PH-Responsive nanoparticles based on ibuprofen prodrug as drug carriers for inhibition of primary tumor growth and metastasis

Article abstract of DOI:10.1039/c7tb01288h

Cancer metastases represent a major determinant of mortality in patients with cancer. Cyclooxygenase-2 (COX-2) and its metabolites play important roles in tumor growth and metastasis. Overexpression of COX-2 have been found in various types of cancers including melanoma. Nonsteroidal anti-inflammatory drugs (NSAIDs) have been widely used to inhibit COX-2 and can be a promising additive for the management of aggressive cancers. A novel pH-sensitive drug delivery carrier based on PEG-derivatized ibuprofen, MPEG-PHEI, was synthesized for dual delivery of anticancer agents and NSAIDs. This amphiphilic and biodegradable copolymer could self-assemble into core-shell nanoparticles (NPs) and the hydrophobic doxorubicin (DOX) could be loaded into the core of the nanoparticles. DOX-loaded MPEG-PHEI nanoparticles (DOX/NPs) could release DOX in endosome microclimate via micelle collapse and ibuprofen via ester bond hydrolysis. In vitro DOX/NPs showed comparable cytotoxicity to DOX·HCl and comparable inhibition of COX-2 to ibuprofen. More importantly, DOX/NPs revealed a significant in vivo therapeutic efficacy in both experimental subcutaneous tumors and lung metastasis model while decreasing the toxicity of DOX. This study demonstrated the advantages of combining NSAIDs with chemotherapy agents and provided a novel nanoparticle system for both primary and metastatic tumor treatment.

Full text of DOI:10.1039/c7tb01288h

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pH-responsive nanoparticle based on Ibuprofen prodrug as
drug carrier for inhibition of primary tumor growth and
metastasis
Received 00th January 20xx,
Accepted 00th January 20xx
Zhi Zeng,a Zeliang Wei,b Limei Ma,a Yao Xu,a Zhihua Xing,a Hai Niu,bc Haibo Wang,*d and Wen
Huang*ab
DOI: 10.1039/x0xx00000x
Cancer metastases represent a major determinant of mortality in patients with cancer. Cyclooxygenaseꢀ2 (COXꢀ2) and its
metabolites play important roles in tumor growth and metastasis. Overexpression of COXꢀ2 have been found in many types
of cancers including melanoma. Nonsteroidal antiꢀinflammatory drugs (NSAIDs) have been widely used to inhibit COXꢀ2,
which could be a promising addictive to the management of aggressive cancers. A novel pHꢀsensitive drug delivery carrier
based on PEGꢀderivatized ibuprofen— MPEGꢀPHEI was synthesized to dualꢀdeliver anticancer agents and NSAIDs. This
amphiphilic and biodegradable copolymer could selfꢀassemble into core–shell nanoparticles (NPs) and payload hydrophobic
doxorubicin (DOX). DOXꢀloaded MPEGꢀPHEI nanoparticles (DOX/NPs) could release DOX in endosome microclimate
via micelle collapse and ibuprofen via ester bond hydrolysis. In vitro DOX/NPs showed comparable cytotoxicity to
DOX·HCl, and comparable inhibition of COXꢀ2 to ibuprofen. More importantly, DOX/NPs revealed a significant in vivo
therapeutic efficacy in both experimental subcutaneous tumors and lung metastasis model, while decreasing the toxicity of
DOX. This study demonstrated the advantages of combining NSAIDs with chemotherapy agents, and provided a novel
nanoparticle system for both primary and metastatic tumor treatment.
www.rsc.org/
nuclear factor κB (NFꢀκB), and cyclooxygenaseꢀ2 (COXꢀ2).8ꢀ10
COXꢀ2 overexpresses in many types of cancers such as
Introduction
Nowadays, cancer is one of the leading causes of morbidity and
mortality in the world. In clinics, chemotherapy is highly
significant in the treatment of solid tumors, but this approach is
usually effective at early stage. Once the tumor has metastases,
12
prostate, colon cancer and melanoma.11,
Cyclooxygenases
(COXs) catalyze the rateꢀlimiting step in the formation of
prostaglandins (PGs) from arachidonic acid (AA). COXꢀ2 is the
inducible isoform of the COX enzyme family. Unlike
chemotherapy is much less successful.1, Clinical researches
2
Cyclooxygenaseꢀ1 (COXꢀ1), which plays
maintaining homeostasis, COXꢀ2 plays
a
a
key role in
key role in
have revealed that metastases accounts for more than 90%
death in cancer patients.3, 4 Furthermore, metastases are highly
resistant to conventional chemotherapies, mainly due to its
biological heterogeneity, as well as the effects of organ
environment on biological behavior of metastatic cells.2,
There is a crucial need to provide new treatment strategies for
inhibiting primary tumor and its metastasis at the time of
chemotherapy.
inflammation, by inducing stimulate proꢀinflammatory
cytokines and growth factors.13, 14 More importantly, COXꢀ2
and its products, especially Prostaglandin E2 (PGE2), were
found to favor tumor growth and metastasis, which has made
5ꢀ7
16
COXꢀ2 an important drug target for cancer treatment.15,
Nonsteroidal antiꢀinflammatory drugs (NSAIDs) such as
ibuprofen and aspirin have been widely used to inhibit COXꢀ2.
Therefore, a combination of NSAIDs and anticancer drug can
be a promising strategy for both primary and metastatic tumors.
To date, NSAIDs have been documented to decrease
migration.17 Using adjuvant NSAIDs, including aspirin,
naproxen, buprofen, celecoxib and piroxicam, has also been
found to decrease the incidence of primary and recurrent
cancers including prostate, colon, breast cancer and melanoma,
thereby decrease mortality.18ꢀ21
The metastatic process is intricate and tightly associated with
enzymes, proꢀinflammatory cytokines and growth factors,
including interleukin (IL), matrix metalloproteinases (MMPs),
Over recent decades, polymeric drug delivery systems have
been developed as carriers for chemotherapeutic agents to
improve drug residence time in tumors, and therapeutic effect,
23
via enhanced permeation and retention (EPR) effect.22,
Numerous investigations have demonstrated that this drug
delivery systems could enhance the solubility and
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bioavailability of drugs, thus, allowing them to reach their Pentamethyldiethylenetriamine
(PMDETA),
pyrene,
25
target sites and reduce side effects.24,
Recently, stimuliꢀ ethanolamine, and 2ꢀbromoꢀ2ꢀmethylpropionyl bromide were
responsive polymeric systems attract much attention. The purchased from Aladdin Chemical Co., Ltd. (Shanghai, China).
payload could be released rapidly in response to external trigger Methanol, tetrahydrofuran (THF), dichloromethane (DCM),
such as pH, temperature, or redox potential.26ꢀ29 Among them, petroleum ether (PE), ethyl acetate (EA), ethyl ether,
pHꢀresponsive drug delivery systems have been widely applied isopropanol, acetone, and N,N′ꢀdicyclohexylcarbodiimide
in cancer therapy due to the presence of a remarkable pH (DCC) were purchased form Chengdu Kelong Chemical Co.,
gradients between intracellular endosome microclimate (~5.0) Ltd. (Chengdu, China). 4ꢀFormylbenzoic acid (pꢀCBA) and 1ꢀ
and normal tissues (7.4).30ꢀ32 The pHꢀresponsive drug delivery [3ꢀ(dimethylamino)propyl]ꢀ3ꢀethylcarbodiimide
methiodide
systems were able to keep stable in the blood circulation and (EDC) were purchased from Energyꢀchemical (Shanghai,
release drug rapidly after internalized by cancer cells.33, 34
China). Doxorubicin hydrochloride (DOX·HCl) was purchased
from Tecoland Corporation (Irvine, CA, USA.). Triethylamine
(TEA), 3ꢀ(4, 5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2, 5ꢀdiphenyl tetrazolium
bromide (MTT) and dimethyl sulfoxide (DMSO) were
purchased from SigmaꢀAldrich (St. Louis, MO, USA). All of
the reagents were used as received. PEG (Mn = 2000 g/mol)
was purchased from Fluka and dried by azeotropic distillation
in the presence of dry toluene.
Dulbecco's modified Eagle's medium (DMEM) was purchased
from HyClone, GE Healthcare Life Science (Logan, UT, USA).
Fetal bovine serum (FBS) was purchased from Gibco Life
Technologies (Carlsbad, CA, USA). Hoechst 33258 was
purchased from Biyuntian Biotechnology Co. (Shanghai,
China). Mouse monoclonal antiꢀGAPDH (1:5,000; G8795)
were purchased from SigmaꢀAldrich (St. Louis, MO, USA).
Rabbit monoclonal antiꢀCOXꢀ2 (1:1,000; ab62331) were
purchased from Abcam (Burlingame, CA, USA). Cleaved
Caspaseꢀ3 (Asp175) Rabbit mAb (1:1000; 9664S) were
purchased from Cell Signaling Technology Inc (Beverly, MA,
USA). Horseradish peroxideꢀconjugated goat antiꢀmouse
immunoglobulin IgG (1:5,000; scꢀ2005) and goat antiꢀrabbit
IgG (1:5,000; scꢀ2004) were purchased from Santa Cruz
Biotechnology Inc. (Santa Cruz, CA, USA).
Scheme 1 Schematic illustration: drug loading process of MPEGꢀPHEI nanoparticles in
aqueous solution and pH triggered drug release.
An innovative pHꢀsensitive, amphiphilic and biodegradable
copolymer methoxy poly (ethylene glycol) – b – poly (2ꢀ([2ꢀ4ꢀ
(2ꢀmethylpropil) phenyl] propionyl] oxy) ethyl methacrylate)
(MPEGꢀPHEI) was synthesized through atom transfer radical
polymerization (ATRP). Because of its amphiphilicity, MPEGꢀ
PHEI could selfꢀassemble and payload hydrophobic drug in
water. Based on previous studies, ibuprofen could be released
via ester bond hydrolysis at physiological conditions (37 °C, pH
7.4), and retain its activity.35, 36 Nonꢀtoxic and biocompatible
Characterization.
1HꢀNMR experiments were conducted on an INOVAꢀ400
spectrometer (400 MHz, Varian, USA), using tetramethylsilane
(TMS) as internal reference, and CDCl3 as solvent. Gel
permeation chromatography (GPC) was performed with
Watersꢀ1515 (USA) using N, Nꢀdimethylformamide
(DMF)/LiBr as eluent. The molecular weights are relative to
narrow polystyrene standards. The flow rate was 1.0 mL/min at
40 °C. The numberꢀaveraged Wn and molecular weight
distributions (PDIs) of the polymers were measured on a
Waters 1515 GPC instrument with a Waters 1515 isocratic
HPLC pump, a Waters 2414 refractive index detector, a Waters
717 plus autosampler and a set of MZꢀGel SD plus columns
(300 × 8.0 mm, 500 °A, 103 °A and 104 °A). TEM was
recorded on a JEOL JEMꢀ100CXꢀII instrument at a voltage of
200 kV. A drop of nanoparticle solution was placed on a
carbonꢀcoated copper grid, and then the liquid was freeze dried
in vacuum before measurement. Sizes of nanoparticles were
measured on a HORIBA DLS particle size analyzer LBꢀ550 at
room temperature. The fluorescence and UVꢀVis spectra were
determined by Fꢀ7000 fluorescence spectrometer (HITACHI,
Tokyo, Japan) and DU 800 UVꢀVis spectrometer (Beckman
Coulter, CA, USA), respectively.
methoxy poly (ethylene glycol) (PEG) was employed as
38
protective shell.37,
DOXꢀloaded MPEGꢀPHEI nanoparticles
(DOX/NPs) possessed excellent biophysical properties
including size, loading capacity, in vitro drug release kinetics
and biocompatibility. Subcutaneous B16 melanoma tumor
model and lung metastasis model were employed to invest in
vivo antiꢀtumor behaviours of DOX/NPs. Among all deadly
types of cancer, melanoma is particularly deadly because of its
propensity to metastasize. Its incidence keeps rising.39 The
overexpression of COXꢀ2 has been frequently observed in both
human melanoma specimens and murine models, as in many
other types of cancer.40ꢀ42 Our results revealed that DOX/NPs
could inhibit the activity of COXꢀ2 in vivo, thusly inhibit tumor
metastasis.
Experimental
Reagents.
Ibuprofen, 2ꢀhydroxyethyl methacrylate (HEMA), 4ꢀ
dimethylaminopyridine
(DMAP),
CuCl,
N,N,N',N',N''ꢀ
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Synthesis of 2-([2-4-(2-methylpropil)phenyl]propionyl]oxy) ethyl isopropanol. The pure PEGꢀCBAꢀOH was dried in vacuum for
methacrylate (HEI). Ibuprofen (2.1 g) and EDC (2.0 g) was 2 days. Yield: 90%.
dissolved in 50 mL of DCM in a single neck round flask. Synthesis of PEG-CBA-Br. PEGꢀCBAꢀOH (4.4 g) and TEA (0.6
HEMA (1.3 g) and DMAP (0.5 g) was added. The mixture was mL) was dissolved in 50 mL of DCM, and 2ꢀbromoꢀ2ꢀ
stirred at room temperature for 24 h. Then the solvent was methylpropionyl bromide (0.5 g) was added drop wise into the
evaporated under vacuum and product was purified by silica gel solution at 0 °C in 20 min. The solution was stirred for 4 h at 0
column chromatograph (EP: EA=15:1, v/v). Yield: 83%. 1H °C and for another 48 h at room temperature. Side product was
NMR (400 MHz, CDCl3) δ 7.18 (d, 2H, ArꢀH), 7.07 (d, 2H, Arꢀ removed by filtration. The solvent was removed under vacuum.
H), 6.03 (s, 1H, C=CH), 5.54 (s, 1H, C=CH), 4.31 (m, 4H, The crude product was precipitated in ethyl ether to remove
COOꢀCH2ꢀCH2), 3.72 (q, 1H, CH), 2.43 (d, 2H, ArꢀCH2), 1.89 impurities, and then in vacuum for 2 days. Yield: 82%. 1H
(s, 3H, CH3), 1.83 (m, 1H, CH), 1.49 (d, 3H, CH3), 0.89 (d, 6H, NMR (400 MHz, CDCl3) δ 8.40 (s, 1H, ꢀCH=Nꢀ), 8.17 – 8.12
CH3).
(d, 2H, ArꢀH), 7.84 – 7.79 (d, 2H, ArꢀH), 4.50 – 4.45 (t, 2H,
CH2), 4.01ꢀ3.96 (m, 2H, ꢀCH2ꢀOꢀ), 3.86ꢀ3.79 (m, 2H, ꢀCNꢀ
CH2ꢀ), 3.65 (m, ꢀCH2ꢀ of PEG), 3.38 (s, 3H, ꢀOꢀCH3), 1.82 (s,
6H, CH3).
Synthesis of MPEG-PHEI. IBUMA (3.0 g) and PEGꢀCBAꢀBr
(2.0 g) was dissolved into THF in a three neck round flask. And
CuCl (0.02 g) and PMDETA (0.07 g) was added under
nitrogen. The reaction was stirred at 50 °C for 24 h under a
nitrogen atmosphere. Reaction mixture was separated by on a
neutral alumina column (THF), and the solvent was evaporated
under reduced pressure to give crude product. The crude
product was further purified by methanol precipitation, and
dried in vacuum for 2 days. Yield: 59%. 1H NMR (400 MHz,
CDCl3) δ 7.36 (d, 4H, ArꢀH), 7.18 – 6.95 (d, 81H, ArꢀH), 4.31
– 4.10 (s, 86H, ꢀCOOꢀCH2), 3.65 (m, ꢀCH2ꢀ of PEG), 3.38 (s,
3H, ꢀOꢀCH3), 2.41 (d, 41H, ArꢀCH2ꢀ), 2.38 – 1.60 (m, 65H, ꢀCꢀ
CH2), 1.60 – 1.34 (m, 69H, CH3), 1.25 (s, 6H, CH3), 0.87 (d, J
= 5.5 Hz, 195H, CH3).
Critical micelle concentration (CMC) measurement.
According to previous study, the CMC was determined by
using pyrene as
a a
fluorescence probe.44 In detail,
predetermined amount of pyrene solution in acetone was added
in a series of 4 mL centrifuge tubes, and acetone was
evaporated overnight completely. The micelle solution in Milliꢀ
Q water in various concentrations were added into each of those
pyrene centrifuge tubes, ensure the final concentrations ranged
from 5.0 × 10−6 to 0.1 g/L, and pyrene’s concentration was 0.12
mg/L. Placed all of the solutions in darkness at room
temperature for 2 h to equilibrate pyrene partition between
water and micelles. For fluorescenceꢀexcitation spectra, the
emission wavelength was set to 390 nm and the excitation
bandwidth was 5.0 nm. The scan speed was 240 nm/min.
Scheme 2 Synthesis route of precursors and MPEGꢀPHEI.
Synthesis of PEG-CBA. PEGꢀCBA was synthesized as the
procedure outlined by Song.43 In briefly, CBA (15.2 g) and
PEG (12.6 g) was dissolved in 200 mL DCM. Then DCC (16.5
g) and DMAP (2.4 g) was added into the solution. The reaction
solution was stirred gently at room temperature for 60 h. Side
product, dicyclohexylurea, was removed by filtration. The
solvent was removed by rotational evaporation. The crude
product was purified by recrystallization in isopropanol, and in
Nanoparticle formation.
10 mg MPEGꢀPHEI was dissolved in 1 mL DMSO. 100 ꢁL of
this solution was added into 400 ꢁL of MilliꢀQ water in drop
wise. The mixture was stirred at room temperature for 2 hours,
and dialyzed by a dialysis bag (MWCO: 3500) against MilliꢀQ
water for one day.
1
vacuum for 2 days. Yield: 85%. H NMR (400 MHz, CDCl3) δ
10.11 (s, 1H, ArꢀCHO), 8.25 – 8.20 (d, 2H, ArꢀH), 7.99 – 7.94
(d, 2H, ArꢀH), 4.55 – 4.49 (t, 2H, CH2), 3.65 (m, ꢀCH2ꢀ of
PEG), 3.38 (s, 3H, ꢀOꢀCH3).
Synthesis of PEG-CBA-OH. Ethanolamine (1.8 g) and PEGꢀ
CBA (6.5 g) was dissolved in 100 mL of THF, and reaction
solution was stirred at 40 °C overnight. The solvent was
removed under reduced pressure. The crude product was
dissolved in methanol, precipitated in ethyl ether to remove
impurities, and further purified by recrystallization in
In vitro ibuprofen release.
Ibuprofen release via hydrolytic degradation of nanoparticles was
evaluated in vitro in phosphate buffered saline (PBS, pH = 7.4 or
5.0), as previous work by Stebbins,35 and Panah45. Briefly, 2 mL of
blank nanoparticles solution were sealed into dialysis bag (MWCO:
3500) against 30 mL of PBS with different pH, and incubated at 37
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°C. At predetermined time intervals, 1 mL of medium was collected added 100 ꢁL of DMSO into each well. The plate was shacked
and 1 mL of fresh PBS was replaced. The ibuprofen concentration in for 10 min at room temperature in dark. The optical density
different samples was calculated by UVꢀVis spectra. The amount of (OD) was then recorded with a SpectraMax M5 microplate
ibuprofen was calculated according to a calibration graph. The reader (Molecular Devices, LLC, Sunnyvale, CA, USA) at 492
maximum absorbance wavelength (λmax) of UVꢀVis spectra was set nm. Cytotoxicity was expressed as percentage relative to the
to 265 nm. Three groups of replicate measurements were carried out unexposed control group. Control values were set at 0%
for each time point.
cytotoxicity.
Drug loading and in vitro DOX release.
In vitro cellular uptake.
DOX/NPs were prepared as previous studies.46 Briefly, 3 mg of B16 cells were seeded on flameꢀdried coverslips placed in 6ꢀ
DOX·HCl and 10 mg of MPEGꢀPHEI were dissolved in 3 mL well culture plates at a density of 1×
105 cells/well. Following
DMSO, and 200 ꢁL of TEA was added. 100 ꢁL of this solution incubation overnight, the culture media was refreshed, the cells
was added to 400 ꢁL of MilliꢀQ water in drop wise. The were cultured with media containing DOX/NPs (containing 10
mixture was stirred at room temperature for 2 hours, and mg/L of DOX) for 2, 4 and 6 h, as well as DOX·HCl
dialyzed by a dialysis bag (MWCO: 3500) against MilliꢀQ (containing 10 mg/L of DOX) for 2 h. The cells were washed
water for one day. The scan speed was 240 nm/min. DOX/NPs with PBS (pH 7.4) for three times. Then Hoechst 33258
were lyophilized to give red powder. The amount of DOX was (Biyuntian Biotechnology Co., Shanghai, China) staining was
calculated according to a calibration graph. The λex/em of performed (37 °C, 10 min). After staining, cells were washed
fluorescenceꢀemission spectra was set to 495/593 nm and the with PBS twice and the uptake was examined by confocal laser
excitation bandwidth was 5.0 nm. Drug loading content (DLC) scanning microscope (CLSM, Imager Z2, Zeiss, Oberkochen,
and loading efficiency (DLE) of DOX was calculated by the Germany).
following formula:
Wound healing assay.
Weight of loaded DOX
Weight of polymer
B16 cells were seeded into 6ꢀwell culture plates at a density of
5 × 10ꢀ cells/well, and then incubated to grow
cell
DLC(%) =
× 100%
× 100%
a
monolayer. The cell monolayer was scratched with a sterile
plastic pipette tip (10 ꢁL) and washed three times with PBS.
Scratched B16 cells were exposed to ibuprofen (46.9 mg/L), or
MPEGꢀPHEI (100 mg/L) for 24 h. The cell migration to the
wound area was photographed and open wound area was
analyzed by ImageJ (Java 1.6.0_24, National Institutes of
Health, Bethesda, MD, USA). The rate of migration was
measured by quantifying the open wound area before and after
treatment.47
Weight of loaded DOX
Weight of feeding DOX
(
)
DLE % =
DOX released by polymer hydrolytic degradation was
evaluated in PBS (pH = 7.4 or 5.0). 2 mL of DOX/NPs solution
were sealed into dialysis bag (MWCO: 3500) against 30 mL of
PBS with different pH, and incubated at 37 °C in dark. At
predetermined time points, 1 mL of the released medium was
withdrawn for fluorescence analysis and 1 mL of PBS was
refreshed. The DOX concentration in different samples was
calculated by fluorescenceꢀemission spectra as the same way in
drug loading section. Three groups of replicate measurements
were carried out for each time point.
Western blot analysis.
B16 cells were seeded into each well of 6ꢀwell culture plates at
a density of 1 × 10ꢁ cells/well, and then incubated overnight.
B16 cells were treated with ibuprofen (46.9 mg/L) or MPEGꢀ
PHEI (100 mg/L) for 24 h and then harvested by cell lysate
(with 1 mM of protease inhibitor). Extracts were centrifuged at
12000 g for 10 min at 4 °C, and the supernatants were
collected, 100 °C water bath heated for 5 min and stored at –80
°C. The protein concentrations were measured using the BCA
protein assay kit (Biyuntian Biotechnology, Co., Shanghai,
China).
20 ꢁg protein were separated by sodium dodecyl sulfate–
polyacrylamide gel electrophoresis (SDS–PAGE) on 10% (w/v)
gels, and then transferred to polyvinylidene difluoride (PVDF)
membranes (EMD Millipore, Billerica, MA, USA). Membranes
were blocked at room temperature for 1 h by blocking buffer
(5% skim milk in TBST, w/v). The membranes were then
probed with primary antibodies for COXꢀ2 or GAPDH
overnight at 4 °C. After washing with TBST (3×5 min),
membranes were incubated with a horseradish peroxidaseꢀ
conjugated goat antiꢀmouse and goat antiꢀrabbit IgG secondary
antibodies at room temperature for 1 h. The reactive bands were
Cell culture.
B16 murine melanoma cells were purchased from the Cell
Bank of the Shanghai Institute of Biochemistry and Cell
Biology, Chinese Academy of Sciences (Shanghai, China). The
cells were cultured in DMEM with 10% of heatꢀinactivated
FBS. The B16 cells were cultured at 37 °C under a humidified
atmosphere of 5% CO2.
In vitro cytotoxicity study.
The cytotoxic behaviors of blank nanoparticles and DOX/NPs
were evaluated using the MTT assay. Cells were seeded into
96ꢀwell culture plates at a density of 5000 cells/well, and
incubated overnight. Then the culture media were replaced.
B16 cells were incubated in media containing MPEGꢀPHEI or
ibuprofen with gradient concentrations of ibuprofen from 0.1 to
1.5 mM (20.6 mg/L to 309.4 mg/L) and media containing
DOX/NPs with gradient concentrations of DOX ranging from
0.01 to 5.0 ꢁg/mL, respectively. After that all cell were
incubated for 24 h. At the end of the incubation, 100 ꢁL of
MTT (5 mg/mL in DMEM) was added to each well and further
incubated for 4 h at 37 °C. The medium was removed and
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detected using Immobilon Western Chemiluminescent HRP substrate. Slides were also counterstained with haematoxylin.
Substrate (EMD Millipore) to enhance chemiluminescence.
Negative controls were treated as previously described, but
primary antibodies were replaced by PBS. All tissue samples
were examined under microscope to observe histopathological
evidence of treatment efficiency.
Animals.
Male BALB/c mice (four weeks old, 18ꢀ22 g), and housed in a
temperature and illumination regulated environment (25±2 °C,
12 h light/dark cycle). Mice were fed with standard food and Statistical analysis.
water. Animal experimental protocol was approved by Ethics All data were reported as the mean ± standard deviation (SD).
Committee of West China Hospital of Sichuan University.
Statistical analysis was performed using SPSS software
package Version 19.0 for Windows (SPSS, Inc., Armonk, NY,
USA). Statistical significance was analyzed using unpaired,
twoꢀtailed Student’s tꢀtest. The significance threshold was
P<0.05.
In vivo antitumor assay.
To investigate the in vivo antitumor growth efficiency of
DOX·HCl and DOX/NPs, a subcutaneous B16 melanoma
tumor models was established. Male BALB/c mice were
subcutaneously injected with 1×
106 B16 cells in 0.1 mL of
Results and discussion
medium. The mice were then reared for a week when tumor
volume reached 100~150 mm3. Before treatment and the mice
were randomly divided into different treatment groups. Mice
were given DOX·HCl or DOX/NPs intravenously via the tail
vein at a dose of 10 mg DOX equiv./kg., or 46.9 mg ibuprofen
equive./kg. every 3ꢀ4 days, physiological saline was
administrated to another group as control. In the meantime the
body weight and the tumor volume of mice were measured.
Tumor sizes were measured with a digital caliper, and tumor
Polymers synthesis and characterization.
Firstly, ibuprofen was linked to HEMA with ester bond, as
shown in Scheme 2, giving a founctional monomer which could
be used for further polymerization. To achieve pHꢀsensitive
property, benzoicꢀimine bond was introduced into the PEG
coating. Since its π−π conjugation, benzoicꢀimine bond is
steady in basic or neutral surrounding, but it will hydrolyze in
acidic environment. Finally, the targeted polymer was
polymerized via ATRP method using PEGꢀCBAꢀBr as the
macroinitiator. With the ibuprofen releasing via ester bond
hydrolytsis, and the pH–sensitive property of benzoicꢀimine
bond, the target delivery and release of both ibuprofen and
DOX into cancer cells could be accomplished.
volumes were calculated by the formula 0.5
the long tumor diameter and is the short one (mm). Relative
× a is
ab2, in which
b
tumor volume (RTV) was V/V0 (V0 is the initial tumor
volume). At the end of the treatment (14 days), mice were
sacrificed.
1H NMR was employed to confirm the chemical structures of
In vivo lung metastasis assay.
1
precursors and MPEGꢀPHEI. H NMR spectrum also verified
To investigate the in vivo antiꢀmetastasis efficiency of
DOX/NPs, a lung metastasis models was employed. Male
BALB/c mice were injected with 1×106 B16 cells in 0.1 mL of
PBS via tail vein. The mice were randomly assigned to several
groups. One hour after injection, the mice were intravenously
administered drugs at a dose of 10 mg DOX equiv./kg., or 46.9
mg ibuprofen equive./kg, control group was treated with
physiological saline. Then mice were treated in every 3–4 days.
Other six mice without injection were raised as normal group.
At the end of the treatment (14 days), mice were sacrificed.
polymer achieved a relatively high drug loading (46.9 wt % of
ibuprofen) as we expected.35 GPC results showed the low
molecular weight distribution (PDI less than 1.2, shown in Fig.
S2) of MPEGꢀPHEI.
Characterizations of blank nanoparticles and DOX/NPs.
To investigate the selfꢀassembly behavior of amphiphilic
MPEGꢀPHEI, pyrene was used as a probe for fluorescence
measurement. The absorption band shifts from 334 nm to 338
nm as the increasing concentration, and the fluorescence
intensity ratio of 338 nm and 334 nm (I338/I334) was calculated.
The function of I338/I334 and the logarithm concentrations (LgC)
is shown in Figure 2A. The concentration, 2.5 × 10ꢂꢃ mg/mL,
at intersection of two tangent lines was the CMC value,
indicating that MPEGꢀPHEI showed decent tendency to selfꢀ
assemble in aqueous environment.
Then nanoparticles were prepared and studied by DLS and
TEM. As shown in Figure 2C, the particle diameters were
measured by DLS, and average diameter was around 190 nm.
The morphology of nanoparticles was observed by TEM,
shown in Figure 2D. The TEM images suggested that the
nanoparticles were spherical, and the diameter was less than
200 nm. The sizes determined by TEM and DLS were different,
since the TEM image is obtained in a dry state. After loaded
with DOX, the diameter of DOX/NPs was measured by the
DLS as a comparison shown in Figure S2. The mean size of
DOX/NPs was 213.7 nm because the loading of DOX in the
Histology and immunohistochemistry.
The heart and tumor tissue of mice with xenograft melanoma
tumor, and lung tissue of lung metastasis mice were collected
after the mice were sacrificed. The dissected organs and tumors
were fixed in 4% paraformaldehyde and processed into paraffin
wax, sectioned at 5 ꢁm. Tissue sections were stained with
hematoxylin & eosin (H&E). For immunohistochemistry (IH),
fixed and embedded tumor and lung tissue specimens were cut
into 5 ꢁm sections, and mounted on polyꢀlysineꢀcoated slides.
TrisꢀEDTA buffer (pH 9.0) was employed for antigen retrieval.
To inactivate endogenous peroxides, sections were treated in
3% hydrogen peroxide for 20 min. Sections were blocked with
normal goat or mouse serum in PBS at 37 °C for 30 min, and
incubated with primary antibodies for COXꢀ2 or Caspaseꢀ3
overnight at
4 °C. The horseradish peroxidaseꢀlabeled
secondary antibody was applied at 37 °C for 30 min.
Diaminobenidine (DAB) was adopted as chromogenic
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hydrophobic core increased the core diameter. Both diameters Drug loading and in vitro release.
Journal Name
Since DOX was employed as the hydrophobic anticancer drug,
DOX loading content was measured by its fluorescence. DOX
was loaded in nanoparticles via dialysis, and then the DOX/NPs
were lyophilized. According to the equations in Materials and
Methods section, the DLC and DLE of nanoparticles were
10.0% and 33.4%. The release behavior of DOX from vehicles
was examined at physiological and acidic conditions. The
amount of released DOX in media was measured by
fluorescence. The DOX releasing was controlled by
nanoparticles collapse from cleavable benzoicꢀimine.
Therefore, under weak acidic condition the release rates were
relatively higher, and the amount of cumulative released DOX
over the same period of time was bigger. As shown in Figure
2B, under physiological conditions, ~35% of DOX was
released in first 10 h, however, more than 55% of DOX was
released at pH 5.0 under the same conditions. After 120 h of
experiment, the release of DOX was 85.0%. These results
suggested the acidic condition reinforced the collapse of
nanoparticles, and release of DOX rapidly at acidic endosome
condition. As for ibuprofen, the release of ibuprofen was
investigated by UVꢀvis spectra. As shown in Figure S4, MPEGꢀ
PHEI exhibited a faster ibuprofen release in acidic condition
throughout the 14ꢀday experiment. Ibuprofen release is
controlled by both nanoparticles collapse and ester bond
hydrolysis. Based on previous studies using NSAIDꢀcontaining
nanoparticles, more labile nanoparticles cleave first, followed
by ester bond hydrolysis.34, 50
of blank nanoparticles and DOX/NPs were amenable to cell
internalization.48, 49
In vitro cytotoxicity of DOX/NPs.
Bioactivity and safety are two important factors in order to
realize further use for synthetic biomaterials. The in vitro
cytotoxicity of blank nanoparticles and DOX/NPs was
evaluated by MTT assay. Figure 3A showed the cytotoxicity of
different concentrations of ibuprofen and blank nanoparticles to
B16 cells. After treatment for 24 h, both blank nanoparticles
and ibuprofen (concentration of ibuprofen ranging from 0.1 to
1.5 mM) showed no significant growth inhibition effect. Then
B16 cells were incubation with DOX and DOX/NPs for 24 h.
As show in Figure 3B, the doseꢀdepend cell inhibition for both
DOX·HCl and DOX/NPs were observed. DOX/NPs showed a
weaker cytotoxicity than DOX·HCl, especially in at low
concentration, because of the prolonged release from the
nanoparticles and different cellular uptake way of DOX. The
results were consistent with the followed cellular uptake results.
The IC50 of DOX·HCl and DOX/NPs was 0.315 and 0.638
ꢁg/mL respectively.
Figure 1 1HꢀNMR spectra of PEGꢀCBAꢀBr (A) and MPEGꢀPHEI (B) in CDCl3.
Figure 2 Critical micelles concentration (A) ascertained by the function of excitation
intensity ration of pyrene at 338 and 334 nm (I338/I334) vs the polymer concentrations
in water. The cumulative DOX release profiles of DOX/NPs (B), in physiological and
acidic conditions (37 °C, pH 7.4 and 5.0). Size distribution (C) and morphology
characteristics (D) of blank nanoparticles. The scale bar of the TEM is 0.2 ꢁm.
Figure
3 Cytotoxicity of blank nanoparticles and ibuprofen (A), DOX/NPs and
DOX·HCl (B) on B16 cells for 24h. Error bars indicate SD (n = 3).
6 | J. Name., 2012, 00, 1-3
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ARTICLE
Cellular uptake of DOX/NPs.
treated group (wound area ~81.1%). The in vitro COXꢀ2
In this work, DOX was employed as the model drug due to its inhibition bioactivity in B16 cells of MPEGꢀPHEI was studied
anticancer activity and intrinsic red fluorescence. Since the by Western blot. As shown in Figure 5C, GAPDH was used as
intracellular fluorescence intensity reflected the DOX amount an internal reference, and the expression of COXꢀ2 in both
absorbed by cells, the DOX uptake and location in B16 cells ibuprofen and MPEGꢀPHEI treated groups were significantly
were visualized by CLSM. With an increasing treated time downꢀregulated compared to control group. These results
(from 2 h to 6 h), the red fluorescence inside the cells intensifies indicated that the MPEGꢀPHEI could effectively inhibit B16
became stronger visually shown in Figure 4A, B and C. But cell migration and COX expression, suggesting that the
even in cells treated with the DOX/NPs for 6 h, the red ibuprofen in MPEGꢀPHEI could be released to the free form
fluorescence intensity was weaker than it in cells treated with retaining its activity.
DOX·HCl for 2 h. In DOX·HCl treated cells, the red
fluorescence mainly overlapped with blue fluorescence in
nucleus, shown in Figure 4D. By comparison, the red
fluorescence signals from NPs mainly distributed in cytoplasm.
It was endocytosis way of nanoparticle internalization that lead
to the difference. These results were impressive evidence of
DOX/NPs got into the tumor cells via endocytosis and released
DOX in cytoplasm.
Figure 5 Wound healing assay in B16 cells treated with ibuprofen and MPEGꢀPHEI for
24 h (A), quantification of wound area in control, ibuprofen and MPEGꢀPHEI treated
B16 cells (B).Western blot (C) and protein expression level of COXꢀ2 in B16 cells (D)
treated with MPEGꢀPHEI and ibuprofen for 24 h, GAPDH was used as internal
reference. Error bars indicate SD (n = 3). *P < 0.01, **P < 0.005, treated group versus
control group. The scale bar is 50 ꢁm.
Figure 4 Fluorescence microscopy images of intracellular uptake of DOX (red), treated
by DOX·HCl for 2 h (A), DOX/NPs for 2 h (B), 4 h (C), and 6 h (D). Nuclei were
labeled with Hoechest (blue). The scale bar is 50 ꢁm.
Inhibition of MPEG-PHEI against COX-2 expression and
migration in B16 cells.
Figure 6 In vivo antitumor efficacy of DOX/NPs. (A) Body weights of mice, (B) tumor
growth curves, (C) tumor morphologies after the mice were sacrificed, (D) H&E
staining of heart sections (a) and tumor sections (b) harvested from each groups, IH
staining of Caspaseꢀ3 (c) and COXꢀ2 (d) in tumor sections from different groups. Error
bars indicate SD (n = 6). *P < 0.01, treated group versus saline group. The scale bar is
500 ꢁm.
The inhibition of MPEGꢀPHEI against B16 cell migration was
tested by the wound healing assay. The effect of MPEGꢀPHEI
treatment on B16 cell migration was shown in Figure 5A, after
treatment with MPEGꢀPHEI for 24 h, cell migration was
notably inhibited with the wound area ~80.1% compared to
wound area ~40.6%in control group. Moreover, the wound area
of MPEGꢀPHEI treated group was comparable to ibuprofen
In vivo antitumor efficacy of DOX/NPs.
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Journal Name
As shown in Figure 6A, DOX/NPs and saline groups showed DOX·HCl and ibuprofen via enhanced permeation and
no obvious changes in body weight, whereas DOX·HCl group retention (EPR) effect.22, 23 H&E staining showed a significant
exhibited a notably decrease, because of inherent systemic reduction of the tumor mass and lungꢀinfiltrating tumor cells in
toxicity of DOX·HCl. Compared to control group, both lungs of DOX/NPs group animals, in Figure 7D a. Moreover,
DOX/NPs and DOX·HCl exhibited tumor inhibition efficacy. immunohistochemistry study revealed that DOX/NPs treatment
The RTV for saline, DOX/NPs and DOX·HCl treated group markedly reduced COXꢀ2 expression in lung, shown in Figure
treated group at day 14 was ~6.0, 2.3 and 3.6, respectively. The 7D b These results confirmed the antimetastatic activity in cell
photo of isolated tumor in figure 6C confirmed DOX/NPs was study, and indicated that DOX/NPs exert antimetastatic activity
more effective than DOX·HCl on tumor growth suppression in in vivo
vivo. Furthermore, the histological assay of tumor and heart
after treatment were performed using H&E staining or
.
Conclusions
immunohistochemistry. H&E assays of hearts indicated that
DOX/NPs caused little damage to heart but widespread
apoptosis in tumor. In DOX·HCl group, more significant heart
damage was observed.
In this paper,
a novel pH responsive amphiphilic and
biodegradable block copolymer, MPEGꢀPHEI based on PEG
and ibuprofen was synthesized successfully. MPEGꢀPHEI
could selfꢀassemble into nanoparticles to form a classic “core–
shell” structure in aqueous solution with an appropriate mean
particle diameter around 200 nm. Moreover, DOX/NPs had
proper drug loading content of DOX and ibuprofen, as well as
pH sensitivity. DOX/NPs could internalize into B16 cells
through endocytosis and kill B16 cells. MPEGꢀPHEI could
release ibuprofen through hydrolysis, and the released
ibuprofen retained activity to inhibit the COXꢀ2 expression and
migration of B16 cells. Furthermore, DOX/NPs exerted
compared antiꢀtumor activity to DOX·HCl, and inhibited
melanoma metastasis to lung in mice. This research provided a
promising drug delivery system combining NASIDs with
antineoplastic drugs, which could be a potential alternative in
chemotherapy for cancer.
Immunohistochemistry study in Figure 6D showed that
both DOX·HCl and DOX/NPs induced Caspaseꢀ3 expression in
tumor tissue and DOX/NPs treatment decreased the COXꢀ2
expression in tumor tissue. These finding indicated that
DOX/NPs could tumorꢀtargeted kill B16 cells by apoptosis
mechanisms as DOX·HCl, with reduced the side effect. These
results revealed that DOX/NPs could enhance antitumor
efficiency, and relieve weight loss issue of DOX·HCl.
Acknowledgements
This study was partly supported by the China National ‘12.5’
Foundation (Grant No. 2011BAJ07B04), and National Natural
Science Foundation of China (Grant No. 81673710 and
51503130).
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