PEG-derivatized embelin as a dual functional carrier for the delivery of paclitaxel

Article abstract of DOI:10.1021/bc3000468

Embelin, identified primarily from the Embelia ribes plant, has been shown to be a natural small molecule inhibitor of X-linked inhibitor of apoptosis protein (XIAP). It is also a potent inhibitor of NF-κB activation, which makes it a potentially effective suppressor of tumor cell survival, proliferation, invasion, angiogenesis, and inflammation. However, embelin itself is insoluble in water, which makes it unsuitable for in vivo applications. In this work, we developed a novel micelle system through conjugating embelin to a hydrophilic polymer, poly(ethylene glycol) 3500 (PEG_3.5K) through an aspartic acid bridge. The PEG_3.5k-embelin₂ (PEG _3.5k-EB₂) conjugate readily forms micelles in aqueous solutions with a CMC of 0.0205 mg/mL. Furthermore, PEG_3.5k-EB ₂ micelles effectively solubilize paclitaxel (PTX), a model hydrophobic drug used in this study. Both drug-free and drug-loaded micelles were small in size (20-30 nm) with low polydispersity indexes. In vitro cytotoxicity studies with several tumor cell lines showed that PEG _3.5k-EB₂ is comparable to embelin in antitumor activity and synergizes with PTX at much lower doses. Our results suggest that PEG-derivatized embelin may represent a novel and dual-functional carrier to facilitate the in vivo applications of poorly water-soluble anticancer drugs such as PTX.

Full text of DOI:10.1021/bc3000468

Article
pubs.acs.org/bc
PEG-Derivatized Embelin as a Dual Functional Carrier for the Delivery
of Paclitaxel
Yixian Huang,#,†,‡ Jianqin Lu,#,†,‡ Xiang Gao,†,‡ Jiang Li,†,‡ Wenchen Zhao,‡ Ming Sun,§
Donna Beer Stolz,§ Raman Venkataramanan,‡ Lisa Cencia Rohan,‡,∥ and Song Li*,†,‡
‡
§
†Center for Pharmacogenetics, Department of Pharmaceutical Sciences, School of Pharmacy; Department of Cell Biology and
∥
Physiology, School of Medicine; Magee Women’s Research Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15261,
United States
S
* Supporting Information
ABSTRACT: Embelin, identified primarily from the Embelia
ribes plant, has been shown to be a natural small molecule
inhibitor of X-linked inhibitor of apoptosis protein (XIAP). It
is also a potent inhibitor of NF-κB activation, which makes it a
potentially effective suppressor of tumor cell survival,
proliferation, invasion, angiogenesis, and inflammation. How-
ever, embelin itself is insoluble in water, which makes it
unsuitable for in vivo applications. In this work, we developed
a novel micelle system through conjugating embelin to a
hydrophilic polymer, poly(ethylene glycol) 3500 (PEG3.5K
)
through an aspartic acid bridge. The PEG3.5k-embelin2
(PEG3.5k-EB2) conjugate readily forms micelles in aqueous
solutions with a CMC of 0.0205 mg/mL. Furthermore, PEG3.5k-EB2 micelles effectively solubilize paclitaxel (PTX), a model
hydrophobic drug used in this study. Both drug-free and drug-loaded micelles were small in size (20−30 nm) with low
polydispersity indexes. In vitro cytotoxicity studies with several tumor cell lines showed that PEG3.5k-EB2 is comparable to
embelin in antitumor activity and synergizes with PTX at much lower doses. Our results suggest that PEG-derivatized embelin
may represent a novel and dual-functional carrier to facilitate the in vivo applications of poorly water-soluble anticancer drugs
such as PTX.
presence of large amounts of carrier materials not only adds to
the cost, but also imposes additional safety issues.8
INTRODUCTION
■
Low water solubility, high protein binding, and relatively short
half-life are major problems in clinical applications of many
potent anticancer drugs such as paclitaxel (PTX).1,2 Currently,
a variety of drug delivery systems such as liposomes,
dendrimers, microcapsules, and polymeric micelles have been
developed to address these problems and further to promote
sustained, controlled, and targeted delivery of poorly water-
soluble anticancer drugs.3 Of all these delivery systems,
polymeric micelles have gained considerable attention as a
versatile nanomedicine platform due to their technical ease,
high biocompatibility, and high efficiency in drug delivery.4,5
Polymer micelles have been demonstrated to improve the
aqueous solubility of chemotherapeutic agents and prolong
their in vivo half-lives, owing to the steric hindrance provided
by a hydrophilic shell.4,5 Moreover, compared with other
delivery systems, micelles show advantages in passive tumor
targeting through the leaky vasculature via the enhanced
permeability and retention (EPR) effect due to their small size
ranging from 10 to 100 nm.6,7 Favorable drug biodistribution
and improved therapeutic index can be achieved by using the
micelle delivery system.3,4 However, most of the polymeric
systems use “inert” excipients that lack therapeutic activity. The
One of the most sophisticated designs of drug delivery
systems is that in which the components forming the carriers
can also have therapeutic effects. The carrier materials may be
capable of counteracting the side effects caused by the loaded
anticancer drugs.9 Also, it is possible that the carrier may
collaborate with the loaded drug to achieve synergistic effects to
better treat the tumor.5 However, the strategy of using highly
water-insoluble drugs themselves as the hydrophobic region of
polymeric micelles is rarely reported. One example is the
pegylated vitamin E, D-α-tocopheryl poly(ethylene glycol)
succinate (Vitamin E TPGS or TPGS).10−12 Vitamin E shows
antitumor activity against a number of types of cancers through
various mechanisms such as induction of apoptosis, inhibition
of tumor cell proliferation and differentiation, suppression of
nuclear factor-kappa B (NF-κB) activation, and so forth.13,14
The pegylated vitamin E is a highly water-soluble amphiphilic
molecule comprising a lipophilic alkyl tail and a hydrophilic
polar head portion. In addition to its antitumor activity, it is
Received: February 2, 2012
Revised: June 4, 2012
Published: June 9, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of PEG3.5K-EB2
a
Conditions: (a) water, Fremy’s salt, KH2PO4, 5 min; Na2S2O4, 30 min; (b) water, Mel NaOH: l h; (c) Boc-Aspartic acid DCC, DMAP, CH2Cl2,
overnight; (d) MeCN, DMF, POCl3; (e) THF, LiHMDS 2 M in THF, decyltriphenylphosphonium bromide, 2 h (f) (1) MeOH, H2, Pd/C; (2)
MeCN, CAN; (3) dioxane, HCl, 2 h; (g) CH2Cl2, DCC, DMAP, 9; overnight; (h) succinic anhydride, DMAP, CH2Cl2, 2 days.
effective in solubilizing various hydrophobic drugs such as PTX.
Synergistic actions between the TPGS-based carrier and
delivered anticancer agents have been reported.5
TRAF1, cFLIP, Bcl-2, and Bcl-xL through the inhibition of
NF-κB activation.25 Embelin is poorly water-soluble, and PEG
modification was originally explored by us as an approach to
increase its solubility. Interestingly, PEG-derivatized embelin
forms micelles that are highly efficient in solubilizing other
compounds such as PTX. Preparation of PEG-derivatized
embelin can be readily achieved with commercially available
embelin. In addition, we have developed an efficient synthesis
strategy to prepare PEG−embelin conjugate. Our in vitro
studies showed that PEG−embelin has similar activity to free
embelin with IC50 in the low micromolar range. More
importantly, PEG−embelin synergizes with PTX at much
lower doses (∼nM) in a number of cancer cell lines tested.
In this study, we report the development of PEG-derivatized
embelin as another novel and dual-functional carrier for
delivery of poorly water-soluble anticancer drugs. Embelin is
a naturally occurring alkyl-substituted hydroxyl benzoquinone
compound and a major constituent of Embelia ribes BURM. It
has been shown to possess antidiabetic, anti-inflammatory, and
hepatoprotective activities.15−17 Embelin also shows antitumor
activity in various types of cancers.15,18−21 One major
mechanism involves the inhibition of the activity of X-linked
inhibitor of apoptosis protein (XIAP).22 XIAP is overexpressed
in various types of cancers cells, particularly drug-resistant
cancer cells, and inhibition of XIAP has been explored as a new
approach for the treatment of cancers.23,24 XIAP plays a
minimal role in normal cells, and therefore, embelin shows
significantly less toxicity on normal cells. Embelin also
downregulates the expression of survivin, XIAP, IAP1/2,
EXPERIMENTAL PROCEDURES
■
Materials. Paclitaxel (98%) was purchased from AK
Scientific. Inc. (CA, USA). 2,5-Dihydroxy-3-undecyl-1,4-
benzoquinone (embelin 98%) was purchased from 3B Scientific
Corporation (IL, USA). Dulbecco’s phosphate buffered saline
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(DPBS) was purchased from Lonza (MD, USA). Methoxy-
PEG3,500-OH, dimethyl sulfoxide (DMSO), 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), trypsin-
EDTA solution, Triton X-100, and Dulbecco’s Modified Eagle’s
Medium (DMEM) were all purchased from Sigma-Aldrich
(MO, USA). Fetal bovine serum (FBS) was purchased from
Gibco Life Technologies (AG, Switzerland). Penicillin−
streptomycin solution was from Invitrogen (NY, USA). All
solvents used in this study were HPLC grade.
Cell Culture. DU145 and PC3 are two androgen-
independent human prostate cancer cell lines. MDA-MB-231
is a human breast adenocarcinoma cell line. 4T1 is a mouse
metastatic breast cancer cell line. All cell lines were cultured in
DMEM containing 10% FBS and 1% penicillin−streptomycin
in a humidified environment at 37 °C with 5% CO2.
was filtered, washed with cold MeCN, dissolved in 20 mL of
water, heated at 50 °C for 0.5 h, and then cooled. The mixture
was extracted with 3 × 40 mL of CH2Cl2, and the combined
organic phase was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuum. The crude residue was
purified by silica gel flash chromatography (MeOH:CH2Cl2,
1
1:10) to give pure 5 as an oil in 80% yield (4.82 g). H NMR
(CDCl3): δ 10.21 (s, 1H), 10.18 (s, 1H), 6.65 (m, 2H), 5.92
(m, 4H), 5.37 (m, 1H), 4.51 (m, 1H), 3.75 (s, 3H), 3.72 (s,
3H), 2.85 (m, 1H), 2.60 (m, 1H), 1.40 (s, 9H). ESI-MS m/z
590.5 ([M+H]+).
Compound 6: A solution of sodium bis(trimethylsilyl)amide
(12 mL, 2 M solution in THF) was added dropwise to a stirred
solution of decanyltriphenylphosphonium bromide (9.67 g, 20
mmol) in 40 mL THF at room temperature. The resulting
mixture was stirred for 30 min at room temperature and then
cooled to −78 °C. To this mixture was added compound 5
(6.03 g, 10 mmol). The reaction mixture was stirred for 2 h at
−78 °C and then warmed up to room temperature. The
reaction mixture was quenched with saturated solution of
NH4Cl, extracted with ethyl acetate. The combined organic
layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated in vacuum. The crude residue was purified by
silica gel flash chromatography (MeOH:CH2Cl2, 1:10) to give
Synthesis of PEG3.5K-Embelin2 (PEG3.5K-EB2). Scheme 1
shows the synthesis sequence of PEG3.5K-EB2 conjugate.
Synthesis of the intermediates and structural characterizations
are detailed below.
Compound 2: Sesamol (1.52 g, 24 mmol) in 30 mL of
methanol was added to a rapidly stirred solution of Fremy’s salt
(7.96 g, 30 mmol) and 5.49 g (40 mmol) of KH2PO4 in 400
mL water at 5 °C. The color of the solution changed from light
brown to bright yellow within 5 min. The mixture was stirred
for another 30 min and then extracted with 4 × 40 mL of ethyl
acetate. The ethyl acetate phase was treated with a solution of
Na2S2O4 (9.0 g, 52 mmol) in water (30 mL), and the yellow
color changed to a colorless solution. The organic layer was
acidified with HCl (1 N), extracted with ethyl acetate (3 × 30
mL), washed with water (20 mL), dried with anhydrous
MgSO4, and concentrated to give 1.1 g (65%) of 2 as a light
pink solid. 1H NMR((CD3)2CO): δ 7.49 (s, 2H), 6.45 (s, 2H),
5.79 (s, 2H).
1
pure 6 as an oil in 90% yield. H NMR (CDCl3): δ 6.48 (m,
2H), 6.39 (m, 2H), 6.02 (m, 2H), 5.86 (m, 4H), 5.35 (m, 1H),
4.53 (m, 1H), 3.75 (s, 3H), 3.72 (s, 3H), 2.83 (m, 1H), 2.61
(m, 1H), 2.15 (m, 4H), 1.40 (s, 9H), 1.29 (m, 28H), 0.91 (m,
6H). ESI-MS m/z 838.4 ([M+H]+).
Compound 7: The double bond in compound 6 (8.37 g, 10
mmol) was saturated by catalytic hydrogenolysis with Pd/C
(10%, 500 mg) under H2 (1 atm) in a methanol solution (50
mL) at room temperature for 2 h. The solution was filtered to
remove Pd/C and concentrated under vacuum. The resulting
product was then dissolved in the solution of 10 mL of water,
10 mL MeCN, and 20 mmol CAN (ammonium ceric nitrate)
(10.96 g). The mixture was cooled to 0 °C and stirred for
another 2 h. MeCN was then removed via evaporation under
reduced pressure, 100 mL CH2Cl2 was added to the remaining
aqueous solution. The organic phase was washed with brine
and then concentrated under vacuum. 10 mL dioxane and 10
mL HCl were then added to the residue. The mixture was
stirred at room temperature for 24 h. The reaction mixture was
quenched with saturated solution of NaHCO3 and extracted
with ethyl acetate. The organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated under vacuum.
The crude product was purified by silica gel flash chromatog-
raphy (MeOH:CH2Cl2, 1:10) to give pure 7 as an oil in 42%
Compound 3: A solution of 2 (1.54 g, 10 mmol) in water
(30 mL) was treated with NaOH (0.4 g, 10 mmol) while the
flask was kept in an ice bath. The reaction mixture was stirred
for 15 min after which MeI (1.41 g, 10 mmol) was added
dropwise. The reaction mixture was then heated under reflux
for 1 h, allowed to cool down to room temperature, and the
solvent was removed via a rotary evaporator. The crude product
was purified by flash chromatography with silica gel (ethyl
acetate:petroleum ether, 1:5) and pure 3 was obtained as an
1
amber oil with a yield of 99% (1.68 g). H NMR(CDCl3): δ
6.11 (m, 2H), 5.88 (s, 2H), 5.35 (s, 1H), 3.72 (s, 3H).
Compound 4: To a solution of N-(tert-butoxycarbonyl)-L-
aspartic acid (Boc-Asp-OH) (2.33 g, 10 mmol) in CH2Cl2 (40
mL) was added dicyclohexylcarbodiimide (DCC) (6.2 g, 30
mmol), 4-dimethyamineopyridine (DMAP) (0.61 g, 5 mmol),
and compound 3 (3.36 g, 20 mmol) at room temperature. The
reaction mixture was stirred overnight at room temperature.
After the reaction was completed, 100 mL Et2O was added to
the mixture. The mixture was filtered to remove the insoluble
DCU byproduct and the organic phase of the filtrate was
concentrated under vacuum. The resulting residue was purified
by silica gel flash chromatography (MeOH:CH2Cl2, 1:10) to
give pure 4 as an oil in 62% yield (3.31 g). 1H NMR (CDCl3):
δ 6.11 (m, 4H), 5.88 (m, 4H), 5.40 (m, 1H), 4.65 (m, 1H),
3.74 (s, 3H), 3.72 (s, 3H), 2.88 (m, 1H), 2.64 (m, 1H), 1.42 (s,
9H). ESI-MS m/z 534.2 ([M+H]+).
1
yield (2.89 g). H NMR(CDCl3): δ 8.16 (m, 2H), 6.75 (m,
2H), 5.35 (m, 2H), 4.53 (m, 1H), 2.85 (m, 1H), 2.60 (m, 1H),
2.43 (m, 4H), 1.25 (m, 36H), 0.89 (m, 6H). ESI-HRMS calcd
for C38H55NO10Na ([M+Na]+) 708.4766, found 708.4747.
Compound 8: A solution of MeO-PEG3.5k-CO2H (3.5 g, 1
mmol) in CH2Cl2 (5 mL) was treated with DCC (0.41 g, 2
mmol), DMAP (0.12 g, 1 mmol), and compound 7 (689 mg, 1
mmol) at room temperature. The reaction mixture was stirred
overnight. After the reaction was completed, 100 mL of Et2O
was added and the mixture was filtered and concentrated under
vacuum. The resulting residue was purified by silica gel flash
chromatography (MeOH:CH2Cl2, 1:10) to give pure 8
(PEG3.5K-EB2) as a wax solid in ∼50% yield (2.1 g). 1H
NMR (CDCl3): δ 8.14 (m, 2H), 6.72 (m, 2H), 5.57 (m, 1H),
Compound 5: To a solution of 4 (5.33 g, 10 mmol) in
acetonitrile (MeCN, 10 mL) at 0−5 °C, dry dimethylforma-
mide (DMF) (0.73 g, 10 mmol) and POCl3 (1.78 g, 11 mmol)
were added with constant stirring over 0.5 h. The salt formed
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4.98 (m, 1H), 3.35 (s, 3H), 2.60 (m, 10H), 1.25 (m, 36H), 0.89
(m, 6H).
and then stained with 1% uranyl acetate. The sample processing
and imaging were performed at room temperature.
Formation of Micelles. PTX-solubilized micelles were
prepared by the following method. PTX (10 mM in
chloroform) was added to PEG3.5K-EB2 (10 mM in chloroform)
with various carrier/drug ratios. The organic solvent was first
removed by nitrogen flow to form a thin film of drug/carrier
mixture. The film was further dried under high vacuum for 2 h
to remove any traces of remaining solvent. Drug-loaded
micelles were formed by suspending the film in DPBS. The
drug-free micelles were similarly prepared as described above.
Measurement of Size and Zeta Potential. Zetasizer
(Zetasizer Nano ZS instrument, Malvern, Worcedtershire, UK)
was used to measure the particle size and zeta potential of drug-
free and drug-loaded micelles. Micelles were stored at 4 °C, and
the samples were tested for changes in particle size and size
distribution.
Determination of PTX Loading Efficiency. PTX-
solubilized micelles were prepared at an input PTX
concentration of 1.07, 2.14, and 3.21 mg/mL, respectively.
Aliquots of samples were filtered through 0.45 μm PVDF
syringe filter. PTX in the filtered and nonfiltered micelles was
extracted using methanol and measured by high-performance
liquid chromatography (HPLC, Waters). A reverse phase
column (C18) was employed. The detection was performed by
using UV detector at 227 nm, 70% methanol as a mobile phase,
flow rate at 1.0 mL/min. Drug loading capacity (DLC) and
drug loading efficiency (DLE) were calculated according to the
following formula:
Hemolysis Assay. Fresh blood samples were collected
through cardiac puncture from rats. Ten milliliters of blood was
added with EDTA-Na2 immediately to prevent coagulation.
Red blood cells (RBCs) were separated from plasma by
centrifugation at 1500 rpm for 10 min at 4 °C. The RBCs were
washed three times with 30 mL ice-cold DPBS. RBCs were
then diluted to 2% w/v with ice-cold DPBS and utilized
immediately for the hemolysis assay. One milliliter of diluted
RBC suspension was treated with various concentrations (0.2
and 1.0 mg/mL) of PEG3.5k-EB2 and PEI, respectively, and then
incubated at 37 °C in an incubator shaker for 4 h. The samples
were centrifuged at 1500 rpm for 10 min at 4 °C, and 100 μL of
supernatant from each sample was transferred into a 96-well
plate. The release of hemoglobin was determined by the
absorbance at 540 nm using a microplate reader. RBCs treated
with Triton X-100 (2%) and DPBS were considered as the
positive and negative controls, respectively. Hemoglobin release
was calculated as (ODsample − ODnegative control)/(ODpositive control
− ODnegative control) × 100%.
In Vitro Cell Cytotoxicity. DU145 (2000 cells/well), PC-3
(5000 cells/well), MDA-MB-231 (2000 cells/well), or 4T1
(1000 cells/well) were seeded in 96-well plates followed by 24
h of incubation in DMEM with 10% FBS and 1%
streptomycin−penicillin. Then, various concentrations of PTX
(dissolved in DMSO or formulated in PEG3.5K-EB2 micelles)
were added in quadruplicate and cells were incubated for 72 h.
20 μL of 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-
bromide (MTT) in PBS (5 mg/mL) was added and cells were
further incubated for 4 h. The medium in the plates was
removed and MTT formazan was solubilized by DMSO. The
absorbance was measured by microplate reader with wavelength
at 550 nm and reference wavelength at 630 nm. Untreated
groups were used as controls. Cell viability was calculated as
[(ODtreat − ODblank)/(ODcontrol − ODblank) × 100%].
DLC(%) = [weight of drug used/(weight of polymer
+ drug used)] × 100%
DLE(%) = (weight of loaded drug/weight of input drug)
× 100%
RESULTS
Determination of the Critical Micelle Concentration
(CMC). The CMC of PEG3.5K-EB2 was determined by
employing pyrene as a fluorescence probe.26 A drug-free
micelle solution in DPBS (2.5 mg/mL) was prepared via
solvent evaporation method. A series of 2-fold dilutions was
then made with PEG3.5K-EB2 concentrations ranging from 7.63
× 10−5 to 2.5 mg/mL. At the same time, aliquots of 50 μL of
4.8 × 10−6 M pyrene in chloroform were added into 15
separate vials. The chloroform was first removed by nitrogen
flow to form a thin film. The film was further dried under high
vacuum for 2 h to remove any traces of remaining solvent.
Then, the preprepared micelle solutions (400 μL in DPBS) of
varying PEG3.5K-EB2 concentrations were added to the pyrene
film to obtain a final pyrene concentration of 6 × 10−7 M for
each vial. The solutions were kept on a shaker at 37 °C for 24 h
to reach equilibrium before fluorescence measurement. The
fluorescence intensity of samples was measured at the excitation
wavelength of 334 nm and emission wavelength of 390 nm by
Synergy H1 Hybrid Multi-Mode Microplate Reader (Winooski,
VT). The CMC is determined from the threshold concen-
tration, where the sharp increase in pyrene fluorescence
intensity is observed.
■
Synthesis of PEG3.5K-EB2 Conjugates. We have devel-
oped a strategy to synthesize PEG3.5K-EB2 conjugate in which
two molecules of embelin were coupled to one molecule of
PEG via a linker of aspartic acid. This is modified from the
scheme reported by Wang’s group27 for the total synthesis of
embelin. This involves the synthesis of benzoquinone followed
by coupling to carboxyl groups of aspartic acid. Undecyl side
chains were then installed onto each of the two benzoquinone
rings. Finally, PEG was coupled to aspartic acid-EB2 through
the deprotected amino group. HPLC shows that the purity of
1
the final product (PEG3.5K-EB2) is 94% (Figure S1). H NMR
spectrum of PEG3.5K-EB2 shows signals at 3.63 ppm attributable
to the methylene protons of PEG, the embelin proton signals at
8.14 and 6.72 ppm, and the carbon chain singles at 1.05−1.25
ppm. The aspartate signals were identified at 5.57, 4.98, and
2.60 ppm (Figure S2). The molecular weight of the PEG3.5K
-
EB2 conjugate from MALDI-TOF MS (4197) is very similar to
the theoretical value (4203) (Figure S3). These results suggest
successful synthesis of PEG3.5K-EB2 conjugate.
Biophysical Characterization of Micelles. Micelles were
readily prepared from PEG3.5K-EB2 conjugate via solvent
evaporation method. PEG3.5K-EB2 conjugate can be dissolved
in water at concentration up to 750 mg/mL (data not shown).
Dynamic light scattering (DLS) measurements showed that
these micelles had hydrodynamic sizes around 22 nm at the
Transmission Electron Microscope (TEM). The mor-
phology of micelles was observed on a Jeol 1011 transmission
electron microscope (TEM). The aqueous micelle solution (1.0
mg/mL) was added onto copper grids coated with Formvar,
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Figure 1. Particle size distribution of PEG3.5K-EB2 (A) and PTX-loaded PEG3.5K-EB2 (C). TEM images of self-assembled micelles of PEG3.5K-EB2
(B) and PTX-loaded PEG3.5K-EB2 (D). The spherical micelles with the diameter of around 20 nm were observed. The drug loading level was 1 mg/
mL (PTX) in PEG3.5K-EB2.
concentration of 20 mg/mL (Figure 1A), which shall ensure
efficient passive targeting to the solid tumors.
in Table 2. DLE was as high as 79.89% when PTX was
formulated in PEG3.5K-EB2 micelles at a carrier/PTX input ratio
PTX, a potent hydrophobic anticancer agent, was readily
loaded into PEG3.5K-EB2 micelles. Figure 1C shows the DLS
size measurement of PTX-loaded PEG3.5K-EB2 micelles at a
drug concentration of 1 mg/mL. There were minor changes in
sizes when PTX was loaded into micelles at a carrier/drug ratio
of 7.5/1 (m/m).
Table 2. Physicochemical Characterization of PTX-Loaded
PPEG3.5K-EB2 Micelles
a
PEG3.5K
EB2:PTX
(m/m)
-
b
c
concentration of PTX in DLC
DLE
(%)
d
micelles (mg/mL)
(%)
zeta (mV)
Figure 1B,D shows the TEM images of drug-free and PTX-
loaded micelles after staining with 1% uranyl acetate. Spherical
particles of uniform size were observed for both drug-free and
PTX-loaded micelles. The sizes of the micelles observed under
TEM are consistent with those measured by DLS.
Table 1 shows the sizes of PTX-loaded micelles at different
carrier/drug molar ratios. PTX-loaded PEG3.5K-EB2 micelles
2.5:1
5:1
1.07
1.07
1.07
2.14
3.21
7.51
3.90
2.63
2.63
2.63
79.9
96.7
98.6
97.5
81.3
1.58 0.37
1.89 0.08
7.5:1
−1.52 0.20
−1.29 0.19
−2.64 0.43
a
b
Values reported are the means SD for triplicate samples. DLC =
drug loading capacity. DLE = drug loading efficiency. Measured by
c
d
dynamic light scattering particle sizer (Zetasizer).
Table 1. DLS Analysis of the Sizes of Free and Drug-Loaded
PEG3.5K-EB2 Micelles
a
of 2.5/1 (m/m) and PTX concentration of 1.07 mg/mL.
Increasing the carrier/PTX input ratios led to further increase
in DLE. PEG3.5K-EB2/PTX formed the most stable particles at a
carrier/drug ratio of 7.5/1. At this ratio, PTX was quantitatively
formulated in the PEG3.5K-EB2 micelles when the PTX
concentration was less than 2.14 mg/mL. Increasing the PTX
concentration to 3.21 mg/mL led to a slight decrease in DLE
(81.3%). The surface charges of PTX-loaded PEG3.5K-EB2
micelles were close to neutral (+1.89 to −2.64) for all particles
examined.
b
c
micelles
molar ratio
size (nm)
PDI
PEG3.5K-EB2

22.8 0.3
143 17
58.7 0.5
27.5 0.2
0.09
0.23
0.32
0.23
d
PEG3.5K-EB2:PTX
PEG3.5K-EB2:PTX
PEG3.5K-EB2:PTX
2.5:1
5:1
7.5:1
a
PTX concentration in micelle was kept at 1 mg/mL. Blank micelle
concentration was 20 mg/mL. Values repotted are the mean SD for
triplicate samples. Measured by dynamic light scattering particle sizer
(Zetasizer). PDI = polydispersity index. PTX = paclitaxel.
b
c
d
CMC Measurements. Figure 2 shows the results of CMC
measurements using pyrene as a fluorescence probe. Upon
incorporation into the micelles, the fluorescence intensity of
pyrene increases substantially at the concentration of micelles
above the CMC.28 On the basis of the partition of the pyrene,
the CMC of PEG3.5K-EB2 could be obtained by plotting the
fluorescence intensity versus logarithm concentration of the
polymer. The CMC of PEG3.5K-EB2 was determined from the
crossover point at the low concentration range. The CMC of
had relatively large size (∼143 nm) at a carrier/drug ratio of
2.5: 1 (m/m) and the particles were stable for less than 1 day.
Increasing the input molar ratio of PEG3.5K-EB2/PTX led to
gradual decrease in the size of PTX-loaded micelles. At the
molar ratio of 7.5/1, the size of the PTX-loaded micelles was
similar to that of drug-free micelles.
Drug Loading Efficiency (DLE). DLE of PTX-loaded
micelles were determined by HPLC and the results are shown
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MB-231 cells. Drug-free PEG3.5K-EB2 did not cause any
cytotoxicity to MDA-MB-231 cells due to its relatively low
concentrations used in this study. Free PTX exhibited
cytotoxicity on MDA-MB-231 cells in a dose-dependent
manner. However, formulation of PTX in PEG3.5K-EB2 micelles
resulted in a significant increase in the cytotoxicity. Similar
results were found with three other cancer cell lines (Figure
5B−D). Table 3 summarizes the IC50 of free PTX and PEG3.5K
-
EB2-formulated PTX in the four different cancer cell lines.
Depending on the cell lines, the IC50 was decreased by 1.5- to
8.7-fold when PTX was formulated in PEG3.5K-EB2 micelles.
DISCUSSION
Figure 2. CMC measurement of the PEG3.5K-EB2 micelles using
pyrene as a hydrophobic fluorescence probe. The fluorescence
intensity of pyrene was collected at the excitation wavelength of 334
nm and the emission wavelength of 390 nm. The fluorescence
intensity was plotted as a function of logarithmic concentration of
PEG3.5K-EB2 micelles, [pyrene] = 6 × 10−7 mol/L. Values reported are
the means SD for triplicate samples.
■
We have developed a new delivery system that consists of an
embelin-based hydrophobic domain and a PEG hydrophilic
segment. The PEG3.5K-EB2 conjugate readily forms micelles in
aqueous solutions. More importantly, hydrophobic drugs such
as PTX can be loaded into PEG3.5K-EB2 micelles.
Various polymeric micelle systems have been reported. Most
micellar systems consist of a hydrophobic core that does not
have any potential therapeutic effect.30 In addition, the
metabolites of the hydrophobic segments might contribute to
some undesired effects, such as inflammation and systemic
toxicity.30,31 The PEG3.5K-EB2 conjugate developed in this study
represents a dual-functional delivery system that may overcome
these limitations. Embelin is a natural product that demon-
strates various biological effects including antitumor activity.15
Embelin also shows excellent safety profiles in animals.32 Thus,
PEG-derivatized embelin may be an attractive delivery system
to achieve synergistic activity with anticancer agents while
minimizing the carrier-associated toxicity. PEG−embelin
conjugates can be synthesized via direct coupling of embelin
to PEG via an ester linkage. However, such synthesis is likely to
yield a mixture of products with PEG randomly linked to the
different hydroxyl groups in the benzene ring. We have
developed a strategy to generate PEG3.5K-EB2 conjugate via
total synthesis (Scheme 1). This method was modified from a
scheme reported by Wang’s group for total synthesis of
embelin.27 Our synthesis ensures generation of a structurally
well-defined conjugate in which PEG is attached to 1-OH
group in the quinone ring. Most of the steps give good yields,
and the synthesis of PEG−embelin conjugate involves a similar
number of steps and cost as that of embelin alone.27
PEG3.5K-EB2 conjugate forms small-sized micelles (20−30
nm), and loading of PTX did not significantly affect the size of
the micelles. It was generally believed that particles in the size
range 100−200 nm can effectively penetrate solid tumors via an
EPR effect.6 A recent study from Lam’s group compared the
passive targeting of nanoparticles of different sizes in a
subcutaneous model of human ovarian cancer xenograft. It
was shown that particles with a size of 154 nm were
significantly taken up by liver and lungs with limited
accumulation at tumor sites. In contrast, particles with
respective size of 17 and 64 nm were much more effective in
passive targeting to the solid tumor.33 Cabral and colleagues
compared the targeting efficiency of polymeric micelles of
different sizes (30, 50, 70, and 100 nm) in both highly and
poorly permeable tumors. While all of the tested polymer
micelles penetrated highly permeable tumors in mice, only the
30 nm micelles could penetrate poorly permeable pancreatic
tumors to achieve an antitumor effect.34 The small size of our
new micelle system suggests its potential for effective tumor
the PEG3.5K-EB2 conjugate is 4.9 μM, which is similar to most
reported micellar delivery systems.
Hemolysis Study. One of the safety concerns for polymeric
micelle systems is the hemolytic activity. To address this issue,
the hemolytic activity of drug-free PEG3.5K-EB2 micelles was
examined and compared to a strong detergent Triton X-100
and polyethylenimine (PEI), a cationic polymer known to have
significant hemolytic effect.29 As shown in Figure 3, PEI
Figure 3. In vitro hemolysis assay of PEG3.5K-EB2 compared with PEL
Both PEG3.5K-EB2 and PEI with two different concentrations [0.2, 1
mg/mL) were incubated with rat red blood cells (RBCs) for 4 h at 37
°C in an incubator shaker. The degree of RBCs lysis was measured
spectrophotometrically (λ = 540 nm) according to the release of
hemoglobin in process (2% Triton X-100 and DPBS were used as a
positive and negative control, respectively). Values reported are the
means SD for triplicate samples.
induced hemolysis in a dose-dependent manner. In contrast, no
observable hemolytic activities (<5%) were found for PEG3.5K
-
EB2 micelles, suggesting the excellent safety of our new delivery
system.
In Vitro Cytotoxicity. Figure 4 shows the cytotoxicity of
PEG3.5K-EB2 in comparison with free embelin (dissolved in
DMSO) in 3 cancer cell lines tested including murine breast
cancer cells 4T1, and two human prostate cancer cell lines PC3
and DU145. PEG3.5K-EB2 was comparable to free embelin in
antitumor activity in all 3 cancer cell lines with IC50 in the low
micromolar range.
Figure 5A compares the cytotoxicity of free PTX (in DMSO)
to that of PEG3.5K-EB2-formulated PTX (5/1, m/m) in MDA-
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Figure 4. Cytotoxicity of free EB and PEG3.5K-EB2 against the 4T1 mouse breast cancer cell line (A) and two androgen-independent human prostate
cancer cell lines PC-3 (B) and DU145 (C). Cells were treated by free EB or PEG3.5K-EB2 for 72 h under the equivalent concentrations of EB.
Cytotoxicity was determined by MTT assay.
Table 3. IC50 of PTX and PTX-Loaded PEG3.5K-EB2 (1/5,
m/m) after 72 h Incubation with Different Cancer Cell Lines
a
IC50 (ng/mL)
MDA-MB-231
4T1
PC-3
DU145
PTX-loaded PEG3.5K-EB2
PTX
13.5
65
51
73
12.7
42.3
8.9
78
a
The concentration of a drug that is required for 50% inhibition in
vitro.
The PEG3.5K-EB2-mediated cytotoxicity is unlikely attributed to
its surface activity, as PEG3.5K-EB2 showed minimal hemolytic
activity even at mM concentrations (Figure 3). Embelin is
coupled to PEG via a cleavable ester linkage. It is likely that
embelin is released from the conjugate following intracellular
delivery and executes the antitumor effect by itself or synergizes
with PTX in antitumor activity. These data are consistent with
the observation that free embelin synergizes with PTX at
subeffective doses (Figure S4). More studies are needed to
better understand the mechanism by which the PEG3.5K-EB2-
based delivery system synergizes with PTX in vitro.
It should be noted that PEG3.5K-EB2 conjugate only
represents a model micelle to demonstrate the utility of PEG-
derivatized embelin as a dual functional delivery system for
hydrophobic anticancer drugs. Considering the flexibility of our
synthesis scheme, more studies on structure−activity relation-
ship (SAR) can be designed to further improve this new
delivery system. These include optimization of the molar ratio
of PEG/embelin in the conjugates, the length and structure of
the acyl chain in the embelin, and the molecular weight of PEG.
Recently, embelin derivatives with improved affinity toward
XIAP have been developed.27 The utility of these new
derivatives as drug carriers can also be examined and compared
to native embelin. Finally, promising candidates identified from
these studies need to be further evaluated in vivo. These studies
are currently ongoing in our laboratory.
Figure 5. Cytotoxicity of free PTX, free PEG3.5K-EB2, and PTX-loaded
PEG3.5K-EB2 (1/5, m/m) nanoparticles against the MDA-MB-231
human breast cancer cell line (A), the 4T1 mouse breast cancer cell
line (B), and two androgen-independent human prostate cancer cell
lines PC-3 (C) and DU145 (D). Cells were treated with free or
formulated PTX for 72 h and cytotoxicity was determined by MTT
assay.
targeting in vivo, which is currently being evaluated in our
laboratory.
In vitro cytotoxicity with several cancer cell lines showed that
PEG3.5K-EB2 is comparable to free embelin in antitumor activity
with IC50 in the low micromolar range. More importantly,
PEG3.5K-EB2 synergizes with PTX in antitumor activity at much
lower concentrations (∼nM) in all 4 cancer cell lines tested.
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(14) Husain, K., Francois, R. A., Yamauchi, T., Perez, M., Sebti, S. M.,
and Malafa, M. P. (2011) Vitamin E δ-Tocotrienol augments the
antitumor activity of gemcitabine and suppresses constitutive NF-κB
activation in pancreatic cancer. Mol. Cancer Ther. 10, 2363−2372.
(15) Chitra, M., Sukumar, E., Suja, V., and Devi, C. S. (1994)
Antitumor, anti-inflammatory and analgesic property of embelin, a
plant product. Chemotherapy 40, 109−113.
(16) Bhandari, U., Jain, N., and Pillai, K. K. (2007) Further studies on
antioxidant potential and protection of pancreatic beta-cells by
Embelia ribes in experimental diabetes. Exp. Diabetes Res. 2007,
15803−15808.
ASSOCIATED CONTENT
■
S
* Supporting Information
1
HPLC trace, H NMR spectra, and MALDI-TOF of PEG3.5K
-
EB2, and the cytotoxicity of embelin, PTX, and the combination
of embelin and PTX against human prostate cancer cells
DU145. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: 412-383-7976. Fax: 412-648-1664. E-mail: sol4@pitt.edu.
■
(17) Singh, D., Singh, R., Singh, P., and Gupta, R. S. (2009) Effect of
embelin on lipid peroxidation and free radical scavenging activity
against liver damage in rats. Basic Clin. Pharmacol. Toxicol. 105, 243−
248.
Author Contributions
#Authors contributed equally to this work.
Notes
(18) Danquah, M., Li, F., Duke, C. B., Miller, D. D., and Mahato, R. I.
(2009) Micellar delivery of bicalutamide and embelin for treating
prostate cancer. Pharm. Res. 26, 2081−2092.
The authors declare no competing financial interest.
(19) Sreepriya, M., and Bali, G. (2005) Chemopreventive effects of
embelin and curcumin against N-nitrosodiethylamine/phenobarbital-
induced hepatocarcinogenesis in Wistar rats. Fitoterapia 76, 549−555.
(20) Dai, Y., Qiao, L., Chan, K. W., Yang, M., Ye, J., Ma, J., Zou, B.,
Gu, Q., Wang, J., Pang, R., Lan, H. Y., and Wong, B. C. (2009)
Peroxisome proliferator-activated receptor-gamma contributes to the
inhibitory effects of Embelin on colon carcinogenesis. Cancer Res. 69,
4776−4783.
ACKNOWLEDGMENTS
■
This work was supported in part by NIH grants R21CA128415
and R21CA155983, and a DOD grant BC09603. We would like
to thank Dr. Gong Chen at Penn State University for critical
reading of this manuscript.
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