-
Cancer Letters 335 (2013) 191200Contents lists available at
SciVerse ScienceDirect
Cancer Letters
journal homepage: www.elsevier .com/locate
/canletOcta-arginine-modified pegylated liposomal doxorubicin: An
effectivetreatment strategy for non-small cell lung
cancer0304-3835/$ - see front matter 2013 Elsevier Ireland Ltd. All
rights reserved.http://dx.doi.org/10.1016/j.canlet.2013.02.020
Corresponding author. Address: Center for Pharmaceutical
Biotechnology andNanomedicine, Northeastern University, 140 The
Fenway (Rm. 225), 360 Hunting-ton Ave., Boston, MA 02115, United
States. Tel.: +1 617 373 3206; fax: +1 617 3737509.
E-mail address: [email protected] (V.P. Torchilin).Swati
Biswas, Pranali P. Deshpande, Federico Perche, Namita S. Dodwadkar,
Shailendra D. Sane,Vladimir P. Torchilin Center for Pharmaceutical
Biotechnology and Nanomedicine, 360 Huntington Avenue, 140 The
Fenway, Northeastern University, Boston, MA 02115, United
States
a r t i c l e i n f o a b s t r a c tArticle history:Received 22
November 2012Received in revised form 9 January 2013Accepted 8
February 2013
Keywords:LiposomesLung
cancerDoxorubicinOcta-arginineSpheroidsTumorThe present study aims
to evaluate the efficacy of octa-arginine (R8)-modified pegylated
liposomal doxo-rubicin (R8-PLD) for the treatment of non-small cell
lung cancer, for which the primary treatment modal-ity currently
consists of surgery and radiotherapy. Cell-penetrating peptide R8
modification ofDoxorubicin-(Dox)-loaded liposomes was performed by
post-insertion of an R8-conjugated amphiphilicPEGPE copolymer
(R8-PEGDOPE) into the liposomal lipid bilayer. In vitro analysis
with the non-smallcell lung cancer cell line, A549 confirmed the
efficient cellular accumulation of Dox, delivered by R8-PLDcompared
to PLD. It led to the early initiation of apoptosis and a 9-fold
higher level of the apoptotic reg-ulator, caspase 3/7 (9.24 0.34)
compared to PLD (1.07 0.19) at Dox concentration of 100 lg/mL.
Thetreatment of A549 monolayers with R8-PLD increased the level of
cell death marker lactate dehydroge-nase (LDH) secretion (1.2 0.1
for PLD and 2.3 0.1 for R8-PLD at Dox concentration of 100 lg/mL)
con-firming higher cytotoxicity of R8-PLD than PLD, which was
ineffective under the same treatment regimen(cell viability 90 6%
in PLD vs. 45 2% in R8-PLD after 24 h). R8-PLD had significantly
higher penetrationinto the hypoxic A549 tumor spheroids compared to
PLD. R8-PLD induced greater level of apoptosis toA549 tumor
xenograft and dramatic inhibition of tumor volume and tumor weight
reduction. The R8-PLD treated tumor lysate had a elevated caspase
3/7 expression than with R8-PLD treatment. This sug-gested system
improved the delivery efficiency of Dox in selected model of cancer
which supports thepotential usefulness of R8-PLD in cancer
treatment, lung cancer in particular.
2013 Elsevier Ireland Ltd. All rights reserved.1.
Introduction
Lung carcinoma is the most frequent cancer in the world, withan
incidence of 1.5 million new cases per year, accounting
forapproximately one-third of all cancer-related deaths [1,2].
Non-small cell lung cancer (NSCLC) is prevalent (85%) among all
lungcancer types. 6580% of all lung cancer patients are diagnosed
atan advanced stage of local carcinoma or metastasis [3,4].
Surgeryremains the most successful treatment option for patients
withearly detection of the disease [1]. However, the rate of
recurrenceis 90% in the first 5 years after surgery [2,5,6]. There
is only a 5%absolute benefit of adding chemotherapy to surgery, a
minimalgain in overall patient survival rates [711]. Another
chemother-apy-related problem is that the relapsed tumors acquire
resistanceto the administered chemotherapy. Therefore, there is an
unmetneed for developing more effective treatment regimens for
NSCLC.
The platinum containing drugs, cisplatin and carboplatin
areconsidered the first choice of chemotherapy for NSCLC [12].
Paclit-axel or docetaxel in combination with either of the two
platinumdrugs is used as a second-line treatment [13,14].
Doxorubicin,alone, or in combination, are currently in clinical
trials for ad-vanced stages of NSCLC [1517]. However, treatment
with Dox isassociated with numerous side-effects including severe
cardiotox-icity [18,19].
The use of liposomes has advantages in cancer treatment due
totheir enhanced permeability and retention related to their
smallsize (100 nm) that allows passage through leaky tumor
bloodvessels and accumulation preferentially in the tumor [20,21].
Inthis regard, liposomes need to be modified to impart the
propertyof long systemic circulation that leads to eventual
accumulation inthe tumor. Nonetheless, the nanocarriers drug load
has to be re-leased at the tumor site, has to be efficiently
cellular-internalizedand followed by release from endosomes to
demonstrate drug effi-cacy [22]. For long circulation, liposomes
are coated with hydro-philic polymers, mainly polyethylene glycol
(PEG). However, thePEG-modified liposomes lead to reduced cellular
internalization.
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192 S. Biswas et al. / Cancer Letters 335 (2013)
191200Long-circulating pegylated liposomal doxorubicin
(commer-cially available as Doxil or Lipodox) is an example of a
stealthliposome, an indispensible treatment means for metastatic,
breastand ovarian cancers [23]. Doxil represents an improved
formula-tion of free drug with better pharmacokinetic profile and
reducedcardiotoxicity. However, its therapeutic efficacy is not
dramaticallyincreased and adverse side-effects remain, indicating a
furtherimprovement of this formulation. Addressing the issue of
poor cel-lular internalization is one of the approaches used to
improve thetherapeutic window of the drug.
Intracellular or organelle-specific targeting is an emerging
con-cept for improvement of drug action of the nanocarriers
[22,2426]. Since the nanocarriers utilize energy-dependent
endocytosisas a major pathway for cellular internalization rather
than randomdiffusion typical for small drug molecules, a cell
penetration en-hancer would dramatically improve their cytoplasmic
delivery[2730]. In this regard, many peptide sequences have been
identi-fied that cause translocation of a variety of cargos across
the cellmembrane [28]. Poly-arginine, a relatively short
cell-penetratingpeptide, with an optimum chain length of 8-arginine
units hasbeen successfully utilized for intracellular delivery of
therapeutics[3134]. A possible internalization pathway of the
R8-modified lip-osomes has also been investigated [35].
In this study, we have utilized R8 modified liposomes toenhance
the penetration of lung cancer cells. R8-conjugatedamphiphilic
poly(ethylene glycol)dioleoyl phosphatidylethanol-amine (PEGDOPE)
conjugate, R8-PEGDOPE was incorporated inthe lipid bilayer of PLD.
PEGDOPE copolymer is much used forliposomes modification due to its
biocompatibility and non-immunogenicity [20]. The hydrophobic lipid
moiety in theconjugate could be easily embedded in the liposomal
bilayer toallow surface modifications. The PEG-spacer imparts easy
R8-accessibility for the cell surface interaction. The
therapeuticefficacy of R8-modified pegylated liposomal Doxorubicin
(PLD)was assessed in human alveolar adenocarcinoma cell line
toevaluate the potential benefit of R8-PLD in the debilitating
NSCLCtreatment.2. Materials and methods
2.1. Materials
Octa-arginine peptide (R8, MW. 1267.46 Da) was synthesized by
the TuftsUniversity Core Facility (Boston, MA). Pegylated liposomal
doxorubicin (Lipodox,2 mg/mL of Dox) was purchased from Sun
Pharmaceutical India Ltd (Gujarat,
India).1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
gly-col)-2000](ammonium salt) (PEG2KDOPE),
1,2-dioleoyl-sn-glycero-3-phosphoeth-anolamine (DOPE) was purchased
from Avanti Polar Lipids (AL, USA).NPCPEG2KNPC was purchased from
Laysan Bio (AL, USA). thiazoyl blue tetrazo-lium bromide (MTT) was
purchased from SigmaAldrich (St. Louis, MO). MicroBCA protein assay
kit, Apo-ONE Homogeneous Caspase-3/7 Assay and CytoTox96
Non-Radioactive Cytotoxicity Assay kits were purchased from
Promega(Madison, WI). Annexin V Alexa Fluor 488 conjugate,
Annexin-binding buffer5 concentrate and Hoechst 33342 were
purchased from Molecular Probes Inc.(Eugene, OR). Para-formaldehyde
was from Electron Microscopy Sciences (Hatfield,PA). Fluoromount-G
was from Southern Biotech (Birmingham, AL). The Trypan bluesolution
was obtained from Hyclone (Logan, UT).2.2. Cell lines
The human alveolar adenocarcinoma cell line, A549 was purchased
from theAmerican Type Culture Collection (Manasas, VA). Dulbeccos
modified Eagles media(DMEM) and heat-inactivated fetal bovine serum
(FBS) were obtained from Gibco(Carlsbad, CA). Concentrated
penicillin/streptomycin stock solution was from Cell-Gro (Herndon,
VA). All other chemical and solvents were of analytical grade,
pur-chased from SigmaAldrich and used without further
purifications. A549 cells weregrown in DMEM with 2 mM L-glutamine,
supplemented with 10% (v/v) heat-inacti-vated fetal bovine serum,
100 units/mL penicillin G and 100 lg/mL streptomycin.Cultures were
maintained in a humidified atmosphere at 37 C and 5% CO2.2.3.
Synthesis of R8-PEG2KDOPE
For this purpose, pNP-PEG2KDOPE was synthesized and purified
according toan established procedure with modification [24,36].
Briefly, into the solution ofNPCPEG2KNPC (1 g, 0.5 mmol) in
chloroform, a DOPE (37.2 mg, 0.05 mmol) solu-tion in chloroform
mixed with 20 lL of triethylamine was added drop wise. Thereaction
mixture was stirred overnight at room temperature. On the
followingday, the reaction mixture was evaporated using a rotary
evaporator and freeze-dried to remove traces of solvent. The dry,
crude reaction mixture was dissolvedin HCl solution (0.01 M) and
purified by gel filtration chromatography using aCl4B column. A HCl
solution was used as an eluent. The pure fractions
werefreeze-dried, weighed and dissolved in chloroform to obtain a
10 mg/mL solutionfor storage at 80 C. For the synthesis of
R8-PEG2KDOPE, into a solution ofpNP-PEG2KDOPE (10 mg, 3.9 lmol) in
chloroform (1.0 mL), R8 (7.4 mg, 5.86 lmol)and triethylamine (10
lL) dissolved in DMF (200 lL) was added. The reaction mix-ture was
stirred overnight at room temperature. The chloroform was
evaporated byrotary evaporation and freeze-dried. The dry reaction
mixture was dissolved in PBS,pH 8.4 and stirred at room temperature
for 4 h and dialyzed against water using acellulose ester membrane
(MWCO. 2000 Da) overnight. The dialysate was freeze-dried to obtain
a solid white fluffy product which was dissolved in methanol at5
mg/mL and stored at 80 C.
2.4. Modification of liposomes
Using the post-insertion method, [3739] we decorated Lipodox
with R8-PEG2KDOPE conjugate. The inserted conjugate was 2 mol% of
the total lipids.Briefly, Lipodox (1.0 mL) was added into the dry
lipid film of PEG2KDSPE(0.90 mg, 0.45 lmol) or R8-PEG2KDOPE (1.64
mg, 0.45 lmol). The liposomal sus-pension, PLD and R8-PLD were
vortexed for complete hydration of the lipid filmand stirred
overnight at 4 C.
2.5. Characterization of liposomes
Surface-modified liposomes (PLD and R8-PLD) were analyzed for
size and zeta-potential. The liposomal size and size distribution
was measured by dynamic lightscattering (DLS) using a Coulter
N4-Plus Submicron Particle Sizer (Coulter Corpo-ration, Miami, FL).
Liposome surface charge was measured using a Zeta Phase Anal-ysis
Light Scattering (PALS) Ultrasensitive Zeta Potential Analyzer
instrument(Brookhaven Instruments, Holtsville, NY).
2.6. Cell association of liposomes
Cell association of liposomes was assessed by FACS analysis.
After the initialpassage in T-75 cm2 tissue culture flasks (Corning
Inc., NY), A549 cells (0.4 106/well) were seeded in 6-well tissue
culture plates. The following day, the cells wereincubated with PLD
or R8-PLD at a Dox concentration of 6 lg/mL in 2 mL of serum-free
media for 1 h and 4 h incubation periods. The media were removed,
the cellswashed several times, trypsinized, suspended in 1 mL PBS
and then centrifugedat 1000 RPM for 5 min. The cell pellet was
suspended in PBS, pH 7.4 before analysisof the cells labeled with
Dox fluorescence using a BD FACS Caliber flow cytometer.The cells
were gated using forward (FSC-H)-vs. side-scatter (SSC-H) to
exclude deb-ris and dead cells before analysis of 10,000 cell
counts.
2.7. Cellular internalization of liposomes
Cellular uptake of liposomes was analyzed by visualization with
confocalmicroscopy. After the initial passage in tissue culture
flasks, A549 cells (4 104)were grown on circular cover glasses
placed in 12-well tissue culture plates in com-plete media. The
following day, cells were incubated with PLD or R8-PLD at a
Doxconcentration of 6 lg/mL for 1.5 h in serum-free media. After
the incubation period,the cells on the cover-slips were washed 4
times with PBS, treated with Hoechst33342 at 5 lg/mL for 5 min,
washed thoroughly and fixed with 4% para-formalde-hyde for 10 min
at room temperature. The cover-slips were again washed with PBSand
mounted cell-side down on superfrost microscope slides with
fluorescence-freeglycerol-based mounting medium (Fluoromount-G) and
viewed with a Zeiss LSM700 Confocal Laser Scanning Microscope
equipped with UV (ex/em. 385/470 nm)and rhodamine filter (ex/em.
548/719 nm) for imaging. The z-stacked images (z115, slice
thickness, 0.75 lm) were obtained by capturing serial images of
thexy planes by varying the focal length of the same to image along
consecutive z-axes. The LSM picture files were analyzed using Image
J software. Nuclear localiza-tion of the Dox signal delivered by
PLD or R8-PLD was assessed by determiningPearsons and Manders
coefficients of colocalization using Image J software.
2.8. Formation of spheroids
Spheroids of 800900 lm diameter were formed from 10,000 A549
cells in 96-well plates according to Yang et al. with modifications
as follows [40,41]. A549 cellswere maintained as monolayers before
detachment with trypsin to generate a sin-gle-cell suspension.
Then, 10,000 cells in 100 lL of media were added to each well
-
S. Biswas et al. / Cancer Letters 335 (2013) 191200 193of a 96
well plate previously coated with 50 lL of DMEM 1.5% agarose.
Finally, theplates were centrifuged 15 min at 1500 rcf to form
spherical cell mass which wasincubated for approximately 4 days
with no medium change. Spheroid formationwas monitored using a
Nikon Eclipse E400 microscope at 10 magnification andwith a Spot
Insight 3.2.0 camera with Spot Advanced software (Spot
Imaging).Spheroids of 800900 lm size were used for experiment.2.9.
Liposomal penetration of spheroids
Spheroids were incubated with PLD and R8-PLD at a Dox
concentration of100 lM for 1 h or 4 h, washed with PBS and viewed
with a Zeiss LSM 700 ConfocalLaser Scanning Microscope equipped
with rhodamine filter (ex/em. 548/719 nm)using a 10 objective. The
Z-stacked images of spheroids (z 116) were obtainedby capturing
serial images of the xy planes by varying the focal length to
imagealong consecutive z-axis. The LSM picture files of spheroids
were analyzed for quan-tifying the mean intensity using Image J
software.2.10. Annexin V assay
The procedure for Annexin V labeling was carried out according
to the manufac-turers protocol. A549 cells were seeded in 12-well
plates at 8 104/well. Afterincubation of A549 cells for 4 h with
PLD or R8-PLD at a Dox concentration of15 lg/mL, the cells were
incubated for an additional 18 h, trypsinized, washed withcold
binding buffer, and re-suspended in Annexin V-Alexa Fluor 488
conjugate(15 lL)-added binding buffer (200 lL) for 15 min in
darkness. The cells were dilutedwith binding buffer at total volume
400 lL and analyzed immediately by flowcytometry.2.11. Cytotoxicity
studies
2.11.1. MTT assayFor cytotoxicity assays including MTT, LDH
release and caspase assay, A549
cells were seeded into 96 well microplates at a density of 5 103
and 3 103cells/well for 24 and 48 h respectively in phenol red-free
DMEM media. On the fol-lowing day, the cells were incubated with
PLD or R8-PLD at Dox concentrations of0100 lg/mL for 4 h. Media was
removed and the cells were incubated for an addi-tional 24 and 48 h
in fresh complete media. After the incubation period, the mediawas
removed and the cells were treated with
3-(4,5-dimethylthiazol-2-yl)-2,5-di-phenyl-2H-tetrazolium bromide
(MTT) solution (5 mg/mL) in serum-free DMEMfor 4 h. At the end of
the incubation period, cell viability was estimated by the abil-ity
of the cells to reduce the yellow dye, MTT to a purple formazan
product. Themedia was replaced with 100 lL of SDS solution (20%) in
0.01 N HCl for 4 h to dis-solve the formazan crystals. The
absorbance was read at 570 nm using a microplatereader (Synergy HT
multimode microplate reader, Biotek Instrument, Winooski,VT). Blank
readings obtained from the treatment well with no cells were
subtractedfrom each reading.2.11.2. LDH releaseThe A549 cells were
treated with PLD or R8-PLD at a Dox concentration range of
0100 lg/mL for 4 h followed by removal of the media and further
incubation for24 and 48 h. Released lactate dehydrogenase (LDH) in
the media was measuredwith a Cytotox 96 Non-Radioactive
Cytotoxicity kit (Promega) following manufac-turers instruction.
LDH release was normalized to total LDH content following celllysis
with a medium containing 0.9% Triton X-100. The plate was read with
a Multi-mode microplate reader (Synergy HT; Biotek; absorbance. 340
nm).2.11.3. Caspase assayAfter the treatment of A549 cells with PLD
or R8-PLD at Dox concentration
range of 0200 lg/mL for 4 h and incubating the cells for
additional 24 and 48 hin phenol red-free complete medium, the
samples were analyzed using theApo-ONE Homogeneous Caspase 3/7
Assay (Promega) following manufacturersinstructions. Briefly,
following the incubation period, caspase substrate was dilutedin
Apo-ONE Homogeneous Caspase-3/7 Buffer and 100 lL added to cells in
100 lLmedia and kept at room temperature with additional gentle
shaking. The plateswere read after 4 h using the microplate reader
at ex/em. 499/521 nm. The valueswere directly proportional to the
amount of apoptotic induction. A blank (allreagents without cells)
reading was subtracted from each reading.
For the determination of caspase 3/7 level in the A549 tumor
xenograft, tumorswere lysed using a hand-held tissue homogenizer
(Qiagen, CA, USA) in PBS, pH 7.4at 0 C and protein content was
measured using micro BCA protein assay usingmanufacturers protocol.
25 lg of protein in 100 lL (PBS, pH 7.4) was added to wellsof a
96-well plate. Samples were analyzed immediately as above.2.12.
Animals
Immunodeficient NU/NU nude mice (46 weeks old) were purchased
fromCharles River Laboratories, MA, USA. All animal procedures were
performed accord-ing to an animal care protocol approved by
Northeastern University InstitutionalAnimal Care and Use Committee.
Mice were housed in groups of 5 at 1923 C witha 12 h lightdark
cycle and allowed free access to food and water.
2.13. In vivo tumor xenograft
A subcutaneous tumor was initiated by inoculating A549 human
alveolar ade-nocarcinoma cells (2 106, suspended in 100 lL PBS)
over the left flank. The timefor the appearance of the tumor was
usually 7 days. However, after an initial phaseof slow growth,
tumors increased grow and reached a volume of 400 mm3 after60 days
from implantation. The length and width of the tumor was measured
bycalipers at 3 days interval and the tumor volume was calculated
using the formula:(width2 length)/2. Treatment was started when the
tumors reached a size of400 mm3.
2.14. Assessment of tumor volume reduction and TUNEL assay
To measure the pro-apoptotic effect following PLD or R8-PLD
treatment, theTUNEL assay was performed on the frozen tumor
sections. Mice (n = 3 in eachgroup) bearing tumor of 400 mm3 volume
were injected twice with PLD or R8-PLD at a Dox dose of 2 and 10
mg/kg at days 1 and 3 respectively. The tumor volumewas measured on
days 1, 3, and 6. The animals were euthanized with CO2, on day 6and
the tumors were isolated. The tumors were washed quickly with PBS,
pH 7.4,weighed and frozen immediately after the extraction by
immersing in tissue freez-ing media and stored at 80 C. Tumor
slices (8 lm) were cryo-sectioned usingCryotome Cryostat, mounted
on superfrost plus slides, fixed in 4% paraformalde-hyde for 10 min
at RT, permeabilized with proteinase K (20 lg/mL) for 15 min atRT.
A TUNEL assay was performed on the sections using the FragElTM DNA
fragmen-tation detection kit following manufacturers instructions
for frozen sections. TheTUNEL positive cells were detected by
fluorescence microscopy equipped with aFITC-filter. Four random
images obtained from two different tumors for each treat-ment group
were analyzed using Spot advanced software.
2.15. Statistical analysis
The data were tested for statistical significance using Students
t-tests. p values,calculated with the Graph Pad prism 5 software
(GraphPad Software, Inc, San Diego,CA). All numerical data are
expressed as mean SD, n = 3 or 4, from 3 differentexperiments. Any
p values less than 0.05 was considered statistically significant.,
, in figures indicated p values
-
A
B
Control PLD-1h R8-PLD-1h PLD-4h R8-PLD-4h
Untreated CellsPLD-1hR8-PLD-1 hPLD-4hR8-PLD-4h
Geo
Mea
n Fl
uore
scen
ce
1 h 4 h0
5
10
15
20Untreated cells
PLD
***
***
R8-PLD
Fig. 1. Comparison of cellular uptake of R8-modified and
unmodified pegylated liposomal doxorubicin by flow cytometry. The
A549 cells were incubated with PLD and R8-PLD at 6 lg/mL of Dox for
1 and 4 h, after which the FACS analysis was performed. The
cell-associated Dox fluorescence was measured. (A) Representative
dot plot obtainedfrom FACS analysis showing the cell population
labeled with Dox after incubation with PLD or R8-PLD at 1 h and 4 h
time points. (B) Comparison of the geometric mean offluorescence of
the cells treated with PLD or R8-PLD (left panel) and the
representative histogram plot obtained from histogram statistics of
the FACS analysis (right panel).The data are mean SD, averaged from
three separate experiments. The significance of difference between
the mean was analyzed by Students t-test, p < 0.001.
194 S. Biswas et al. / Cancer Letters 335 (2013)
191200internalization of Dox in R8-PLD compared to PLD.
Localization ofthe Dox signal to the nucleus was observed in the
merged picturesas indicated by purple signal, whereas most of the
Dox signal deliv-ered via PLD was in the cytosolic compartment. The
averaged Pear-sons and Manders coefficients obtained by the Image J
analysis ofthe three center z-stacked images were 0.40 0.10, 0.49
0.03 forPLD and 0.71 0.04, 0.9 0.11, respectively (Fig. 2B).
3.4. Accumulation of liposomes in spheroids
Spheroids are a robust model to study the tumor
penetrationbehavior of drugs/drug delivery systems in cancer since
spheroidsto a great extent mimic both the architecture of tumors
and thepenetration barriers of solid tumors where drugs are mostly
con-fined to the outer cell layers. The Z-stacked images of
spheroidsshowed higher accumulation of R8-PLD compared to PLD at
boththe time points (Fig. 3). The formulations had limited
penetrationin 1 h. However, R8-PLD demonstrated very high
accumulation inthe center zone of the spheroids at 4 h, as shown by
the center slice(100 lm from top). Mean intensity (arbitrary units)
of the Dox sig-nal in the center slice of the PLD and R8-PLD
treated spheroids,quantified by Image J analysis was 29.7 1.2, 71.7
1.5 for 1 hand 105 5, 178.6 3.2 for 4 h, respectively.
3.5. Assessment of early apoptosis
Binding of the fluorescently labeled Annexin V to the cell
sur-face-bound early apoptotic marker, phosphatidyl serine is
usedto detect early apoptosis. Initiation of apoptosis causes
transloca-tion of phosphatidyl serine from inner cytoplasmic
membrane tothe outer surface. The FACS analysis result
demonstrating the com-parison of up-regulation of this early
apoptotic marker upon treat-ment with PLD and R8-PLD has been
illustrated in Fig. 4. Aftertreatment, both PLD and R8-PLD
initiated apoptosis, demonstrat-ing a shift in the cell population
as shown by the increased labelingof the cells with the Annexin
V-Alexa Fluor-488 conjugate. How-ever, R8-PLD treated cells had
significantly higher total geometricmean fluorescence (182.67 10.8)
compared to PLD treated cells(129.80 5.36). The lower right
quadrant was additionally gatedto analyze the phosphatidyl serine
expression, 8.7 1.5% of the cellpopulation was there for R8-PLD
compared to 3.6 0.5% for PLD.3.6. Assessment of cell viability by
MTT assay
To determine whether the initiation of apoptosis leads to
de-creased cell viability, an MTT assay was performed (Fig. 5). A
statis-tically significant decrease in cell viability was observed
with R8-PLD treatment compared to PLD at Dox concentration of 6.25
lg/mL and greater. R8-PLD treatment showed a 55% decrease in
cellviability compared to 10% with PLD at the highest used Dox
con-centration of 100 lg/mL (% cell viability for R8-PLD treatment
was45.35 2.14%, compared to 89.6 5.9% for PLD at 100 lg/mL ofDox
concentration). Slow decrease in the cell viability(62.89 1.95% to
45.35 2.14% at 24 h and 53.5 5.71% to46.10 2.56% at 48 h) was
observed with R8-PLD treatment atDox concentration between 6.25 and
100 lg/mL.3.7. Assessment of other cell death markers
Release of lactate dehydrogenase (LDH) in the media by
apopto-tic/necrotic cells and up-regulation of apoptotic protein,
caspase 3/7 in the cells were quantified (Fig. 6). The
R8-PLD-treated cells hadsignificantly higher level of LDH release
compared to PLD at alltested Dox concentrations. There was a
Dox-concentration depen-dent increase in LDH release in
R8-PLD-treated cells. No noticeableincrease in LDH release was
observed in PLD treatment even athighest Dox concentration of 100
lg/mL (Fig. 6A). There was 1.9-fold increase in the level of LDH
release in R8-PLD compared toPLD at Dox concentration of 100 lg/mL.
Fig. 6B illustrates caspase3/7 activity after PLD and R8-PLD
treatment. At a Dox concentra-tion of 200 lg/mL, the R8-PLD
treatment increased caspase 3/7 le-vel 5.5-fold compared to PLD
treatment (13.72 0.15 for R8-PLDvs. 2.48 0.54 for PLD). Likewise, a
LDH release profile of the Doxconcentration-dependent increase in
the caspase 3/7 protein levelwas observed in R8-PLD.
-
A
B
PLD
R8-
PLD
Top BottomZ-Slices
115 97
Dox
Mer
ged
Dox
Mer
ged
Arb
itrar
y un
its(C
oloc
aliz
atio
n co
effic
ient
s)
Pears
on's
Mand
er's
0.0
0.2
0.4
0.6
0.8
1.0PLDR8-PLD
**
Fig. 2. Assessment of cellular internalization by confocal
microscopy. (A) Confocal laser scanning micrographs of A549 cells
obtained from z-experiment. A549 cells wereincubated with PLD or
R8-PLD for 90 min, were taken at a fixed XY plane along a
consecutive Z-axes (Z 5, 7, 9, 11 from total 16 slices). The nuclei
were stained with Hoechst33342. The lower panel represents merged
pictures of Dox and Hoechst 33342 fluorescence. Scale bar: 20 lm.
(B) Assessment of nuclear localization of Doxorubicin deliveredby
PLD and R8-PLD. The merged (nuclei stained and PLD/R8-PLD treated)
pictures of the center z-slices were analyzed with Image J software
and the colocalizationcoefficients were obtained. The data are mean
SD, averaged from three images of the same treatment. p < 0.05,
paired Students t-test.
S. Biswas et al. / Cancer Letters 335 (2013) 191200 1953.8.
Assessment of in vivo therapeutic efficacy
The tumor volumes and weights of post-mortem tumors
weremeasured. The initial tumor volume at day 1 was 397.4 5.9,405.0
3.5, 397.8 4.7 mm3, which was increased at day 3 to496.38 10.5,
475.70 15.2 and 451.25 8.2 mm3 for PBS, PLDand R8-PLD treated mice,
respectively. On the day of euthanize(day 6), strong suppression of
tumor volume was observed withR8-PLD treatment. On day 6, the tumor
volumes reached to674.16 20.5, 600.00 25.0, 368.56 18.5 mm3 for
PBS, PLD andR8-PLD treatment, respectively. The tumor weight after
isolationwas 0.5802 0.20, 0.5561 0.1 and 0.355 0.05 g for PBS,
PLDand R8-PLD treatment, respectively. Detection of apoptotic
cellsby TUNEL assay, the measurement of the level of caspase 3/7
inPLD, R8-PLD or PBS-treated solid A549 tumors were detected inthe
apoptotic cells in tumor sections by TUNEL staining. The
cell-nuclei of tumors treated with PBS or PLD treatment exhibited
nogreen fluorescence attributable to FITC-labeled TdT, while the
tu-mors treated with R8-PLD had significantly higher amounts
ofgreen dots representative of apoptotic nuclei (Fig. 7). The level
ofcaspase 3/7 expression in R8-PLD-treated tumor lysate was
signif-icantly higher (1.1-fold) compared to PBS and PLD treatment.
Thus,R8-modified PLD allowed for a strong pro-apoptotic effect of
Dox inan in vivo mouse A549 tumor xenograft model.
-
A
B
PLD
-1h
R8-
PLD
-1h
PLD
-4h
R8-
PLD
-4h
m 100 Top 60 m 140 m 180 m
Mea
n In
tens
ity o
f Dox
sig
nal
(Arb
itrar
y un
its)
1 h 4 h0
50
100
150
200PLDR8-PLD
***
***
Fig. 3. Accumulation of PLD and R8-PLD in A549 spheroids by
confocal laser scanning microscopy. (A) Selected Z-stacked
micrographs of A549 spheroids, taken at consecutiveZ-axes were
shown. (B) Graphical representation of mean intensity of the
Doxorubicin signal of the center slice (100 lm inside from top) of
PLD and R8-PLD dosed spheroidsquantified with Image J software. The
cells were treated with PLD and R8-PLD at 100 lM of Dox for 1 and 4
h. Images were selected out of 16 Z-slices. Scale bar: 200 lm.
Thedata in figure B are mean SD, averaged from nine different
regions of interest from the images of two spheroids. p < 0.001,
paired Students t-test.
196 S. Biswas et al. / Cancer Letters 335 (2013) 1912004.
Discussion
Determining an effective treatment strategy for cancer
includ-ing NSCLC is challenging as among all the patients with lung
can-cer, 55% are diagnosed at an advanced stage. Surgery,
radiotherapyand chemotherapy are being utilized as treatment
options for lungcancer with high chances of relapse [42].
Platinum-based chemo-therapy is the first-line chemotherapy for
NSCLC patients. Othercytotoxic agents such as docetaxel,
pemetrexed, gefetinib are op-tions for second or third-line
therapy. However, the high incidenceof relapsed tumor growth
necessitates work on development of aneffective chemotherapeutic
treatment regimen [4]. Dox, combinedwith platinum-based therapy or
radiotherapy is currently in clini-cal trials for locally advanced
NSCLC [4345]. Dox is one of themost effective anticancer drugs ever
developed and considered afirst-line chemotherapeutic [46].
However, Dox, like other chemo-therapeutic drugs, has many adverse
effects, the most debilitatingis cumulative dose-dependant
cardiotoxicity [18,19]. Pegylatedliposomal Doxorubicin (PLD)
(commercially available as Doxil,Lipodox), is a nano-sized
Dox-loaded carrier, which accumulatesspecifically in tumor sites by
the enhanced permeability and reten-tion effect, reduces the
toxicity of free Dox [2023,47,48]. Coatingof liposomes with
biocompatible, non-immunogenic polymer PEGimparts non-recognition
by plasma serum opsonins that rendersprolonged plasma circulation,
which is advantageous for tumortargeted nanocarrier accumulation
via EPR effect. However,
-
A Annexin V-Negative Cells
Annexin V-Labeled Cells
Unt
reat
ed C
ells
PLD
R8 -
PLD
3.6 %
8.7 %
ControlPLDR8-PLD
C
Geo
Mea
n Fl
uore
scen
ce
Contr
olPL
D
R8-PL
D0
50
100
150
200
250
AnnexinV-Labeled Cells
AnnexinV-Negative Cells **
B
Fig. 4. Comparison of up-regulation of an early apoptotic
marker, phosphatidyl serine, upon treatment with PLD and R8-PLD by
flow cytometry. The A549 cells were treatedwith PLD or R8-PLD at 30
lg/mL of Dox for 4 h, then incubated for 18 h in complete media.
The cells were treated with Annexin V-Alexa Fluor 488 conjugate,
the ligand ofphosphatidyl serine. The phosphatidyl serine receptor
translocates from cytoplasm to the outer cell surface on the
initiation of apoptosis. The labeling of cells with Annexin V-Alexa
Fluor 488 conjugate was measured by FACS analysis. (A)
Representative dot plot showing the cell population with or without
Annexin V labeling. (B) Geometric mean offluorescence of PLD/R8-PLD
dosed cells treated with or without Annexin V Alexa Fluor 488
conjugate, and (C) Representative histogram plot of cells treated
with PLD or R8-PLD followed by Annexin V-Alexa 488. Data in (B) are
mean SD, averaged from three separate experiments. p < 0.01,
analyzed by the paired Students t-test.
24 h-A549
Doxorubicin (g/mL)
% C
ell v
iabi
lity
03.1
25 6.25
12.5 25 50 10
00
20
40
60
80
100
120
PLD
R8-PLD
*** *** *** *** ***
48 h-A549
Doxorubicin (g/mL)
% C
ell v
iabi
lity
03.1
25 6.25
12.5 25 50 10
00
20
40
60
80
100
120PLD
R8-PLD
*** *** *** *** ***
Fig. 5. Assessment of Dox-induced cell death of A549 cells
treated with PLD and R8-PLD. Cells were incubated with PLD or
R8-PLD at a Dox concentration of 0100 lg/mL for4 h followed by the
incubation period of 24 and 48 h before cell viability was
measured. The data are mean SD, averaged from three separate
experiments. p < 0.001,paired Students t-test.
Doxorubicin (g/mL)
LDH
rele
ased
(48
h)
06.2
512
.5 25 50 100
0
1
2
3
R8-PLDPLD
******
***
***A
Doxorubicin (g/mL)
Cas
pase
3/7
act
ivity
(arb
itrar
y un
its)
0 25 50 100
200
0
5
10
15
R8-PLD
PLD***
***
******
B
Fig. 6. The effect of PLD and R8-PLD on Dox-induced LDH release
(A) and caspase 3/7 activity (B) in A549 cells. The cells were
treated with PLD or R8-PLD at varied Doxconcentration for 4 h,
media removed and cells incubated for 48 h in complete media before
analyzing the media for LDH release and the cell lysate for caspase
3/7 activity.Each bar represents mean SD of n = 3 from three
separate experiments. The significance of differences between the
means was analyzed by the paired Students t-test.p < 0.001.
S. Biswas et al. / Cancer Letters 335 (2013) 191200
197PEGylation hinders intracellular delivery in the cancer cells
afteraccumulation at the tumor site and the maximum therapeutic
ben-efit of Dox is not achieved. This necessitates further
modification ofPLD for cytosolic delivery to improve the
effectiveness of thishighly potent anticancer drug.In an attempt to
promote cellular internalization, we surface-modified PLD with the
cell-penetrating octa-arginine (R8) peptide.The relatively short,
synthetic R8 peptide resembles the peptide se-quence of HIV-1TAT
peptide. This highly charged basic peptide hasdemonstrated
efficient cell transport properties [31,32,34,35]. The
-
A
PBS R8-PLDPLD
DA
PITU
NEL
B
Cas
pase
3/7
act
ivity
(arb
itrar
y un
its)
PBS
PLD
R8-PL
D25
30
35
40
45
50
**
C
******
Tum
or W
eigh
t (g)
PBS
PLD
R8-PL
D0.0
0.2
0.4
0.6
0.8
1.0
**
Days post-injection
Tum
or V
olum
e (m
m3)
1 3 6300
400
500
600
700
800PBSPLDR8-PLD
***
Fig. 7. Assessment of the in vivo therapeutic efficacy of
R8-modified PLD compared to unmodified PLD i.v. in A549-tumor
xenograft. (A) Tumor volume and weight (arrowsindicate treatment).
(B) Apoptosis analysis. Apoptotic cells were detected in frozen
tumor sections, determined by TUNEL assay and visualized by
fluorescence microscopy.The upper panel shows the sections stained
with Hoechst 33342 and the right panel shows the TUNEL staining.
Magnifications-20 objectives. (C) Measurement of caspase 3/7 level
in A549 tumor. The tumors isolated from the treatment groups of
PBS, PLD or R8-PLD were homogenized, centrifuged and a tumor lysate
equivalent to 25 lg ofproteins was analyzed using the Apo-ONE
Homogeneous Caspase 3/7 Assay kit. and , P < 0.01and 0.001,
unpaired Students t-test. indicates p < 0.001 compared to
PBS.
198 S. Biswas et al. / Cancer Letters 335 (2013)
191200R8-conjugated amphiphilic PEGDOPE co-polymer was
success-fully inserted into the liposomes lipid bilayer. This
modificationof the surface of PLD with a cationic peptide increased
the positivecharge on the surface of R8-PLD compared to PLD.
However, thesize remained unchanged. As expected, R8-PLD had a
time-depen-dent higher cell association compared to PLD, as
estimated bytracking the cell associated Dox fluorescence. The
cellular internal-ization, PLD- and R8-PLD-treated cells visualized
under confocalmicroscopy using z-stacked imaging indicate that
R8-PLD had ahigher cytosolic Dox delivery compared to PLD and
indicated theco-localization of Dox at its nuclear site of action.
Higher co-local-ization coefficients (Pearsons and Manders) in
R8-PLD treatedcells obtained by Image J analysis of the center
slices clearlypointed out the higher intracellular Dox-delivery
efficiency of R8-PLD compared to PLD. Even though, R8 has been
recognized as acell penetration enhancer in previous studies,
however, the intra-cellular trafficking of R8 after cellular
internalization remain rela-tively unexplored. While investigating
intracellular trafficking ofD, and L-enantiomer of R8, Fretz et al.
reported that D-R8 was addi-tionally bound to the nucleolus,
whereas both of them translocatedin the cytoplasm and labeled
nucleus [49]. Therefore, the enhancedDox delivery to the nucleus by
R8-PLD compared to PLD could bedue to enhanced intracellular
delivery coupled with R8-mediatednuclear targeting. However, the
role of R8 as a promoter for nucleardelivery needs detailed
investigation.
Over the past several years, spheroids, a 3D multilayer,
spheredcell culture system has garnered much attention as an
importanttool to study several drug developmental stages including
drugtransport, binding, therapy resistance as well as cell invasion
incancer [41,5053]. Spheroids supplement monolayer-based assaysas
they better mimic the complexities of the tumor tissues.
Themulticellular spheroids resemble avascular tumor modules of
solidtumors, the cells in spheroids resorting to an in vivo-like
differen-tiation pattern due to the resemblance in
pathophysiological mili-eu conditions such as extracellular matrix
assembly as well as cellmatrix and cellcell interactions [50]. Due
to similarities of spher-oids with in vivo tumor tissues, spheroids
have been considered asan inevitable tool to utilize in the
therapeutic development beforeturning to whole animal studies
[50,51,5456]. We studied liposo-mal accumulation in spheroids and
demonstrated that R8-PLD hadallowed for much higher Dox penetration
compared to PLD as fol-lows from the enhanced Dox signal in the
center slices of the z-stacked images (Fig. 3). The higher cellular
uptake of R8-PLD ledto higher apoptosis mediated by Dox compared to
PLD. The resultdemonstrated that the R8-PLD treated cells have
higher expressionof the protein phosphatidyl serine compared to
PLD. The resultantinduction of early apoptosis was consistent with
higher cytotoxic-ity. The result demonstrated that R8-PLD induced
higher Dox-med-iated cytotoxicity compared to PLD. The induction of
apoptosis isindicated by the elevated release of cell death marker
LDH andthe up-regulation of the pro-apoptotic protein, caspase 3/7.
R8-PLD demonstrated higher release of LDH and up-regulation of
cas-pase 3/7 in A549 monolayer compared to PLD. The in vivo
experi-ment using A549 tumor xenograft demonstrated that
R8-PLDsignificantly reduces the tumor growth compared to the PLD
treat-ment. Higher induction of apoptosis by R8-PLD treatment
wasdemonstrated by TUNEL assay compared to PLD. Tumor lysate
ofR8-PLD treated A549 tumor bearing mice had increased caspase3/7
expression compared to PLD treatment due to the higher
intra-cellular Dox delivery achieved by R8-PLD over PLD.
-
S. Biswas et al. / Cancer Letters 335 (2013) 191200 199In
conclusion, this study has identified a novel liposomal
drugdelivery system, R8-modified PLD, for improved chemotherapy
ofNSCLC. R8-PLD addresses the issue of poor cell penetration byPLD
to increase its therapeutic efficacy. The study provides a
ratio-nale for the continued investigation of R8-PLD as a promising
anticancer therapy.Acknowledgements
The work was supported in part by NIH Grants RO1 CA121838and RO1
CA128486 to Vladimir P. Torchilin. We thank Dr. WilliamHartner for
his help in editing the manuscript.References
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Octa-arginine-modified pegylated liposomal doxorubicin: An
effective treatment strategy for non-small cell lung cancer1
Introduction2 Materials and methods2.1 Materials2.2 Cell lines2.3
Synthesis of R8-PEG2KDOPE2.4 Modification of liposomes2.5
Characterization of liposomes2.6 Cell association of liposomes2.7
Cellular internalization of liposomes2.8 Formation of spheroids2.9
Liposomal penetration of spheroids2.10 Annexin V assay2.11
Cytotoxicity studies2.11.1 MTT assay2.11.2 LDH release2.11.3
Caspase assay
2.12 Animals2.13 In vivo tumor xenograft2.14 Assessment of tumor
volume reduction and TUNEL assay2.15 Statistical analysis
3 Results3.1 Physico-chemical characterization of liposomes3.2
Cell association of liposomes3.3 Cellular internalization of
liposomes3.4 Accumulation of liposomes in spheroids3.5 Assessment
of early apoptosis3.6 Assessment of cell viability by MTT assay3.7
Assessment of other cell death markers3.8 Assessment of in vivo
therapeutic efficacy
4 DiscussionAcknowledgementsReferences