Ketal cross-linked poly(ethylene glycol)-poly(amino acid)s copolymer micelles for efficient intracellular delivery of doxorubicin

Article abstract of DOI:10.1021/bm101517x

A biocompatible, robust polymer micelle bearing pH-hydrolyzable shell cross-links was developed for efficient intracellular delivery of doxorubicin (DOX). The rationally designed triblock copolymer of poly(ethylene glycol)-poly(l-aspartic acid)-poly(l-phenylalanine) (PEG-PAsp-PPhe) self-assembled to form polymer micelles with three distinct domains of the PEG outer corona, the PAsp middle shell, and the PPhe inner core. Shell cross-linking was performed by the reaction of ketal-containing cross-linkers with Asp moieties in the middle shells. The shell cross-linking did not change the micelle size and the spherical morphology. Fluorescence quenching experiments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced SternVolmer quenching constant (K_SV). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study showed that the hydrolysis half-life (t_1/2) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH, compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.

Full text of DOI:10.1021/bm101517x

ARTICLE
pubs.acs.org/Biomac
Ketal Cross-Linked Poly(ethylene glycol)-Poly(amino acid)s
Copolymer Micelles for Efficient Intracellular Delivery of Doxorubicin
Sang Jin Lee,†,‡ Kyung Hyun Min,†,‡ Hong Jae Lee,§ Ahn Na Koo,§ Hwa Pyeong Rim,‡ Byeong Jin Jeon,‡ Seo
Young Jeong,*,‡ Jung Sun Heo,§ and Sang Cheon Lee*,§
‡Department of Life and Nanopharmaceutical Science, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Korea
§Department of Maxillofacial Biomedical Engineering and Institute of Oral Biology, School of Dentistry, Kyung Hee University, 1 Hoegi-
dong, Dongdaemun-gu, Seoul 130-701, Korea
S Supporting Information
b
ABSTRACT: A biocompatible, robust polymer micelle bearing
pH-hydrolyzable shell cross-links was developed for efficient
intracellular delivery of doxorubicin (DOX). The rationally
designed triblock copolymer of poly(ethylene glycol)-poly(L-
aspartic acid)-poly(L-phenylalanine) (PEG-PAsp-PPhe) self-
assembled to form polymer micelles with three distinct domains
of the PEG outer corona, the PAsp middle shell, and the PPhe
inner core. Shell cross-linking was performed by the reaction of
ketal-containing cross-linkers with Asp moieties in the middle
shells. The shell cross-linking did not change the micelle size
and the spherical morphology. Fluorescence quenching experi-
ments confirmed the formation of shell cross-linked diffusion barrier, as judged by the reduced Stern-Volmer quenching constant
(KSV). Dynamic light scattering and fluorescence spectroscopy experiments showed that shell cross-linking improved the micellar
physical stability even in the presence of micelle disrupting surfactants, sodium dodecyl sulfate (SDS). The hydrolysis kinetics study
showed that the hydrolysis half-life (t1/2) of ketal cross-links was estimated to be 52 h at pH 7.4, whereas 0.7 h at pH 5.0, indicating
the 74-fold faster hydrolysis at endosomal pH. Ketal cross-linked micelles showed the rapid DOX release at endosomal pH,
compared to physiological pH. Confocal laser scanning microscopy (CLSM) showed that ketal cross-linked micelles were taken up
by the MCF-7 breast cancer cells via endocytosis and transferred into endosomes to hydrolyze the cross-links by lowered pH and
finally facilitate the DOX release to inhibit proliferation of cancer cells. This ketal cross-linked polymer micelle is promising for
enhanced intracellular delivery efficiency of many hydrophobic anticancer drugs.
’ INTRODUCTION
caused by the dilution to below critical micelle concentration or
adverse shift in the micelle-unimer equilibrium. This resulted in
the low delivery efficiency to target sites, and furthermore, the
EPR effect cannot be expected.
Recent advances in functional self-assembled polymer mi-
celles based on amphiphilic block copolymers have led to the
development of smart nanocarriers that can enhance the delivery
efficiency of anticancer drugs, genetic drugs, and imaging
agents.1-6 Polymer micelles have expressed many benefits for
cancer chemotherapy due to the effective resistance to rapid renal
clearance and nonspecific uptake by the reticuloendothelial
system (RES).7,8 This unique property enables the micellar
passive targeting and enhanced permeability and retention
(EPR) effect for effective tumor therapy.9-11 For the polymer
micelles to be an ideal carrier, they should meet the following
three requisites: high stability in blood, minimized drug loss
before reaching target tissues, and the facilitated release of loaded
drugs within target cells. However, most polymer micelles suffer
from low structural stability, and furthermore, the drug release is
initiated upon intravenous administration, resulting in drug loss
at unwanted sites.12,13 Recently, increasing the micellar stability
is an issue of a great interest, because the polymer micelle tends
to lose its carrier properties by disintegration in vivo conditions,
To date, cross-linking of micellar shells or cores has been
recognized as a useful approach to stabilize micelle structure,
because the cross-linking holds micelles in a thermodynamically
frozen state.14-19 However, within target cells, the cross-links
should be cleaved to release entrapped drugs, because the
maintenance of cross-links can act as a barrier to drug release.
For this reason, shell or core cross-linked polymer micelles
(CLMs) satisfying not only enhanced stability but also specific
intracellular drug-releasing property are an important target.
Our recent research has been focused on disulfide cross-linked
micelles and mineral-reinforced micelles, and we demonstrated
that they are good examples for enhanced robustness and
Received: December 15, 2010
Revised:
February 4, 2011
Published: February 23, 2011
r
2011 American Chemical Society
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Scheme 1. Illustration of Shell Cross-Linking of DOX-Loaded Polymer Micelles with a pH-Labile Ketal Cross-Linker and
Intracellular Release of Dox Triggered by Endosomal pH
the facilitated release of entrapped anticancer drugs at
cytoplasm.20,21
with rational sequence of poly(ethylene glycol)-b-poly(L-aspartic
acid)-b-poly(L-phenylalanine) (PEG-PAsp-PPhe) was utilized.
PEG-PAsp-PPhe is expected to form polymer micelles featuring
the PEG corona, the PAsp middle shell, and the PPhe inner core.
The three domains of the core, the shell, and the corona, are each
expected to play a role in generating a useful CLM nanocarrier.
The PEG outer corona can keep the micelles stable by protecting
against the formation of intermicellar bridges during the shell
cross-linking process, which is a problem associated with core-
shell type micelles of AB diblock copolymers.30 In addition, the
PEG corona enables the prolonged circulation of CLMs for
effective EPR effects. The PPhe core serves as a reservoir of
liphophilic drugs. The PAsp middle shells with carboxylic acid
groups provide a region for shell cross-linking with a ketal-
containing cross-linker. The cross-links in the middle shells not
only can enhance micellar stability against micelle-destabilizing
conditions but also can protect drug release efficiently at extra-
cellular environments (pH 7.4). Upon endocytosis, the hydro-
lysis of ketal linkages in cross-links can be promoted by an acidic
endosomal pH (∼5.0) to trigger intracellular drug release.
Scheme 1 illustrates the key idea and working principle of our
ketal cross-linked micelles as an intracellular carrier of DOX.
In this study, we describe the rational synthetic approach of
ketal cross-linked micelles (KCLMs) and the cross-linking effect
on the micellar robustness. The hydrolysis kinetics of ketal cross-
links was investigated at endosomal and physiological pH. The
inhibited DOX release at pH 7.4 and the facilitated DOX release
in response to endosomal pH were demonstrated. The intracel-
lular release of DOX from KCLMs was visualized using breast
cancer cells (MCF-7) and correlated with the inhibitory effect of
cancer cell proliferation.
It is well-known that intracellular endosomes (pH 5.0-5.5)
and lysosomes (pH 4.0-4.5) have mildly acidic pH.21-23 These
environments with lowered pH than extracellular compartments
have provided an opportunity for designing specific intracellular
drug delivery systems. Therefore, the incorporation of pH-
breakable shell cross-links into polymer micelles may enable
the design of the useful intracellular nanocarriers.
Herein, we describe the synthesis of CLMs using a cross-
linking agent containing pH-hydrolyzable ketal groups to pro-
duce a biocompatible, robust, and smart nanocarrier that can
preferentially release doxorubicin (DOX) in response to endo-
somal pH. Recently, a fundamental study of pH-sensitive shell
cross-linked nanoparticles with hydrolytically labile cross-links
was reported, and the research scope focused on the feasibility of
shell cross-linking and pH-dependent breakage of cross-links.24
As another example, an acid-labile core cross-linked micelle was
reported for pH-triggered release of antitumor drugs.25 As most
CLMs, these systems were based on nondegradable polyacrylate
or polystyrene with limited biocompatibility.14-19
For CLMs to be practically useful, the first consideration is to
tailor the polymer design for nontoxic biodegradable CLMs.
Another point of view is the design of micelle structures suitable
for intravenous administration. For long-circulation for en-
hanced EPR effects, the use of PEG as micellar outer corona
was recommended.26,27 Based on these consideration, we se-
lected poly(ethylene glycol) (PEG) and poly(amino acid)s as
building blocks for CLMs due to their low toxicity and
immunogenicity.28,29 In contrast to current CLM systems based
on nondegradable polyacrylate and polystyrenes, our system can
be eliminated from body through renal excretion as nontoxic
PEG and degraded amino acid molecules (L-aspartic acid and L-
phenylalanine) after expressing its roles for drug delivery. As a
polymer micelle for shell cross-linking, a ABC triblock copolymer
The ketal cross-linking is designed to occur by the reaction of
ketal-containing cross-linkers with Asp moieties within middle
shells, and thus, the maintenance of PEG outer corona may lead
to the enhanced accumulation of micelles at target tissues.
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’ EXPERIMENTAL SECTION
operating at an acceleration voltage of 200 kV. For the observation of
size and distribution of micellar particles, a drop of sample solution
(concentration = 0.5 g/L) was placed onto a 200-mesh copper grid
coated with carbon. About 2 min after deposition, the grid was tapped
with a filter paper to remove surface water, followed by air-drying.
Negative staining was performed using a droplet of a 5 wt % uranyl
acetate solution.
Materials. R-Methoxy-ω-amino-poly(ethylene glycol) (CH3O-
PEG-NH2) with number average molecular weight (Mn) of 2000 g/
mol and polydispersity index (PDI) of 1.06 (GPC) was purchased from
SunBio Inc. (Seoul, Korea) and used as received. β-Benzyl L-aspartate
(BAsp), L-phenylalanine (Phe), doxorubicin hydrochloride
(DOX HCl), pyridinium p-toluenesulfonate (PPTS), N-hydroxysucci-
3
Synthesis of a Ketal-Containing Cross-Linker. The ketal-
containing bifunctional cross-linker was synthesized by a two-step
literature process.34 In brief, to a THF solution (250 mL) of HETFA
(10.9 g, 69.3 mmol), PPTS (0.7 g, 2.8 mmol) was added. Molecular
sieves (5 Å) was then added, and the reaction mixture was stirred. After
20 min, 2-methoxypropene (2 g, 27.7 mmol) was added, and the
reaction was maintained for 24 h at room temperature. The reaction
product was purified by column chromatography. Yield: 55%. 1H NMR
(DMSO-d6): δ 1.30 (s, 6H), 3.5 (s, 8H), 6.7 (s, 2H). The reaction
product (3 g, 8.48 mmol) was dissolved in aqueous NaOH (6 M, 20 mL)
solution and stirred for 3 h. Finally, the ketal cross-linker was isolated by
extraction with CH2Cl2 (3 ꢀ 200 mL) and dried with anhydrous
MgSO4, followed by evaporation under vacuum. Yield: 45%. 1H NMR
(DMSO-d6): δ 1.38 (s, 6H), 1.65 (s, 4H,), 2.86 (t, 4H), 3.47 (t, 4H).
Synthesis of a PEG-PAsp-PPhe Triblock Copolymer. PEG45-
PBAsp8-PPhe19 that has PEG units of 45, BAsp units of 8, and Phe units
of 19 was synthesized by a procedure established in our laboratory.21 To
a stirred solution of CH3O-PEG-NH2 (5 g, 0.0025 mol) in dry DMF
(50 mL) was added BAsp-NCA (6.23 g, 0.025 mol) at 35 °C under
nitrogen. After 24 h, Phe-NCA (9.56 g, 0.050 mol) and dry DMF
(100 mL) were added to the reaction mixture, and the reaction was
maintained for an additional 24 h. PEG45-PAsp8-PPhe19 was isolated by
repeated precipitation from DMF into diethyl ether. Yield: 90%. The
deprotection of PEG45-PBAsp8-PPhe19 was performed by treating the
block copolymer (5 g) with 0.1 N NaOH (200 mL) to remove benzyl
groups. The aqueous solution was then dialyzed using a membrane
(molecular weight cutoff (MWCO): 1000) for 24 h, followed by
freeze-drying.
Shell Cross-Linking of PEG-PAsp-PPhe Micelles. Polymer
micelles of PEG45-PAsp8-b-PPhe19, consisting of PEG coronas, PAsp
shells, and PPhe cores, were prepared by dialyzing the polymer solution
in DMSO against doubly distilled water. Micelles were immersed into
acidic aqueous solution (pH 4.0, 5 mg/4.8 mL) of EDC (21 mM) and
NHS (21 mM) for 3 h. Shell cross-linking was carried out by adding a
solution of the amine-terminated bifunctional ketal cross-linker
(10.5 mM, 0.2 mL, water/THF (1:1, v/v)) to a NHS-activated micellar
solution (1 g/L) at pH 9.0. The reaction mixture was stirred for 10 h at
room temperature and then dialyzed against doubly distilled water (pH
9.0) for 3 h to remove unreacted cross-linkers. The dialyzate was
lyophilized to obtain the KCLMs.
nimide (NHS), N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide
(EDC), triphosgene, pyrene, 1-dodecylpyridinium chloride (DPC),
N-(2-hydroxyethyl)-2,2,2-trifluoroacetamide (HETFA), 2-methoxypro-
pene, and 2,20-(ethylenedioxy)bis(ethylamine) were purchased from
Aldrich Co. (Milwaukee, WI) and used as received. Tetrahydrofuran
(THF) was distilled from Na/benzophenone under N2, prior to use. N,
N-Dimethylformamide (DMF) was dried and distilled over calcium
hydride. β-Benzyl L-aspartate N-carboxyanhydride (BAsp-NCA) and L-
phenylalanine N-carboxyanhydride (Phe-NCA) of high purity were
synthesized by the Fuchs-Farthing method using triphosgene.31 Anal.
Calcd for BAsp-NCA (C12H11NO5): C, 57.83; H, 4.45; N, 5.62. Found:
C, 57.93; H, 4.49; N, 5.64. Anal. Calcd for Phe-NCA (C10H9NO3): C,
62.82; H, 4.74; N, 7.33. Found: C, 62.29; H, 4.90; N, 7.28.
Instrumentation. Nuclear Magnetic Resonance Spectroscopy
(NMR). 1H NMR spectra were recorded at 400 MHz on a Varian
INOVA400 NMR spectrometer with a sample spinning rate of 5 kHz at
25 °C. Samples for NMR measurement were prepared by dissolving 10
mg of samples in 0.5 mL of DMSO-d6.
Gel Permeation Chromatography (GPC). Molecular weight distribu-
tions were determined using a GPC equipped with a Waters 2414
refractive index detector, 515 HPLC pump, and three consecutive
Styragel columns (HR1, HR2, and HR4). The eluent was DMF with a
flow rate of 1 mL/min. The molecular weights were calibrated with
polystyrene standards.
Elemental Analysis. Elemental analysis was performed on a Perkin
Elmer Series II CHNS/O Analyzer 2400.
Zeta Potential Measurement. The zeta potential (ζ) was measured in
a phosphate buffered saline (PBS) solution (10 mM, pH 7.4) using a 90
PLUS (Brookhaven Instruments Cooperation, New York, U.S.A.)
particle size analyzer.
Fluorescence Measurements. Pyrene fluorescence was recorded on a
JASCO FP-6500 spectrofluorometer for determination of critical mi-
celle concentration (cmc). For micellar solutions, doubly distilled water
(20 mL) was added dropwise to a vigorously stirred THF solution of the
block copolymer. After THF was evaporated in vacuo, the micellar
solution was diluted to obtain a concentration range from 2 to 1 ꢀ 10-4
g/L. An aqueous pyrene solution (12 ꢀ 10-7 M) was mixed with
micellar solutions to obtain copolymer concentrations from 1 to 5 ꢀ
10-5 g/L. The pyrene concentration in the samples was 6.0 ꢀ 10-7 M.
All the samples were sonicated for 10 min and were allowed to stand
for 1 day at 25 °C before measurements.32 The cmc was calculated
based on a red shift of (0,0) band from 332 to 336 nm in pyrene
excitation spectra.32
Light Scattering Measurements. Dynamic light scattering measure-
ments were performed using a 90 Plus particle size analyzer
(Brookhaven Instruments Corporation). The sample solutions were
purified by passing through a Millipore 0.45 μm filter. The scattered light
of a vertically polarized He-Ne laser (632.8 nm) was measured at an
angle of 90° and was collected on an autocorrelator. The hydrodynamic
diameters (d) of micelles were calculated by using the Stokes-Einstein
equation.33 The polydispersity factor of micelles, represented as μ2/Γ2,
where μ2 is the second cumulant of the decay function and Γ is
the average characteristic line width, was calculated from the cumulant
method.33
Steady-State Fluorescence Quenching. Fluorescence quench-
ing experiments were performed by adding aqueous solutions of DPC
(0.75 mL) with a various concentration (0.0016-0.5 mM) to a pyrene-
loaded noncross-linked micelles (NCLMs) and KCLMs (pyrene con-
centration = 1 ꢀ 10-6 M, polymer concentration = 0.75 g/L, 0.75 mL).
In a Stern-Volmer equation (I0/I - 1 = kqτ0[Q] = KSV[Q]), I0 and I
represent the integrated fluorescence intensity of pyrene in the absence
and in the presence of quencher, respectively. [Q] is the molar
concentration of quencher and τ0 is the fluorescence lifetime in the
absence of the quencher. Time-resolved fluorescence measurement in
our previous study showed that pyrene had a lifetime (τ0) of 128 ns in
aqueous phase.21 The sample solution containing pyrene was excited at
338 nm. The pyrene fluorescence was detected at 393 nm.
Stability of Cross-Linked Micelles. Kinetic stability of KCLM
was investigated by adding SDS. The effect of SDS on KCLMs in
aqueous media was estimated by dynamic light scattering analysis and
fluorescence spectroscopy. A SDS solution (1 mL, 7.5 g/L) was added to
Transmission Electron Microscopy. Transmission electron micro-
scopy (TEM) was performed on a JEM-2000EX (JEOL Tokyo, Japan),
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Figure 1. Synthetic route for the PEG45-PAsp8-PPhe19 triblock copolymer.
the KCLM solution (2 mL, 0.75 g/L), and the solution was stirred at
500 rpm. At predetermined time periods, scattered light intensity (SLI)
was monitored and compared to the initial scattered light intensity
(SLI0) by dynamic light scattering analysis. For time-dependent stability
of DOX-loaded micelles, fluorescence spectroscopy was utilized to
monitor the change of DOX fluorescence intensity ratio (FI/FI0) in
the presence of SDS, where FI is the intensity at predetermined times,
and FI0 is the intensity at initial time. For comparison, the SDS treatment
of the NCLM solution was conducted under the same condition.
Hydrolysis Kinetics of Ketal Cross-Links in KCLMs. Hydro-
lysis kinetics of ketal cross-links in KCLMs was estimated using 1H NMR
spectroscopy. The KCLMs (1 mL, 10 mg/mL) were dissolved in a
mixture of D2O (0.5 mL) and each buffer solution (0.5 mL, pH 7.4
phosphate buffer, pH 5.0 acetate buffer). At predetermined time
intervals, the hydrolysis kinetics was monitored by time-dependent
quantification of the decrease in ketal integration at 1.34 ppm. Half-
lives of ketal hydrolysis at different pH were calculated using the
Arrhenius equation.
DOX-KCLMs, except that 2,20-(ethylenedioxy)bis(ethylamine) was
used as a nondegradable cross-linker instead of the ketal cross-linker.
Controlled DOX Release from DOX-Loaded KCLMs. In vitro
release profiles of DOX from DOX-loaded NCLMs and KCLMs were
investigated in the aqueous buffer solutions (pH 7.4 phosphate buffer
and pH 5.0 acetate buffer). Various DOX-loaded micelles were dis-
persed in aqueous buffer solutions (1 mL, 0.75 g/L), and transferred to a
dialysis membrane bag (MWCO: 1000). The release experiment was
initiated by placing the dialysis bag in 10 mL of release media. The
release medium was shaken at a speed of 150 rpm at 37 °C. At
predetermined time intervals, samples (10 mL) were withdrawn and
replaced with an equal volume of the fresh medium. The concentration
of released DOX in the samples was determined by measurement of
fluorescence emission intensity at 588 nm (excitation at 480 nm) based
on the standard curve obtained using DOX in pH 7.4 phosphate buffer
and pH 5.0 acetate buffer.
Cell Culture. Human breast cancer MCF-7 cells were obtained from
the Korean Cell Line Bank (KCLB, Seoul). Cells were propagated in
Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL, Gaithers-
burg, MD) supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (FBS, Gibco BRL), and 1% (v/v) penicillin-streptomycin (Gibco
BRL). Cells were cultured in a humidified incubator at 37 °C with 5%
CO2. The culture medium was replaced every two days.
Biocompatibility of NCLMs and KCLMs. MCF-7 cells were
seeded into 96-well flat-bottomed tissue-culture plate at 2000 cells/well,
and incubated for 24 h in a humidified atmosphere of 5% CO2 at 37 °C.
The micelles solution was diluted with culture medium to obtain a
concentration range from 1 to 300 μg/mL. After the incubation for
24 and 48 h, medium was removed, and cells were washed with PBS, and
the medium was replaced with cell counting kit-8 (CCK-8) solutions
(Dojindo Laboratories, Kumamoto). The absorbance of individual
wells was measured at 450 nm by a microplate reader (Biorad Elizer,
PA).
Preparation of DOX-Loaded Non-Cross-Linked Polymer
Micelles (DOX-NCLMs). DOX was loaded into PEG45-PAsp8-PPhe19
micelles by the dialysis method. Before loading DOX to the PEG45-
PAsp8-PPhe19 micelles, DOX HCl (2 mg, 0.0034 mmol) was stirred
3
with TEA (0.6 μL, 0.0044 mmol) in DMF (0.6 mL) overnight in the
dark. The triblock copolymer (20 mg) was dissolved in 4 mL of DMF for
3 h at 70 °C, and then the DOX solution was subsequently added and
stirred in the dark at room temperature. The solution was dialyzed using
a membrane (Spectrapor, MWCO: 1000) for 12 h and followed by
lyophilization in the dark. To determine the drug loading content and
loading efficiency, DOX-loaded polymer micelles were dissolved in
DMF, and then measured by fluorescence emission intensity at 588 nm
(excitation at 480 nm). The drug loading content and loading efficiency,
based on the standard curve of DOX in DMF, were calculated to be 8.5
wt % and 94.1%, respectively.
Preparation of DOX-Loaded Ketal Cross-Linked Polymer
Micelles (DOX-KCLMs). DOX-KCLMs were prepared based on an
identical process used for shell cross-linking of DOX-free PEG45-PAsp8-
PPhe19 micelles, except that the DOX-loaded micelles were used as a
template for ketal cross-linking instead of DOX-free micelles.
Preparation of DOX-Loaded Shell Cross-Linked Micelles
with Nondegradable Cross-Links (DOX-CLMs-ND). DOX-
CLMs-ND were prepared following the identical process used for
Cytotoxicity. In vitro cytotoxicity of DOX-loaded KCLMs were
evaluated by measuring the half maximal inhibitory concentration
(IC50) using the CCK assay. Briefly, MCF-7 cells were seeded onto a
96-well plate at a density of 5 ꢀ 103 cells per well in 200 μL of medium
and incubated at 37 °C and 5% CO2. After 24 h, the medium of each well
was then replaced by 200 μL of fresh medium containing free DOX,
DOX-loaded NCLMs, DOX-loaded KCLMs at various drug concentra-
tions from 0.01 to 50 μg/mL, and then the plates were incubated (37 °C,
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was 45:8:19. The conversion of monomeric BAsp and Phe to
polymeric PBAsp and PPhe was found to be 80 and 95%,
respectively (Table 1). The Mn of PEG45-PAsp8-PPhe19, calcu-
1
lated by H NMR, was 5570 g/mol. GPC analyses showed a
narrow molecular weight distribution (Mw/Mn = 1.08; Table 1).
To produce the block copolymer with narrow molecular weight
distribution, we used PEG45 with an low polydispersity index
(PDI) of 1.06 (GPC). This initial low molecular weight
distribution may contribute to the low PDI of resulting PEG45-
PAsp8-PPhe19. Besides, N-carboxyanhydride (NCA)-based
polymerization for poly(amino acid) blocks was known to follow
pseudoliving polymerization, in case the ratio of the monomer to
the amine-based initiator ([M]0/[I]0) is below about 20.35 The
choice of starting PEG and the inherent feature of NCA
polymerization may afford such a narrow molecular weight
distribution of PEG45-PAsp8-PPhe19. We considered the specific
composition for a design of PEG45-PAsp8-PPhe19. First, the
choice of PEG45 with Mn of 2000 g/mol has been acceptable
as nanocarriers for intravenous administration.21,26 In addition,
PEG45 has a small hydrodynamic diameter and, thus, can be
easily excreted through renal filtration after carrying out its role.36
Second, the PAsp composition of 8 units is selected, since
this length would be enough for shell cross-linking reaction.
Third, the 19 units of Phe can induce the self-assembly by
hydrophobic interaction to form micelles. In case the length of
hydrophobic PPhe is not enough, the micellar structure cannot
be formed, because the block copolymer exists in a water-soluble
state. In contrast, too much composition of hydrophobic
PPhe makes the block copolymer lose its dispersibility and
resulted in precipitation in water. For this reason, we reasonably
selected the block composition of PEG, PAsp, and PPhe for
PEG45-PAsp8-PPhe19.
Figure 2. 1H NMR spectra of CH3O-PEG-NH2 (a), PEG45-PBAsp8
(b), PEG45-PBAsp8-PPhe19 (c), and PEG45-PAsp8-PPhe19 (d) in
DMSO-d6. Arrows in (c) and (d) show the complete deprotection of
benzyl groups of PBAsp blocks.
5% CO2). After 24 h, the medium was removed, and cells were washed
with the fresh medium, followed by the CCK assay. IC50 was calculated
as the concentration of DOX yielding 50% inhibition of cell prolifera-
tion, compared to the untreated control.
Rationally designed PEG45-PAsp8-PPhe19 self-assembled to
form core-shell-corona polymer micelles with three well-
defined, distinct domains: the inner hydrophobic PPhe core,
the anionic PAsp middle shell, and the solvated PEG outer
corona. The mean hydrodynamic diameter was 55.3 nm.
The cmc of the block copolymer, estimated by pyrene excitation
spectra,27 was 15.3 mg/L (Table 1). The zeta potential (ζ) of
PEG45-PAsp8-PPhe19 micelles was reasonably negative (ζ = -
29.8 mV).
Formation of Ketal Cross-Linked Polymer Micelles. Ketal
cross-linking of PAsp middle shells was performed by adding the
ketal cross-linkers to an aqueous solution of PEG45-PAsp8-
PPha19, in which Asp groups were preactivated with 2-fold excess
EDC at pH 4.0. Bifunctional ketal cross-linkers with the primary
amines reacted with O-acylurea to form intramicellar cross-
linking bridges via amide bond formation. The feed molar ratio
of the ketal cross-linker to Asp repeating units of the PAsp middle
shells was 2:1. It is noteworthy that the zeta potential of KCLMs
are -19.2 mV, which is much higher than that of NCLMs (ζ = -
29.8 mV; Table 2). The increased zeta potential reflects the
conversion of some carboxylic acid groups of middle PAsp shells
to amide linkages formed during shell cross-linking reaction.
KCLMs maintain a reasonable size of precursor polymer mi-
celles. TEM images show the spherical nature of polymer
micelles, and that the shell cross-linking reaction did not induce
the intermicellar aggregation, which is consistent with dynamic
light scattering results (Figure 3). This indicates that the PEG
outer corona confines the shell cross-linking reaction within the
PAsp middle shells, preventing the formation of intermicellar
aggregates.
Intracellular Distribution of DOX-Loaded KCLMs. MCF-7
cells (5 ꢀ 103 cells/mL per well) were seeded onto a cover glass-bottom
dish in 2 mL of DMEM medium supplemented with 10% FBS,
1% antibiotics (penicillin 100 U/mM, streptomycin 0.1 mg/mL). After
incubation for 24 h (37 °C, 5% CO2), the medium was carefully
aspirated and replaced with 1 mL of medium containing 5 μg/mL
DOX equivalent of DOX-KCLM and LysoTracker (50 nM). The cells
were incubated for 1 and 5 h, then washed three times with PBS.
Formaldehyde (1 mL, 3.7%) was added, and the cells were fixed for
5 min. Finally, the solution was aspirated, and the cells were rinsed
three times with PBS, and then the coverslips were transferred to glass
slides. The confocal laser scanning microscopy (CLSM) images of
LysoTracker labeled MCF-7 cells treated with DOX-KCLMs were
obtained using a confocal laser scanning microscope (C1si, Nikon,
Japan) by green-fluorescing (λex = 470-490 nm) and red-fluorescing
(λex = 520-550 nm).
’ RESULTS AND DISCUSSION
Preparation and Characterization of PEG-PAsp-PPhe Mi-
celles. The PEG45-PAsp8-PPhe19 copolymer was synthesized via
the one-pot two-step polymerization of β-benzyl L-aspartate N-
carboxyanhydride (BAsp-NCA) and L-phenylalanine N-carbox-
yanhydride (Phe-NCA) in the presence of a CH3O-PEG-NH2
macroinitiator and
a subsequent deprotection process
(Figure 1). The composition ratio of PEG-PAsp-PPhe was
calculated by the peak integration ratio of -OCH2CH2- protons
of PEG at 3.49 ppm, PhCH2- of PBAsp at 5.05 ppm, and phenyl
protons in of PPhe at 7.16 ppm (Figure 2). The molar composi-
tion ratio of monomeric repeating units in PEG, PAsp, and PPhe
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Figure 3. Size distributions and morphology of NCLMs (a and c) and KCLMs (b and d) estimated by dynamic light scattering and TEM. Scale bars =
100 nm.
Table 1. Characteristics of the PEG45-PAsp8-PPhe19 Copolymer
feed ratio
composition ratioa
conversion of BAspa
(%)
conversion of Phea
(%)
Mw/
cmc c
a
b
copolymer
([EG]/[BAsp]/[Phe])
([EG]/[Asp]/[Phe])
Mn
Mn
(mg/L)
PEG45-PAsp8-PPhe19
45:10:20
45:8:19
80
95
5570
1.08
15.8
a Calculated by 1H NMR spectra b Estimated by GPC. c Critical micelle concentration at 25 °C.
Table 2. Micelle Sizes, Zeta Potentials, and Polydispersity
Factors of Various Micelles
because the shell cross-links may inhibit the penetration of
quenchers to micellar core to quench the pyrene fluorescence.
The diffusion rate across the cross-linked shells was estimated
using fluorescence quenching of pyrene loaded within micellar
cores by DPC.38 As shown in Figure 4, the quenching constant
(KSV) and the bimolecular rate constant (kq), calculated by a
Stern-Volmer kinetic model of steady-state quenching experi-
ments: I0/I - 1 = kqτ0[Q] = KSV[Q], substantially decreased for
KCLMs with cross-linked shells. The KSV value of KCLMs was
lowered by 54% over NCLMs, reflecting that the shell cross-links
act as a diffusion barrier for low molecular weight compounds.
Normally, the drug release behavior from the micellar core is
dependent on the diffusion-controlled process. Therefore, this
result indicates that shell cross-linking may be effective in
reducing the drug release at pH 7.4, where the ketal cross-links
are intact.
micelles
da (nm)
zeta potentialb (mV)
μ2/Γ2c
NCLM
55.3
54.5
54.7
52.5
-29.8
-19.2
-34.6
-18.1
0.15
0.20
0.18
0.15
KCLM
DOX-NCLM
DOX-KCLM
a Mean hydrodynamic diameters at 25 °C. b Estimated at pH 7.4 at
25 °C. c Polydispersity factor estimated by dynamic light scattering.
Steady-State Fluorescence Quenching. Shell cross-links of
KCLMs can act as a diffusion-inhibiting barrier. This indicates
that the diffusion of small molecules across the cross-linked
networks within middle shells would be prevented to a certain
degree. Steady-state quenching analysis is a useful tool to
estimate the accessability of small molecules to micellar hydro-
phobic core domains.37 We monitored the access of DPC, a
quencher molecule to a hydrophobic fluorescent pyrene loaded
in KCLMs. For KCLMs, it is expected that the collisional
fluorescence quenching of pyrene by DPC would be inhibited,
Stability of Cross-Linked Micelles. The kinetic stability of
KCLMs and NCLMs was monitored using light scattering
analysis in the presence of SDS, a strong micelle-disrupting
agent.21,39 For NCLMs, the relative scattering intensity was
dramatically decreased to 70% after 1 h, which indicates SDS-
induced dissociation of micelles (Figure 5(a)). On the other
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Figure 4. Stern-Volmer plots for micelle-entrapped pyrene (6.0 ꢀ
10-6 M) quenched by DPC.
Figure 6. Arrhenius plots for hydrolysis kinetics of ketal cross-links
within KCLMs at pH 7.4 and 5.0. (A and A0 represent the integrations of
(CH3)2 peaks of ketal cross-links after a prefixed period of incubation
time and before incubation, respectively).
Figure 7. 1H NMR spectra of KCLMs in D2O at pH 5.0 at various
incubation period and the postulated corresoponding structure of ketal
cross-linked shell domains. Arrows indicate the gradual disappearance of
ketal peaks.
fluorescence, and, within 1 h, increased up to 40% of the initial
fluorescence intensity (FI/FI0). On the other hand, KCLMs
treated with SDS exhibited slowed increase, and, within 2 h,
increased only to 20% of initial fluorescence intensity. This
indicats that NCLMs released the more amount of DOX than
KCLMs in an identical time period. These results indicate that
shell cross-linking is effective not only in enhancing micellar
stability but also in inhibiting the DOX release from the micelles.
Hydrolysis Kinetic of Ketal Cross-Links within KCLMs.
Hydrolysis kinetics of the ketal cross-links within KCLMs at
physiological pH (7.4) and endosomal pH (5.0) was investigated
by incubating KCLMs in a mixture of D2O and each buffer
solution (phosphate buffer and acetate buffer). The hydrolysis
kinetics was monitored by the integration ratio of the integration
at a certain period of incubation of the ketal peak ((CH3)2CO2-,
1.34 ppm) to the integration before hydrolysis. The half-lives of
ketal hydrolysis at different pH were calculated using the
Arrhenius equation. As shown in Figure 6, the hydrolysis rate
was much faster at pH 5.0 than at pH 7.4. The half-life of ketal
Figure 5. (a) Kinetic changes in relative scattered light intensity (SLI/
SLI0) for NCLMs and KCLMs, and (b) the relative fluorescence
intensity (FI/FI0) of DOX-NCLMs and DOX-KCLMs in the presence
of SDS (2.5 g/L).
hand, the scattered intensity of KCLM solutions was minimally
decreased. Even after 3 h, KCLMs maintained the initial scatter-
ing intensity over 90% (Figure 5(a)). This indicates that KCLMs
could remain their assembled structure even in the harsh condi-
tion for micelle dissociation, because shell cross-linkers enable
polymer micelles to be resistant to SDS effects. The stability of
polymer micelles in a drug-loaded state was estimated by
monitoring DOX fluorescence. This study was based on the
photophysical property of DOX. The DOX fluorescence is self-
quenched when DOX are in a densely packed status in the core of
micelles, whearas the DOX fluorescence was recovered upon
DOX releasing form the micelles. As shown in Figure 5(b),
NCLMs treated with SDS exhibited abrupt increase in DOX
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Figure 9. In vitro cytotoxicity of DOX, DOX-NCLMs, and DOX-
KCLMs with MCF-7 cells after 24 h (n = 3).
Figure 8. pH-Controlled DOX release profiles from DOX-NCLMs and
DOX-KCLMs (n = 3).
the reported solubility was 370 ug/mL.40 This indicates that sink
conditions were maintained during the release experiments.
One may have question of whether the enhanced rate of the
DOX release from KCLMs at pH 5.0 over at pH 7.4 is caused
mainly by the enhanced DOX solubility. For DOX-NCLMs, the
pH-dependent DOX release behavior was observed. As shown in
Figure 8, the release amount at pH 5.0 from DOX-NCLMs was
higher by 2.2% at 24 h, over at pH 7.4. In order to examine the pH
effect on the DOX release from the shell cross-linked micelles,
the DOX release from DOX-loaded micelles with nondegradable
cross-links (DOX-CLMs-ND) was examined (Figure S1 in the
Supporting Information). The release amount at pH 5.0 was
higher as low as 0.3% at 24 h, over at pH 7.4, and the difference
was not significant. This shows that the pH effect on the DOX
release was negligible, in case the cross-linked structure was
maintained. This strongly supports that the enhanced release rate
of DOX at pH 5.0 from KCLMs was primarily due to the
hydrolysis of ketal cross-links, not due to the increased solubility
of DOX.
In Vitro Cytotoxicity. The cytotoxicities of DOX-free
NCLMs and KCLMs were evaluated using a MCF-7 human
breast cancer cell line with various concentrations. NCLMs and
KCLMs showed no noticeable cytotoxicity up to 300 μg/mL
(Figure S2 in the Supporting Information). In vitro cytotoxicities
of DOX, DOX-NCLMs and DOX-KCLMs for MCF-7 cells were
estimated. As shown in Figure 9, DOX-KCLMs showed the
effective inhibitory effect on the proliferation of cancer cells. The
somewhat lower toxicity of DOX-KCLMs (IC50 = 6.96 μg/mL)
compared to free DOX (IC50 = 0.48 μg/mL) and DOX-NCLMs
(IC50 = 2.55 μg/mL) was probably due to the gradual release of
DOX within the endosomes. DOX-NCLMs without ketal cross-
links began releasing DOX immediately upon placing in the
culture media. For this reason, the released DOX could be
accumulated within cell nucleus in a short time period. In
contrast, the DOX release rate from DOX-KCLMs was slower
because the DOX release was accelerated after hydrolysis of ketal
cross-links within cellular endosomes. The potential not only for
minimizing drug loss in blood but also for selective accumulation
in tumor tissue by the EPR effect may enhance its overall
therapeutic efficacy in vivo relative to free DOX and DOX-
NCLMs.
hydrolysis at pH 5.0 was calculated to be 0.7 h, whereas the half-
life at pH 7.4 was 52 h. This indicates the 74-fold faster hydrolysis
of cross-links at endosomal pH. Figure 7 shows the correlation
between the postulated structure changes at pH 5.0 within cross-
linked shells and the disappearance of ketal peaks in NMR
spectra. After 1 min incubation, the hydrolysis process already
began to occur, but the most ketal linkages at 1.34 ppm remained
intact. As the incubation time increased, the ketal integration
gradually decreased, whereas the peak intensity of acetone peaks
at 2.12 ppm increased. After 3 h, the ketal resonance peak almost
completely disappeared. This pH-dependent hydolysis analysis
indicates that the ketal cross-links can maintain its structure to
strengthen the micelle structure, reduce the drug loss at blood-
stream, and finally undergo the cleavage at endosomes to
facilitate the loaded anticancer drugs.
Controlled Drug Release from DOX-Loaded KCLMs. To
estimate controlled DOX-releasing properties, DOX-NCPMs
and DOX-KCLMs with identical drug loading contents (8.5 wt
%) were used. Figure 8 shows that the pH-dependent DOX
release rate from NCLMs and KCLMs. The DOX release from
NCLMS did not notably depend on the differences between
extracellular (pH 7.4) and endosomal pH (pH 5.0). On the other
hand, DOX-KCLMs showed a dramatic pH-dependent DOX
release behavior. At pH 7.4, DOX-KCLMs effectively inhibited
the DOX release in the extracellular media at 48 h by 43.8% over
polymer micelles without ketal cross-links (NCLMs). Although
shell cross-linking did not totally block the release of DOX from
KCLMs, it displayed the inhibitory effect to some extent. In
contrast, at pH 5.0, the DOX release from KCLMs was acceler-
ated by 68.7% at 48 h, compared with the DOX release from
KCLMs at pH 7.4. As described in hydrolysis kinetics study, the
structural integrity of ketal cross-links within micellar shells could
be maintained at pH 7.4, and thus the diffusion-dependent DOX
release could be inhibited to a certain degree. On the other hand,
the fast breakage of shell cross-links at pH 5.0 would facilitate the
diffusion of DOX molecules across the middle shells of micelles.
The percentage of DOX release from KCLM at pH 5.0 is around
35% at 48 h, and the continuous DOX release is expected over
longer period of times.
In our experiments, the DOX concentration in the release
medium at each collection time was below 10 ug/mL, irrespec-
tive of medium pH. It was reported that the aqueous solubility of
DOX at pH 7.4 was reported to be 62 ug/mL. At pH 5.0, the
degree of DOX protonation increases as does its solubility, and
Intracellular Distribution of DOX-Loaded KCLMs. The
endocytosis of DOX-KCLMs by MCF-7 cells was monitored
by confocal laser scanning microscopy (CLSM). The acidic
cellular endosome was labeled with green-fluorescent
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disrupt the cross-links, and finally triggered the facilitated DOX
release to inhibit proliferation of cancer cells.
’ CONCLUSIONS
We have developed a novel, robust, biocompatible shell cross-
linked micelles that could preferentially release entrapped DOX
under intracellular endosomal compartments The introduction
of acid-labile ketal cross-links into PEG-poly(amino acid)s
micelles may meet the major requirements of targeted nanocar-
riers with a high delivery efficiency: (i) high stability in the
bloodstream, (ii) prolonged blood circulation due to the PEG
outer corona (minimized uptake by RES), (iii) ability to mini-
mize drug loss before reaching target disease tissues, (iv) passive
targeting and cellular uptake, and (v) preferential drug release
within target cells. This new shell cross-linked micelle can serve
as a useful intracellular nanocarrier of many poorly water-soluble
anticancer drugs.
Figure 10. CLSM images of MCF-7 cells incubated with Lysotracker
(50 nM), free DOX (5 μg/mL), DOX-KCLMs (DOX = 5 μg/mL), and
DOX-CLMs-ND (DOX = 5 μg/mL) for 5 h exposure. (a) Free DOX for
1 h exposure, (b) DOX-KCLMs for 1 h exposure, (c) DOX-KCLMs for
5 h exposure, (d) DOX-CLMs-ND for 1 h exposure, and (e) DOX-
CLMs-ND for 5 h exposure (green fluorescence is associated with
LysoTracker and the red fluorescence is expressed by free DOX and
released DOX). Scale bar = 20 μm.
’ ASSOCIATED CONTENT
S
Supporting Information. Cell viability of DOX-free
b
NCLMs and KCLMs, pH-dependent DOX release from DOX-
CLMs-ND, and the confocal laser scanning microscope images
of intracellular DOX release from NCLMs. This material is
available free of charge via the Internet at http://pubs.acs.org.
LysoTracker as an indicator. We monitored first the intracellular
DOX release from NCLMs and localization of DOX within cells
using CLSM. As shown in Figure S3 (Supporting Information),
even after 1 h incubation with MCF-7 cells, DOX released from
NCLMs showed the strong red florescence within the endo-
somes and also a larger population of DOX was found within the
nuclei. This indicates that DOX-NCLMs began to release DOX
right upon addition into the cell culture media. In other words,
DOX-NCLMs tended to release DOX in the extracellular area,
and the released DOX was localized within the endosomes and
nuclei in a short time period. After 5 h incubation, the DOX
population within nuclei was more pronounced than within
endosomes.
’ AUTHOR INFORMATION
Corresponding Author
*Tel.: þ82-2-961-2317. Fax: þ82-2-960-1457. E-mail: schlee@
khu.ac.kr; syjeong@khu.ac.kr.
Author Contributions
†These authors contributed equally to this work.
’ ACKNOWLEDGMENT
To verify that facilitated DOX release in cell culture experi-
ments is indeed related to the degradation of cross-links at
endosomal pH, we performed the cell experiments using
DOX-KCLMs with degradable cross-links and also with DOX-
CLMs-ND with nondegradable cross-links. In Figure 10, the
DOX distribution was visualized for DOX-KCLMs and DOX-
CLMs-ND. For DOX-KCLMs, after 1 h incubation, DOX was
released from KCLMs with low florescence intensity within the
endosomes, and the small population of DOX was found within
nuclei (Figure 10b). After a 5 h incubation, DOX of a strong
fluorescence was released from DOX-KCLMs. DOX not only
spread out in endosomes but also accumulated in the nuclei
(Figure 10c). For DOX-CLMs-ND, the DOX fluorescence was
confined within the endosome at 1 h incubation (Figure 10d).
After a 5 h incubation, it is noteworthy that the DOX fluores-
cence intensity within nuclei was much lower than DOX-KCLMs
(Figure 10e). This reflects that the nondegradable cross-links of
DOX-CLMs-ND acted as barriers of DOX release even within
acidic endosomes and thereby could not trigger the enhanced
DOX release. This is consistent with the negligible enhancement
of DOX release rate at endosomal pH (Figure S1 in the
Supporting Information). These results strongly supports that
the facilitated DOX release within cells was primarily related to
the hydrolysis of ketal cross-links of DOX-KCLMs. Based on in
vitro results, we confirmed that DOX-KCLMs was taken up by
the cells via endocytosis, and transferred into endosomes to
This research was supported by Basic Science Research
Program through the National Research Foundation of Korea
(NRF) funded by the Ministry of Education, Science and
Technology (2010-0007721) and the Fundamental R&D Pro-
gram for Core Technology of Materials (K0006028) by Ministry
of Knowledge Economy, Korea.
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