposomes respectively). The 3.4-fold difference in
tumor accumulation cannot be explained fully by the 1.9 fold
difference in plasma exposure which should reflect the driving
force for tumor uptake by EPR (plasma AUC 71473 ng/mL*h
and 37,787 ng/mL*h, for control- and anti-GD2 liposomes
respectively). Additionally, one may expect a faster uptake by
tumor cells of anti-GD2-liposomes via receptor mediated
uptake and intracellular processing of anti-GD2 liposomes, which
will liberate YM155 from the liposomal carrier. In contrast,
control liposomes will be internalized to a lesser extent in the
tumor, and YM155 will remain entrapped inside the liposomal
vesicles. Although it was not possible in the context of this pilot
study, it would also be very interesting to do an additional study
comparing the anti-tumor effects of both liposomal
formulations, in order to assess the added value of anti-GD2
immunoliposomes over conventional long-circulating
PEG-liposomes for the delivery of YM155.
Liposomal formulations of YM155 have been previously
studied, for example in papers by Kawana et al. (
Shakushiro et al. (
). In the study by Kawana and coworkers,
the authors developed a liposomal YM155 formulation
composed of DSPC, cholesterol and DSPE-PEG2000 lipids using
phosphate buffer as liposomal ‘inner’ phase. The reported
blood circulation half-life (t1/2) was 14.6 and 26.5 h for
YM155 liposomes at a dosage of 3 and 9 mg/kg, respectively
). In another study by Shakushiro and coworkers, the
authors mainly focused on evaluating the effect of different
liposomal formulations on the pharmacokinetics and efficacy. They
reported a t1/2 of 24 h at 3 mg/kg dose for a liposomal YM155
formulation composed of DPPC, cholesterol and
DSPEPEG2000, and a t1/2 of 48 h at 3 mg/kg dose for a formulation
composed of DSPC, cholesterol and DSPE-PEG2000, both
containing phosphate buffer as liposomal inner phase (
). We now
observe shorter half-lives of 5.3 h and 6.8 h for non-targeted
and targeted liposomal YM155 formulations, respectively,
which have been determined at relative lower lipid doses, which
may have resulted in relative faster clearance of the liposomes.
The present study reports on the encapsulation of the survivin
inhibitor YM155 in immunoliposomes directed to
neuroblastoma cancer cells. Surface bound anti-GD2 antibodies render
the liposomes neuroblastoma cell specific (i.e. targeted) which
has been confirmed by binding and uptake studies with GD2
expressing tumor cells. Both the immunoliposomes and the
non-targeted control liposomes showed similar stability under
all conditions, excluding detrimental effects of the
surfaceconjugated antibodies on liposome stability. The liposomal
formulations were studied in a pilot pharmacokinetic experiment
which demonstrated their long-circulating character and
capability to accumulate intra-tumor. When compared to free
YM155 after single bolus intravenous (i.v.) injection, prolonged
intratumoral levels were obtained. A clear added value of using
anti-GD2 immunoliposomes could not be assessed in the
conducted pilot study. Further studies are required to evaluate the
pharmacological effects (efficacy) of YM155-loaded anti-GD2
immunoliposomes at different YM155 dose ranges which may
disclose more details on the potential of the liposomal YM155
formulations for clinical therapeutic applications.
ACKNOWLEDGMENTS AND DISCLOSURES
This work has been supported by NanoNextNL, a micro and
n a n o t e c h n o l o g y c o n s or t i u m o f t h e g o v e r n m e n t o f
The Netherlands and 130 partners (project 03D.07). The
authors would like to thank Charlene Ogu for her helps with
characterization of SATA modified anti-GD2 antibody.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License
permits unrestricted use, distribution, and reproduction in any
medium, provided you give appropriate credit to the original
author(s) and the source, provide a link to the Creative
Commons license, and indicate if changes were made.
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