Hyperbranched double hydrophilic block copolymer micelles of poly(ethylene oxide) and polyglycerol for pH-responsive drug delivery

Article abstract of DOI:10.1021/bm300151m

We report the synthesis of a well-defined hyperbranched double hydrophilic block copolymer of poly-(ethylene oxide)-hyperbranched-polyglycerol (PEO-hb-PG) to develop an efficient drug delivery system. In specific, we demonstrate the hyperbranched PEO-hb-PG can form a selfassembled micellar structure on conjugation with the hydrophobic anticancer agent doxorubicin, which is linked to the polymer by pH-sensitive hydrazone bonds, resulting in a pHresponsive controlled release of doxorubicin. Dynamic light scattering, atomic force microscopy, and transmission electron microscopy demonstrated successful formation of the spherical core-shell type micelles with an average size of about 200 nm. Moreover, the pH-responsive release of doxorubicin and in vitro cytotoxicity studies revealed the controlled stimuli-responsive drug delivery system desirable for enhanced efficiency. Benefiting from many desirable features of hyperbranched double hydrophilic block copolymers such as enhanced biocompatibility, increased water solubility, and drug loading efficiency as well as improved clearance of the polymer after drug release, we believe that double hydrophilic block copolymer will provide a versatile platform to develop excellent drug delivery systems for effective treatment of cancer.

Full text of DOI:10.1021/bm300151m

Article
pubs.acs.org/Biomac
Hyperbranched Double Hydrophilic Block Copolymer Micelles of
Poly(ethylene oxide) and Polyglycerol for pH-Responsive Drug
Delivery
†
‡
§
§
‡
‡
Sueun Lee, Kyohei Saito, Hye-Ra Lee, Min Jae Lee, Yuji Shibasaki, Yoshiyuki Oishi,
,†
and Byeong-Su Kim*
†
Interdisciplinary School of Green Energy, Ulsan National Institute of Science and Technology (UNIST), Ulsan 689-798, Korea
Department of Chemistry and Bioengineering, Iwate University, 4-3-5 Ueda, Morioka, Iwate 020-8551, Japan
Department of Applied Chemistry, Kyung Hee University, Yongin 446-701, Korea
‡
§
*
S Supporting Information
ABSTRACT: We report the synthesis of a well-defined
hyperbranched double hydrophilic block copolymer of poly-
(
ethylene oxide)-hyperbranched-polyglycerol (PEO-hb-PG) to
develop an efficient drug delivery system. In specific, we
demonstrate the hyperbranched PEO-hb-PG can form a self-
assembled micellar structure on conjugation with the hydro-
phobic anticancer agent doxorubicin, which is linked to the
polymer by pH-sensitive hydrazone bonds, resulting in a pH-
responsive controlled release of doxorubicin. Dynamic light
scattering, atomic force microscopy, and transmission electron
microscopy demonstrated successful formation of the spherical
core−shell type micelles with an average size of about 200 nm.
Moreover, the pH-responsive release of doxorubicin and in vitro cytotoxicity studies revealed the controlled stimuli-responsive
drug delivery system desirable for enhanced efficiency. Benefiting from many desirable features of hyperbranched double
hydrophilic block copolymers such as enhanced biocompatibility, increased water solubility, and drug loading efficiency as well as
improved clearance of the polymer after drug release, we believe that double hydrophilic block copolymer will provide a versatile
platform to develop excellent drug delivery systems for effective treatment of cancer.
INTRODUCTION
conjugated with various chemotherapeutic agents in a series of
■
10−13
papers.
One approach for the development of pH-sensitive
Various drug delivery approaches have been developed to
achieve site-specific and time-controlled delivery of therapeutics
to improve therapeutic efficacy while minimizing undesired side
polymer micelles is to covalently conjugate drugs to the
polymer via acid degradable linkages such as hydrazone, ester,
14−16
1
and carbamate.
When the pH changes, the linkages
effects. Self-assembled nanostructures have long been studied
degrade with concomitantly releasing the drug from the micelle
at a specific location. Compared to the traditional approaches in
which the drug is physically entrapped in the hydrophobic core
of micelles by hydrophobic interactions, the aforementioned
approach enables control and modification of the drug release
rate from these micelles, as proved in the case of smart
as promising vehicles for the delivery of active therapeutics.
Recently, self-assembled polymeric micelles have been exten-
sively utilized in drug delivery systems because of their unique
features, such as high loading capacity and enhanced solubility
of drugs, reduced systemic adverse effects, preferential
accumulation at the tumor site owing to enhanced permeability
and retention (EPR) effect, and perhaps, most importantly,
high tunability of chemical and physical characteristics with
respect to the field of applications. Despite these advantages
of polymeric micelles, the sophisticated delivery of active
therapeutics from the carriers remains a challenge in the
development of advanced drug delivery systems.
17
nanocarrier systems.
To date, various pH-responsive polymeric micelles have been
successfully reported; however, only a few examples have
advanced to the clinical settings, mostly due to their intrinsic
toxicity. To resolve the issues associated with toxicity of the
carrier, many biocompatible polymers such as poly(ethylene
oxide), polyglycerols (or polyglycidols), and sugar derivatives
together with biodegradable polymers like polyester, poly-
2
−5
As alternatives to traditional micelle systems, smart nano-
carriers are actively pursued that can stably encapsulate
therapeutics and release them at a desired site in response to
6
−9
external stimuli such as pH, temperature, redox, and light.
Received: January 28, 2012
Revised: March 10, 2012
Published: March 13, 2012
For example, the Kataoka group has extensively studied the
development of intracellular pH-triggered polymeric micelles
©
2012 American Chemical Society
1190
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Scheme 1. Illustration of Hyperbranched Double Hydrophilic Block Copolymer Conjugated with Doxorubicin (DOX) and Its
Self-Assembled Micelle for the pH-Responsive Intracellular Release of Active Therapeutics
carbonate, and polypeptides are often employed as the
polymeric segments to form micelles in aqueous solutions.
Polyglycerols are a class of hyperbranched polyethers with
excellent biocompatibility. Although initial synthetic ap-
proaches for constructing polyglycerols were rather challenging,
the recent advancement by Sunder and co-workers allows the
synthesis of well-defined and complex architectures of
Acros) and methoxy ethanol (99.5%, Acros) were purified by
distillation from CaH2 directly prior to use. The cation-exchange
resin, Dowex 50W × 2 (50−100 mesh) was purchased from Wako
Pure Chemical (Japan) and used after washing with methanol. Other
reagents and solvents were used as received.
1
Measurements. H NMR spectroscopy (VNMRS 600 spectrom-
eter 600 MHz, Varian, U.S.A.) was used with DMSO-d as the solvent.
6
Mn and Mw were measured by a gel permeation chromatography
polyglycerols with relatively low polydispersity (M /M
=
w
n
(
GPC) on Tosoh HLC-8120 GPC equipped with a consecutive
polystyrene gel column (TSK-GEL GMHHR-M and GMHHR-N) at
0 °C by eluting with N-methyl-2-pyrrolidone (NMP) containing 0.01
1
8,19
1
.2−1.9).
They also exhibit flexibility in physicochemical
properties by the convenient end-group functionalizations.
Recently, polyglycerols with varying architectures have been
4
M lithium bromide at a flow rate of 1.0 mL/min, which was calibrated
by standard polystyrene samples. Transmission electron microscopy
20−25
designed and synthesized for biomedical applications.
Brooks and co-workers have extensively investigated the
suitability of hydrophobically functionalized hyperbranched
polyglycerols for use as synthetic albumin substitutes and as
(
TEM, JEM-2100, JEOL, Japan) and atomic force microscopy (AFM,
Dimension 3100, Veeco, U.S.A.) were performed to investigate the
morphology of the PEO-hb-PG micelles. Size distribution analysis was
studied using dynamic light scattering (DLS, Nano ZS, Malvern, U.K.,
and BI-APD, Brookhaven Instrument, New York, U.S.A.). Two
instruments were used to crosscheck the reliability of the obtained
data. The amount of the released DOX was measured by high
performance liquid chromatography (HPLC, 1200 series, Agilent,
U.S.A.) with a mobile phase of a mixture of CH CN and H O (4:6, v/
v) at a rate of 0.80 mL/min and 20 °C. The detection of DOX was
performed using a UV detector at a wavelength of 480 nm.
Synthesis of Ethoxyethyl Glycidol Ether (EEGE). This
compound was prepared by modifying a previously reported
23−25
general drug delivery vehicles.
In this article, we designed the hyperbranched double
hydrophilic block copolymer of poly(ethylene oxide)-hyper-
branched-polyglycerols (PEO-hb-PG) with an aim to enhance
biocompatibility, to increase water solubility, and to improve
25
3
2
the clearance of the polymer after drug release. Moreover, the
hyperbranched architecture of polyglycerol can potentially
increase the loading capacity of active therapeutics that are
linked by the pH-responsive hydrazone moiety for an effective
26
drug delivery system. We found that hyperbranched PEO-hb-
PG can form a self-assembled micellar structure upon
conjugation with hydrophobic therapeutics, which in turn
exhibits a pH-responsive controlled release of doxorubicin
27
method. Briefly, 85.00 g (1.147 mol) glycidol and 225.9 g (3.133
mol) ethyl vinyl ether were added to a 500 mL two-neck flask with a
magnetic stirring bar. This mixture was cooled to −30 °C, and 1.915 g
(
11.12 mmol, 1.0 mol % to glycidol) p-toluene sulfonic acid
(
Scheme 1).
monohydrate (TsOH) was slowly added. After the addition, the
mixture was kept stirring at 25 °C for another 3 h. The reaction
EXPERIMENTAL SECTION
Materials. Cesium hydroxide monohydrate, poly(ethylene oxide)
monomethyl ether (nominal number average molecular weight (M )
of 2000) (PEO), doxorubicin hydrochloride, N,N′-dimethylformamide
mixture was washed with saturated aqueous NaHCO solution, and
3
■
the organic layer was separated and dried with MgSO . After filtration,
4
the residual ethyl vinyl ether was removed under reduced pressure, and
the remainder was distilled in vacuo to yield EEGE as a colorless liquid
n
1
(
DMF), triethylamine, dimethylsulfoxide, and hydrazine monohydrate
product. Yield: 74%, b.p. 50 °C/0.6 Torr. H NMR (400 MHz,
were purchased from Sigma-Aldrich. p-Nitrophenyl chloroformate (p-
NPC) was purchased from TCI (Japan). 1,4-Dioxane was dried over
sodium and distilled prior to use. For polymerization, diglyme (99%,
DMSO-d
2.53−2.73 (m, 2H, CH
the epoxy ring), 3.25−3.77 (m, 4H, -OCH CH and −OCH −), 4.69
6
, ppm): δ 1.10 (t, 3H, −CH
3 2 3
), 1.19 (q, 2H, -OCH CH ),
of the epoxy ring), 3.07−3.09 (m, 1H, CH of
2
2
3
2
1
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Scheme 2. Synthetic Approach for Preparation of PEO-hb-PG Copolymer
(
1
m, -OCH(CH )O-) ppm. 13C NMR (101 MHz, DMSO-d , ppm): δ
5.2, 19.8, 39.5, 43.6, 50.4, 60.3, 66.0 ppm.
an acidic cation exchange resin. This mixture was filtered and poured
into cold diethyl ether; the precipitate was dried at 40 °C for 2 days to
obtain the hypergrafted PEO-hb-PG copolymer with a yield of 1.88 g
(65%). From the yield we calculated the weight of PG in PEO-hb-PG
to be 1.026 g (13.85 mmol) by using the following equation: mass of
PG in PEO-hb-PG = (mass of PEO-hb-PG) − (mass of used PEO-b-
PG) = 1.88 − 0.854 g = 1.026 g. The numbers of OH group in PEO-b-
PG was determined to be 6.788 mmol, which was calculated from the
3
6
Synthesis of PEO-b-PG. The linear block copolymer was prepared
28
by the modified method in literature. Briefly, 1.001 g (0.5826 mmol)
of PEO (monomethyl ether, M = 1720, determined by GPC) and
0
n
.072 g (0.429 mmol) of cesium hydroxide monohydrate were placed
in 4 mL of benzene under nitrogen. The heterogeneous solution was
stirred at 60 °C for 30 min, and the solvents were removed by a rotary
evaporator. The opaque viscous liquid was heated at 90 °C, and dried
under reduced pressure for 1 h. After nitrogen was introduced into this
flask, 1 mL of dry 1,4-dioxane was added. Then, 0.980 g (6.70 mmol)
of EEGE was added dropwise using a syringe in one portion, and the
temperature was raised to 90 °C for 2 days. The reaction mixture was
cooled to 50 °C, and the polymerization was terminated by the
addition of 10 mL of methanol containing an acidic ion-exchange resin
equation: 0.854 g (weight of PEO-b-PG)/3900 (M of PEO-b-PG) ×
n
1
00 × (29.5 + 1) (repeating unit of PG + terminal OH). Therefore,
the M of the final polymer can be estimated to be 8580, from 3900
n
(
M of PEO-b-PG) + 13.85 mmol/0.2190 mmol × 74.08 (number of
n
PG unit per prepolymer × Mw of (glycidol)), which shows good
agreement with the GPC data of 9300. Finally, we determined the
number of OH group per polymer chain to be 29.5 + 63.2 + 1 = 93.
Conjugation of Doxorubicin to PEO-hb-PG. PEO-hb-PG
copolymer (100 mg, 1.09 mmol based on the number of OH groups)
was dissolved in 5 mL of DMF and triethylamine (0.160 mL, 1.15
mmol) was added in the solution. The copolymer solution was
dropped into p-NPC (11.5 mmol, 2.32 g, 10 equiv) solution in 20 mL
of DMF for 1 h, and reacted for additional 24 h with stirring. After
methanol addition to remove unreacted p-NPC, the solvent was
removed by a rotary evaporator. The product was then precipitated in
ether and dried. The p-NPC conjugated PEO-hb-PG (100 mg, 1.09
mmol) dissolved in 10 mL of DMF was added slowly into hydrazine
monohydrate solution (11.5 mmol, 3.6 mL, 10 equiv) in 5 mL of DMF
and stirred overnight. After DMF was removed in vacuo, the product
was dissolved in deionized water, followed by dialyzing against water.
After removal of deionized water, the residue and 10 mg (0.017 mmol)
doxorubicin (DOX) were dissolved in 10 mL of DMF. The mixture
(
ca. 9 g). This mixture was filtered and poured into cold diethyl ether
to yield the acetal-protected block copolymer. HCl solution (1.0 M, ca.
00 mL) in methanol was added dropwise into 4 mL of polymer
1
solution in ethanol (ca. 20 wt %), and the mixture was stirred for 1 h.
Then, potassium carbonate was carefully added to neutralize the
solution (pH was checked with pH test paper). Filtration and
precipitation in diethyl ether yielded the pure PEO-b-PG block
copolymer which was dried in vacuo at 80 °C for 2 days with a yield of
0
.888 g (59%). The block efficiency (BE) of PEO-b-PG is calculated
using the following equation: BE = reacted PEO/used PEO = (0.888
0.497 g)/1.001 g = 0.39. In addition, the M of the block copolymer
−
n
PEO-b-PG can be calculated to be 3900 g/mol, obtained from the
following equation: M (PEO) + M (PG) = 1720 + EEGE/reacted
n
n
PEO (in mol) × M (glycidol) = 1720 + 6.70/(0.391 g/1720 g/mol ×
1
w
000) × 74.08.
Synthesis of PEO-hb-PG. The hypergrafting of the linear block
2
9
2
8
was kept stirring for 48 h at room temperature.
copolymer PEO-b-PG was performed by using the Frey’s method.
Preparation of PEO-hb-PG-DOX Micelles. DOX-loaded PEO-
hb-PG micelles (PEO-hb-PG-DOX) were prepared by dialysis against
deionized water pH-adjusted to 9 for 12 h, to remove any residual
solvent and unreacted DOX.
Doxorubicin Release. A 4 mL solution of PEO-hb-PG-DOX
micelles was placed inside a dialysis membrane of molecular weight
cutoff (3500−5000 Da) and dialyzed against either pH 5.0 or pH 7.4
phosphate buffer solution (solution mixture of appropriate volume of
The linear macroinitiator PEO-b-PG (0.854 g, 6.45 mmol as OH
function) was placed in a two-neck flask and dissolved in benzene (3.5
mL, ca. 20 wt %). Cesium hydroxide monohydrate (0.270 g, 1.61
mmol) was added to achieve deprotonation of 25% of the hydroxyl
groups along the backbone. The heterogeneous solution was stirred at
6
0 °C for 30 min, and the solvents were removed by a rotary
evaporator. The opaque viscous liquid was heated at 90 °C and dried
under reduced pressure for 1 h. After nitrogen was introduced into this
flask, dry 3.5 mL of diglyme was added (ca. 20 wt %). The flask was
placed in an ultrasonic bath for 30 min, and the mixture was heated to
10 mM KH PO and K HPO ). The micelles solution inside the
2 4 2 4
membrane was collected at predetermined time points and the amount
of the released doxorubicin was measured using HPLC with a standard
calibration curve constructed using a UV/vis detector at a wavelength
of 480 nm.
9
0 °C. Then, glycidol (2.062 g, 27.83 mmol) in 8.5 mL of diglyme was
slowly added with a syringe over a period of approximately 24 h. The
reaction was terminated by the addition of excess methanol containing
1
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In Vitro Cell Cytotoxicity. Cell viability was assessed by the
stage of polymerization. We verified the above assumption by
30
standard MTT assay with slight modifications. Briefly, cells were
NMR of the soluble product in cold ether. Again, the M of the
n
4
grown in 96 well plates at a density of 2 × 10 cells per well. After the
resulting hypergrafting copolymer PEO -hb-PG was measured
39
z
treatment of the micelles as indicated concentrations, thiazolyl blue
tetrazolium bromide (MTT, Sigma-Aldrich) was added to each well of
cells (final conc. 0.5 mg/mL) and incubated for 1.5 h at 37 °C in a
by GPC to be 9300 g/mol (M /M = 1.42), which shows good
w
n
agreement with that from the calculated value 8580 g/mol. The
repeated number of z was determined to be 93. Therefore, the
number of OH function per a polymer chain can be calculated
to be 93.
humidified atmosphere of 95% air/5% CO . A solution of 0.08 N HCl
2
in 2-isopropanol was added to solubilize the blue MTT-formazan
product and the sample was incubated for further 30 min at room
temperature. Absorbance of the solution was read at a test wavelength
of 550 nm. Half maximal inhibitory concentration (IC ) values were
The conjugation of DOX to the prepared hyperbranched
polymer to afford PEO-hb-PG-DOX was achieved by a
50
determined using a sigmoidal dose−response model from GraphPad
Prism v. 4.0 (GraphPad Software, Inc.).
29
procedure similarly described in a literature. After the
preparation of the PEO-hb-PG copolymer, the multiple
hydroxyl groups on the PG segment were further modified
with DOX via an acid-labile hydrazone linkage. The hydroxyl
groups were first modified using p-NPC mediated hydroxyl
amine coupling reaction followed by an amine-ketone reaction
with DOX to provide a pH-responsive feature of PEO-hb-PG-
DOX as shown in Scheme 3. The chemical structure of PEO-
RESULTS AND DISCUSSION
Hyperbranched PEO-hb-PG copolymers were prepared accord-
ing to the modified Frey’s method (Scheme 2). Initially, the M
■
n
of the commercially obtained PEO monomethyl ether was
determined by GPC in DMF calibrated with polystyrene
standard samples, and NMR analysis in deuteriated chloroform.
The M , polydispersity (M /M ), and the degree of polymer-
1
hb-PG-DOX was characterized with H NMR spectra to
n
w
n
confirm the presence of the aromatic DOX (See Supporting
Information). Moreover, the degree of conjugation was
determined to be around 2.1%, on the basis of HPLC
measurement.
Once the conjugation of DOX was confirmed, the PEO-hb-
PG-DOX was dialyzed against the pH-adjusted deionized water
to form the micellar structures that consist of the core of
hydrophobically modified PG-DOX and hydrophilic PEO shell,
which can stabilize the resulting micellar structures. The size of
micelles measured by dynamic light scattering (DLS) was 183.3
ization determined by GPC were 1720 g/mol, 1.20, and 39,
respectively, and these results agreed well with those obtained
by NMR analysis. The hydroxyl functional group at the
terminal of PEO was transformed into the macroinitiator of
PEO-OCs for the polymerization of ethoxyethyl glycidol ether
(
EEGE) by dehydration with cesium hydroxide. After the
removal of unreacted PEO by precipitation in cold diethyl
ether, the acetal group was cleaved by HCl. After block
copolymerization, we found no EEGE was remained in the final
mixture by NMR, suggesting the presence of unreacted PEO,
because the yield of the purified block copolymer was not
quantitative (59%). Thus, the block efficiency was calculated to
be 0.39 (see Experimental Section for details). The relatively
low block efficiency might be attributed to the heterogeneous
±
6.03 nm, confirming the formation of the micelles after to
conjugation of DOX, while that of unmodified plain polymer
PEO-hb-PG) was 4.91 ± 1.96 nm (Figure 1). This significant
(
difference indicates the successful formation of a core−shell
type micellar nanostructure on conjugation with hydrophobic
DOX, while plain double hydrophilic block copolymer of PEO-
hb-PG does not induce any micelle formation. This feature is
important because after the delivery of the drug to a site from
micelles of PEO-hb-PG-DOX, the structures can potentially
disassemble to all-biocompatible, double hydrophilic block
copolymer of PEO-hb-PG, which can be readily cleared and/or
biodegraded. According to the study by Brooks and co-workers,
the hydrophobically modified PEO containing PG could
degrade particularly well under acidic conditions of an
macroinitiator in 1,4-dioxane solution. The M of the resulting
n
block copolymer PEO -b-PG was measured by GPC to be
x
y
3
685 g/mol (M /M = 1.19) (Table 1), which showed a good
w n
Table 1. Characterization Data for Polymers Used in This
Study
a
b)
Mn
c)
b)
run
polymer
yield (%)
Mn(calc)
M /Mn
w
1
2
3
PEO39
1720
3685
9300
1.20
1.19
1.25
PEO -b-PG
30
59
65
3900
8580
39
23
PEO -hb-PG
39
93
intracellular environment. It should also be noted that the
hyperbranched morphology of PG has a great potential for
a
b)
Commercially available PEO of nominal M of 2000 (Aldrich).
n
c)
31
Determined by GPC (CHCl , PS standards). Calculated values from
3
developing a promising system for enhanced drug loading.
the yield of the polymer (see Experimental Section for details).
The morphology of micelles was observed by transmission
electron microscopy (TEM) and atomic force microscopy
agreement with the calculated value of 3900 g/mol (see
Experimental Section for details). Thus, the block efficiency
was calculated to be 0.39. The repeating numbers of each block
were determined to be 39 for PEO and 30 for PG.
(
AFM) (Figure 2). Figure 2a showed the spherical micelles
consisting of a dark core (average diameter of 240 nm) and a
relatively brighter shell layer (average thickness of 70 nm) in an
aqueous solution. The overall average diameter of 310 nm was
relatively larger than that obtained from the DLS measurement,
which could be attributed to the flattening effect induced
during sample preparation. In addition, AFM image in Figure
The hypergrafting of linear PEO -b-PG with glycidol was
3
9
30
then performed. About 4.1 equiv of glycidol to OH function on
the linear macroinitiator was hypergrafted in solution. As a
result, the hypergrafted copolymer was obtained in 65% yield.
Imperfect yield might be attributed to the presence of glycidol
homopolymer, which can be removed by precipitation in cold
diethyl ether. We assumed that the PEO-hb-PG was
quantitatively collected during the process of repeated
precipitation, and therefore, the nonquantitative yield should
come from the formation of homo-PG during the hypergrafting
2
b indicates the hydrophobic core is surrounded by hydrophilic
shell. The core/shell morphology of the PEO-hb-PG-DOX
micelle is also promising for cancer therapy, since the
biocompatible PEO shell can provide a stealth property with
prolonged circulation life while the stabilized internal core can
effectively prevent early burst release of encapsulated
therapeutics from the micelle during circulation.
1
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Scheme 3. Synthetic Approach for the Formation of Doxorubicin Conjugated PEO-hb-PG-DOX
Figure 1. Dynamic light scattering (DLS) analysis of free PEO-hb-PG
and PEO-hb-PG-DOX micelles.
To verify the acid-labile cleavage of the hydrazone bond, the
release of DOX from the micelles was studied at pH 5.0 and 7.4
conditions (Figure 3). As expected, PEO-hb-PG-DOX micelles
exhibited the pH-responsive DOX release profile due to the
hydrazone bond linkage between DOX and polymer backbone.
The PEO-hb-PG-DOX micelles released 57.7% of DOX within
3
h and liberated 71% after 48 h. In contrast, the amount of
released DOX was 53.6% after 48 h in the case of samples
subjected to pH 7.4. As suggested in other reports, the
accelerated release of DOX under acid conditions is desirable
for an effective cancer therapy, because intracellular endosomal
pH within the tumor cells is considerably lower than that of the
Figure 2. Representative (a, b) TEM and (c) AFM images of PEO-hb-
PG-DOX micelles. Inset shows the line scan of one of the micelles
with an average diameter of 235 nm, which is corresponding to the
results from DLS.
3
2
normal tissue.
In vitro efficacy of PEO-hb-PG-DOX micelles was
investigated by cell viability assay using a human cervical
cancer HeLa cell line (Figure 4). A modified MTT assay was
performed by subjecting release aliquots to HeLa cells to
ascertain that DOX retained its activity. As shown in Figure 4,
PEO-hb-PG-DOX micelles induced significant cytotoxicity in
HeLa cells, while virtually no toxicity was observed from PEO-
hb-PG treated cells at the entire concentration ranges tested.
The IC50 value of PEO-hb-PG-DOX was estimated to be
around 6.9 μM of polymer concentration. In addition, it is
evident that the plain double hydrophilic copolymer of PEO-
hb-PG is highly biocompatible and nontoxic, confirming the
desired feature of our system.
The cellular uptake efficiency of PEO-hb-PG-DOX micelles
was further studied by confocal laser fluorescence microscopy.
HeLa cells were also incubated with the same dose of plain
PEO-hb-PG polymers for 4 h as a control. Figure 5 shows that
the clear red fluorescence of DOX appears in the cells treated
DOX-loaded micelles, suggesting the successful internalization
1
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free DOX, because of the rapid transport of intracellular DOX
from the cytosol to the nucleus and avid binding of DOX to
chromosomal DNA. In contrast, it appears that the PEO-hb-
PG-DOX micelles were initially located within the endosomal
vesicles, releasing cleaved DOX in a controlled manner.
CONCLUSION
■
In summary, we designed and synthesized a new class of
hyperbranched double hydrophilic block copolymer of poly-
(
ethylene oxide)-hyperbranched-polyglycerol (PEO-hb-PG)
with an aim to enhance biocompatibility, increase water
solubility, and improve the clearance of the polymer after
delivery of the drug. The model anticancer drug, doxorubicin
(
DOX), was conjugated to PEO-hb-PG via an acid-labile
hydrazone linkage. The doxorubicin-conjugated copolymer of
PEO-hb-PG-DOX underwent a spontaneous self-assembly
process in aqueous solution to create core−shell type micellar
aggregates. Upon exposure to an acidic condition, the PEO-hb-
PG-DOX micelles released DOX to a greater extent through
the cleavage of acid-labile hydrazone bonds. The cytotoxicity of
the PEO-hb-PG-DOX micelles was also evaluated with HeLa
cells, which showed a marked increase of efficacy with the
concentration, whereas the plain PEO-hb-PG polymer does not
show any noticeable cytotoxicity, confirming the highly
biocompatible and nontoxic features of double hydrophilic
block copolymer. We believe that this double hydrophilic block
copolymer will provide a platform not only to improve the
solubility of the drugs in aqueous solution, but also to enhance
the targeting capacity to the solid tumor.
Figure 3. Release profiles of doxorubicin from PEO-hb-PG-DOX
micelles under different pH conditions. (open circle) pH 5.0 and
(
open triangle) pH 7.4. The release profile was normalized to the
amount of doxorubicin initially loaded into the micelle core.
ASSOCIATED CONTENT
■
*
S
Supporting Information
Additional H and 13C NMR spectra of the polymers are
presented. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
Figure 4. MTT-based cell viability assay of (blue square) plain PEO-
hb-PG and (red triangle) PEO-hb-PG-DOX micelles.
of micelles within the cells. Interestingly, more DOX was
accumulated than in the cytosol. According to other report,
DOX accumulated only in the nuclei of HeLa cells treated with
AUTHOR INFORMATION
■
Corresponding Author
E-mail: bskim19@unist.ac.kr.
*
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study is supported by a grant of the Korea Healthcare
Technology R&D Project, Ministry of Health and Welfare,
Republic of Korea (A091047), National Research Foundation
of Korea Grant (NRF-2010-000-D00094), the Basic Science
Research Program (2011-0007990), and by a grant of the Japan
Society for the Promotion of Science (JSPS) Joint Research
Projects in 2010.
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