Tumor-targeting, pH-responsive, and stable unimolecular micelles as drug nanocarriers for targeted cancer therapy

Article abstract of DOI:10.1021/bc900422j

A new type of multifunctional unimolecular micelle drug nanocarrier based on amphiphilic hyperbranched block copolymer for targeted cancer therapy was developed. The core of the unimolecular micelle was a hyperbranched aliphatic polyester, Boltorn H40. The inner hydrophobic layer was composed of random copolymer of poly(εcaprolactone) and poly(malic acid) (PMA-co-PCL) segments, while the outer hydrophilic shell was composed of poly(ethylene glycol) (PEG) segments. Active tumor-targeting ligands, i.e., folate (FA), were selectively conjugated to the distal ends of the PEG segments. An anticancer drug, i.e., doxorubicin. (DOX) molecules, was conjugated onto the PMA segments with pH-sensitive drug binding linkers for pH-triggered drug release. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) analysis showed that the unimolecular micelles were uniform with a mean hydrodynamic diameter around 25 nm. The drug loading content was determined to be 14.2%. The drug release profile, cell uptake and distribution, and cytotoxicity of the unimolecular micelles were evaluated in vitro. The folate-conjugated micelles can be internalized by the cancer cells via folate-receptormediated endocytosis; thus, they exhibited enhanced cell uptake and cytotoxicity. At pH 7.4, the physiological condition of bloodstream, DOX conjugated onto the unimolecular micelles exhibited excellent stability; however, once the micelles were internalized by the cancer cells, the pH-sensitive hydrazone linkages were cleavable by the intracellular acidic environment, which initially caused a rapid release of DOX. These findings indicate that these unique unimolecular micelles may offer a very promising approach, for targeted cancer therapy.

Full text of DOI:10.1021/bc900422j

496
Bioconjugate Chem. 2010, 21, 496–504
Tumor-Targeting, pH-Responsive, and Stable Unimolecular Micelles as
Drug Nanocarriers for Targeted Cancer Therapy
Xiaoqiang Yang,† Jamison J. Grailer,‡ Srikanth Pilla,† Douglas A. Steeber,‡ and Shaoqin Gong*,†,§,|
Department of Mechanical Engineering, Department of Biological Sciences, and Department of Materials, University of
Wisconsin-Milwaukee, Milwaukee, Wisconsin 53211, and Department of Biomedical Engineering and Wisconsin Institute for
Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53706. Received September 23, 2009;
Revised Manuscript Received January 31, 2010
A new type of multifunctional unimolecular micelle drug nanocarrier based on amphiphilic hyperbranched block
copolymer for targeted cancer therapy was developed. The core of the unimolecular micelle was a hyperbranched
aliphatic polyester, Boltorn H40. The inner hydrophobic layer was composed of random copolymer of poly(ε-
caprolactone) and poly(malic acid) (PMA-co-PCL) segments, while the outer hydrophilic shell was composed of
poly(ethylene glycol) (PEG) segments. Active tumor-targeting ligands, i.e., folate (FA), were selectively conjugated
to the distal ends of the PEG segments. An anticancer drug, i.e., doxorubicin (DOX) molecules, was conjugated
onto the PMA segments with pH-sensitive drug binding linkers for pH-triggered drug release. Transmission electron
microscopy (TEM) and dynamic light scattering (DLS) analysis showed that the unimolecular micelles were
uniform with a mean hydrodynamic diameter around 25 nm. The drug loading content was determined to be
14.2%. The drug release profile, cell uptake and distribution, and cytotoxicity of the unimolecular micelles were
evaluated in Vitro. The folate-conjugated micelles can be internalized by the cancer cells Via folate-receptor-
mediated endocytosis; thus, they exhibited enhanced cell uptake and cytotoxicity. At pH 7.4, the physiological
condition of bloodstream, DOX conjugated onto the unimolecular micelles exhibited excellent stability; however,
once the micelles were internalized by the cancer cells, the pH-sensitive hydrazone linkages were cleavable by
the intracellular acidic environment, which initially caused a rapid release of DOX. These findings indicate that
these unique unimolecular micelles may offer a very promising approach for targeted cancer therapy.
The in ViVo instability problem associated with multimolecular
micelles can be overcome by developing unimolecular micelles
based on amphiphilic copolymers with a dendritic or hyper-
branched structure (6, 7). Unimolecular micelles do not disas-
semble upon dilution and are stable to environmental changes
due to their covalent nature, thereby providing excellent in ViVo
stability. Unimolecular micelles also offer other advantages,
including high drug loading capacity and narrow nanoparticle
size distribution. Furthermore, the highly branched structure of
the copolymers can provide many end groups for further
functionalization (8, 9).
To improve the efficacy of cancer therapy using drug
nanocarriers such as unimolecular micelles, the unimolecular
micelles must exhibit a number of desirable properties, including
in ViVo stability, high drug loading capacity, and well-controlled
nanoparticle sizes, as described previously. In addition, the
ability to actively target the tumor cells Via specific targeting
ligands and the ability for controlled drug release Via stimulus-
responsive drug-copolymer linkers are essential.
Drug nanocarriers are desirable for cancer therapy because
they can target the tumor tissue through the enhanced perme-
ability and retention (EPR) effect (i.e., passive targeting) (10).
To further improve the delivery efficiency and cancer specificity,
active tumor-targeting ability is also required. Active targeting
can be achieved by functionalizing the nanocarriers with
targeting ligands such as small molecules (e.g., folate (FA)),
peptides (e.g., cRGD) and antibodies, which can recognize and
bind to specific receptors/integrins that are unique to or
positively expressed on cancer cells (11). In terms of controlled
drug release, ideally, anticancer drug should not be released
during circulation in the bloodstream; however, once the drug
nanocarriers are internalized by the cancer cells, a sufficient
INTRODUCTION
Micelles formed by self-assembly of amphiphilic block
copolymers have attracted significant attention in various
biomedical fields, including drug and gene delivery (1–4).
Micelle drug delivery systems can improve the solubility of
hydrophobic drugs, stabilize and protect drugs that are sensitive
to the surrounding environment, reduce the nonspecific uptake
by the reticuloendothelial system (RES), and facilitate targeted
drug delivery; however, conventional multimolecuclar micelle
drug delivery systems based on the linear amphiphilic block
copolymers suffer from instability in ViVo. Multimolecular
micelles are only thermodynamically stable above the critical
micelle concentration (cmc) of the amphiphilic copolymers.
Above the cmc, micelles are in dynamic equilibrium with the
free copolymer molecules in solution, continuously breaking
and reforming (5). When the concentration of the copolymer is
below the cmc, micelles disassemble, thereby releasing the drug
prematurely before reaching the tumor target. Furthermore, there
are a number of other factors that may affect the in ViVo stability
of the multimolecular micelles including flow stress, interactions
between the micelles and the serum components, and variations
in temperature, pH, and ionic strength.
* To whom correspondence should be addressed. University of
Wisconsin-Madison, Madison, WI 53706, USA; Tel: +1-(608) 890-
3463, E-mail: sgong@engr.wisc.edu.
† Department of Mechanical Engineering, University of Wisconsin-
Milwaukee.
‡ Department of Biological Sciences, University of Wisconsin-
Milwaukee.
§ Department of Materials, University of Wisconsin-Milwaukee.
| University of Wisconsin-Madison.
10.1021/bc900422j  2010 American Chemical Society
Published on Web 02/17/2010

Unimolecular Micelles as Drug Nanocarriers
Bioconjugate Chem., Vol. 21, No. 3, 2010 497
Scheme 1. Schematic Structure of Unimolecular Micelle Based
on H40-(poly(ꢀ-malic acid)-hydrazone-doxorubicin-co-
poly(ε-caprolactone))-methoxy-poly(ethylene
Scheme 2. Synthesis Procedure of Benzyl Malolactonate
Monomer
glycol)-poly(ethylene glycol)-folate
(H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA) Polymer
biocompatibility and low immunogenicity (19). The anticancer
drug, doxorubicin (DOX), is widely used as an antitumor
therapeutic agent and was conjugated onto the PMA-co-PCL
segments through an acid-sensitive hydrazone bond. The random
copolymer of PMA and PCL allows the DOX molecules to
effectively conjugate onto the PMA segments due to the much
reduced steric hindrance from the neighboring MA unit in the
random copolymer. The acid-cleavable hydrazone linkages
reduce the unwanted DOX release during blood circulation, yet
provide quick DOX release initially once inside the tumor cells.
This tumor-targeting unimolecular micelle nanosystem with a
pH-triggered drug release profile and potentially excellent in
ViVo stability will open many exciting opportunities for targeted
cancer therapy.
amount of anticancer drug should be released relatively quickly
initially in order to effectively kill the cancer cells. The pH value
of the bloodstream is approximately 7.4, while that of the
endocytic compartments of the cells generally ranges from 4.5
to 6.5. This difference in pH value makes pH-triggered drug
release possible (12).
EXPERIMENTAL PROCEDURES
Boltorn H40 has received much attention in the design of
amphiphilic hyperbranched block copolymers for drug delivery
application because of its biodegradability, biocompatibility,
globular architecture, and high number of chain end functional-
ities (13, 14). Several excellent reports on drug nanocarriers
using the hyperbranched aliphatic polyester, Boltorn H40 as the
core are available in the literature. For instance, Prabaharan et
al. reported a unimolecular micelle system made of a hyper-
branched amphiphilic block copolymer based on Bolton H40
and copolymer of polylactide (PLA) and poly(ethylene glycol)
(PEG) using FA as the active tumor-targeting ligands (15). Chen
et al. reported a drug nanocarrier based on Bolton H40 and
copolymer of polycaprolactone (PCL) and PEG, also using FA
as the tumor-targeting ligand (16); however, in these systems,
the anticancer drug, doxrubincin (DOX), was physically en-
capsulated only in the drug nanocarriers Via hydrophobic
interaction. As such, a significant amount of DOX may be
released in the bloodstream during circulation, leading to poor
targeting and reduced therapeutic effect.
In this paper, we developed a new type of multifunctional
unimolecular micelle based on Boltron H40 as drug nanocarrier
for targeted cancer therapy, which exhibited a pH-triggered drug
release profile (Scheme 1). These unimolecular micelles were
formed by amphiphilic hyperbranched copolymer, H40-((poly(ꢀ-
malic acid)-hydrazone-doxorubicin)-co-poly(ε-caprolactone))-
methoxy-poly(ethylene glycol)/poly(ethylene glycol)-folate (H40-
(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA) in aqueous solution.
As shown in Scheme 1, Bolton H40 was used as the core of
the amphiphilic hyperbranched copolymer. The hydrophobic
segments of the amphiphilic copolymer arms were formed by
random copolymerization of poly(ꢀ-malic acid) (PMA) and
poly(ε-caprolactone) (PCL). The hydrophilic segments of the
amphiphilic copolymer arms were formed by methoxy poly-
(ethylene glycol) (MPEG) or FA conjugated PEG (MPEG/PEG-
FA). FA was used as the active tumor-targeting agent. FA is a
vitamin with low molecular weight (Mw ) 441.4) and behaves
as a ligand because it has high affinity for its receptor, folate-
binding protein, which is selectively overexpressed on the
surface of many types of human cancer cells (17, 18). The PMA
and PCL are important biodegradable aliphatic polyesters and
of interest for biomedical applications owing to their good
Materials. L-Aspartic acid, sodium bromide, sodium nitrite,
trifluoroacetic anhydride, benzyl alcohol, and stannous octanoate
(Sn(Oct)2) were purchased from Fisher Scientific Inc., USA.
Boltron H40 (64 hydroxyl groups per molecule theoretically
and Mn of 2830 Da) was provided by Perstorp Polyols Inc.,
USA, and purified with tetrahydrofuran (THF). ε-Caprolactone
(ε-CL, from Sigma-Aldrich) was purified by vacuum distillation
over calcium hydride (CaH2). Folic acid, dicyclohexylcarbodi-
imide (DCC), 4-dimethylamino pyridine (DMAP), and anhy-
drous hydrazine were purchased from Sigma-Aldrich, USA.
MPEG was obtained from Fluka. R-Hydroxy-ω-amino poly-
(ethylene glycol) (HO-PEG-NH2) with a Mw of 2000 was
purchased from Iris Biotech GmbH and methoxy poly(ethylene
glycol) (MPEG, Mw: 2000) was purchased from Fisher Scientific
Inc., USA. Doxorubicin hydrochloride (DOX ·HCl) was supplied
by Tecoland Corporation, USA, and used as supplied. All other
chemicals used were of analytical reagent grade. Phosphate and
acetate buffered solutions were prepared in our laboratory.
RPMI-1640 was purchased from Gibco BRL, USA. The mouse
mammary carcinoma, 4T1 (ATCC), was cultured in RPMI
medium supplemented with 10% fetal calf serum.
Synthesis of Benzyl Malolactonate Monomer (Scheme
2). Benzyl malolactonate monomer was prepared by a reported
simple and reproducible method following Scheme 2 (20). First,
to an ice-cold clear solution of L-aspartic acid (1, 50 g) and NaBr
(200 g) in 2 N H2SO4 (300 mL), NaNO2 was added progressively
over a period of 2 h. The whole mixture was further stirred for
1.5 h at room temperature. After the excess reagents were
decomposed by adding urea, the bromosuccinic acid was extracted
with ethyl acetate twice. The extracts were combined and washed
with slightly acidic water and dried over anhydrous sodium sulfate.
The product 2 was recovered after evaporation of ethyl acetate.
Second, the product 2 (20 g) was dissolved in anhydrous tetrahy-
drofuran (THF) (100 mL) containing trifluoroactetic anhydride
(23.5 g) in a three-neck flask at 10 °C. The mixture was allowed
to stir for 1.5 h at 10 °C and 2 h at room temperature. The THF
and trifluoroacetic acid were then distillated under vacuum to yield
bromosuccinic anhydride (3), a white crystalline compound. Then,
benzyl alcohol (13 g) was immediately added to the flask containing
3.

498 Bioconjugate Chem., Vol. 21, No. 3, 2010
Yang et al.
Scheme 3. Synthesis Procedure of H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA Polymer
The mixture was allowed to react for 4 h at room temperature
and 1 h at 60 °C yielding a mixture of bromosuccinic acid
monobenzyl ester isomers (4 and 5). Finally, the resulting
mixture was dissolved in sodium hydroxide water with a pH
value of 7.8. Subsequently, 200 mL of benzene was added into
the mixture, which was stirred overnight at 50 °C. After cooling,
the organic layer was separated from the aqueous phase and
crude benzyl malolactonate was recovered after evaporation of
the solvent. The product 6 was purified using column chroma-
tography and further vacuum distilled.
solution was washed three times with aqueous hydrochloric acid
(10% in v/v) and then four times with a saturated NaCl solution.
Then, the organic phase was isolated, dried over magnesium
sulfate, and filtered. The final product, FA-PEG-COOH, was
recovered by precipitation into cold diethyl ether and dried under
vacuum.
MPEG-COOH was prepared from MPEG-OH using a pro-
cedure similar to the one described above.
Synthesis of H40-(PMABz-co-PCL)-MPEG/PEG-FA. H40-
(PMABz-co-PCL)-MPEG/PEG-FA copolymer was synthesized
by reacting the terminal hydroxyl groups of H40-(PMABz-co-
PCL)-OH with the terminal carboxyl groups of anhydrous
MPEG-COOH and FA-PEG-COOH. The molar ratio of MPEG-
COOH and FA-PEG-COOH was 19:1 (the number of PMABz-
co-PCL arms was 20, which was calculated according to the
GPC analysis). For example, H40-(PMABz-co-PCL)-OH (5.52
g, 0.044 mmol) was dissolved in 30 mL anhydrous chloroform.
Then, a mixture of MPEG-COOH (2.51 g, 1.26 mmol) and FA-
PEG-COOH (152 mg, 0.06 mmol), DCC (272 mg, 1.32 mmol)
and DMAP (161 mg, 1.32 mmol) in 20 mL anhydrous
chloroform was added into the chloroform solution of H40-
(PMABz-co-PCL)-OH. The reaction was carried out at room
temperature for 24 h under stirring. The resulting solution was
precipitated with cold diethyl ether twice. The precipitate was
dried under vacuum. The impurities and unreacted materials of
the product were removed by dialysis against deionized water
using cellulose tube (Mw cutoff: 12 000 Da) for 48 h. After
dialysis, the H40-(PMABz-co-PCL)-MPEG/PEG-FA copolymer
was obtained by freeze-drying.
Synthesis of H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-
FA. The benzyl groups of H40-(PMABz-co-PCL)-MPEG/PEG-
FA were substituted with hydrazide groups for DOX binding
by an ester-amide exchange aminolysis reaction (Scheme 3).
H40-(PMABz-co-PCL)-MPEG/PEG-FA (2 g, 0.012 mmol) was
dissolved in 100 mL of anhydrous DMF. Then, anhydrous
hydrazine (5 mg, 0.156 mmol) was added to this solution. The
reaction was allowed to proceed at 40 °C for 24 h, followed by
dialysis against 0.25% ammonia solution using cellulose tube
Random Copolymerization of Benzyl Malolactonate and
ε-Caprolactone Using H40 as Macroinitiator (H40-(PMABz-
co-PCL)-OH). H40-(PMABz-co-PCL)-OH was prepared by the
ring-opening polymerization of benzyl malolactonate and ε-ca-
prolactone using H40 as a macroinitiator and Sn(Oct)2 as a
catalyst (Scheme 3). H40 (200 mg, 4.5 mmol of hydroxyl
groups), benzyl malolactonate (0.5 g 2.43 mmol), ε-caprolactone
(3.0 g 26.3 mmol), and catalyst ([catalyst]/[ε-caprolactone] )
1:1000) were added into a 250 mL flame-dried flask under N2.
The reaction was carried out under stirring at 110 °C for 24 h.
After the reaction was complete, the mixture was diluted with
THF, precipitated into diethyl ether, and washed with diethyl
ether to produce a white H40-(PMABz-co-PCL)-OH powder.
Synthesis of r-Carboxyl-ω-FA Poly(ethylene glycol) (FA-
PEG-COOH). FA-PEG-HO was prepared according to our
previous work with a slight modification (15). The γ-NHS-FA
was dissolved into 5 mL of a mixture of anhydrous DMSO and
triethylamine (TEA) with a volume ratio of 10:1. An equimolar
amount of HO-PEG-NH2 was added to the mixture and the
reaction was carried out under an anhydrous condition overnight.
The product, FA-PEG-HO, was freeze-dried after dialysis
against deionized water using cellulose tubing (Mw cutoff: 2000
Da). FA-PEG-COOH was prepared by reacting FA-PEG-OH
(2 g, 0.57 mmol) with succinic anhydride (170 mg, 1.7 mmol)
in the presence of pyridine. The reaction was carried out in
anhydrous toluene under N2 atmosphere at 70 °C for 24 h. Crude
polymer obtained by precipitation into cold diethyl ether was
redissolved in dichloromethane (DCM). The resulting DCM

Unimolecular Micelles as Drug Nanocarriers
Bioconjugate Chem., Vol. 21, No. 3, 2010 499
1
Figure 1. H NMR spectrum of benzyl malolactonate monomer.
(Mw cutoff: 2000 Da) and freeze-drying. To conjugate DOX
onto the amphiphilic hyperbranched copolymer, H40-(PMA-
Hyd-co-PCL)-MPEG/PEG-FA (1.5 g, 0.009 mmol) was dis-
solved in 50 mL of DMF, and excess amount of DOX ·HCl
(150 mg, 0.26 mmol) was added. The mixture solution was
stirred at room temperature for 24 h while being protected from
light. The reaction solution was concentrated to a volume of
10 mL under reduced pressure and dialyzed against deionized
water using cellulose tube (Mw cutoff: 2000 Da) for 48 h,
followed by gel purification using Sephadex LH-20 to com-
pletely remove unbound DOX. For comparison, the H40-(PMA-
Hyd-DOX-co-PCL)-MPEG copolymer without FA targeting
ligands was synthesized following the same procedure as
described above.
Evaluation of pH Sensitivity. The unimolecular micelle’s
pH-responsive DOX release behavior was studied using an UV
spectrophotometer. First, 50 mg freeze-dried micelle samples
were dispersed in 5 mL of medium, placed in a dialysis bag
with a molecular weight cutoff of 2000 Da, and then placed
into various pH conditions from 7.4 to 4.0 (10 mM phosphate
buffer (pH 7.4-6.0), 10 mM acetate buffer (pH 5.8-4.0)). The
dialysis bag was then immersed in 100 mL of the release
medium and kept in a horizontal laboratory shaker maintaining
a constant temperature and stirring. At selected time intervals,
the buffered solution (5 mL) outside the dialysis bag was
removed for UV-vis analysis and replaced by fresh buffered
solution with same volume. The amount of released DOX was
analyzed with UV spectrophotometer at 485 nm. Each sample
was measured three times.
Cellular Uptake and Cytotoxicity. The cellular uptake and
distribution of micelles were performed using flow cytometry
and confocal laser scanning microscopy (CLSM). For CLSM
studies, 4T1 cells (1 × 106) were seeded onto 22 mm round
glass coverslips placed in 6-well plates and grown overnight.
Cells were treated with free DOX, DOX-conjugated H40-(PMA-
Hyd-DOX-co-PCL)-MPEG/PEG-FA (i.e., FA-targeted), or H40-
(PMA-Hyd-DOX-co-PCL)-MPEG (i.e., nontargeted) micelles
for 2 h (DOX concentration 20 µg/mL). Then the cells were
washed and fixed with 1.5% formaldehyde. Coverslips were
placed onto glass microscope slides and DOX uptake was
analyzed using a Leica TCS SP2 Confocal System installed on
an upright compound microscope (Leica, Wetzler, Germany).
Digital monochromatic images were acquired using Leica
Confocal Software (v 2.61).
To determine the DOX distribution in the nucleus and
cytoplasm of the 4T1 cells, the DOX mean fluorescence intensity
(MFI) in the CLSM images was measured in a 4 µm2 area
located in the nucleus or cytoplasm (n ) 10 cells) for each
sample using ImageJ software (http://rsb.info.nih.gov/ij). For
flow cytometry, 4T1 cells (1 × 106) were seeded in 6-well
culture plates and grown overnight. The cells were then treated
with free DOX, DOX-conjugated H40-(PMA-Hyd-DOX-co-
PCL)-MPEG/PEG-FA, or H40-(PMA-Hyd-DOX-co-PCL)-
MPEG micelles for 2 h (DOX concentration 20 µg/mL).
Thereafter, the cells were lifted using a cell stripper (Media
Tech, Inc.) and washed. The DOX uptake was analyzed using
an FACSCalibur flow cytometer (BD bioscience). A minimum
of 2 × 104 cells were analyzed from each sample with the
fluorescence intensity shown on a four-decade log scale.
The cytotoxicity of the unimolecular micelles against 4T1
cells was studied using the MTT assay. First, 4T1 cells (1 ×
104) were seeded in 96-well plates and incubated for 24 h. The
media were replaced with fresh media containing free DOX,
H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA, H40-(PMA-
Hyd-DOX-co-PCL)-MPEG micelles with different DOX con-
centrations or control media and incubated for 48 h. Thereafter,
the wells were washed three times with warm phosphate buffer
Preparation of H40-(PMA-Hyd-DOX-co-PCL)-MPEG/
PEG-FA Micelles. H40-(PMA-Hyd-DOX-co-PCL)-MPEG/
PEG-FA micelles were prepared using the dialysis method.
Briefly, 10 mg of H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-
FA polymer was dissolved in 3 mL of DMF under stirring. Then,
5 mL of deionized water was added dropwise into the above
solution. Thereafter, the mixture was dialyzed against deionized
water using a dialysis tube (Mw cutoff: 2000 Da) for three days
followed by freeze-drying.
1
Characterization. H NMR spectrum was recorded on a
Bruker DPX 300 spectrometer. Gel permeation chromatography
(GPC) analysis was carried out at ambient temperature using
the 270max Viscotek (Viscotek, USA) GPC system equipped
with triple detectors, including a refractive index detector, a
viscometer detector, and a light-scattering detector. THF was
used as an eluent with a flow rate of 1 mL/min and polystyrene
(PS) standards were used for column calibration. The am-
phiphilic hyperbranched block copolymer solutions in THF (5
mg/mL) were prepared. Absorbance measurements were carried
out using a Varian Cary 100 Bio UV-visible spectrophotometer.
The size and size distribution of the unimolecular micelles were
determined by dynamic light scattering (DLS) (Beckman Coulter
PCS submicrometer particle size analyzer, USA) with an angle
detection at 90°. Samples for transmission electron microscopy
(TEM, Hitachi H-600) analysis were prepared by drying a
dispersion of the unimolecular micelles on a copper grid coated
with amorphous carbon. Subsequently, a small drop of phos-
photungstic acid (PTA) solution (2 wt % in water) was added
to the copper grid; after 30 s the grid was blotted with filter
paper for TEM observation. The DOX-loading content (DLC),
defined as the weight percentage of DOX in unimolecular
micelles, was quantified by UV-vis analysis using a Varian
Cary model 100 Bio UV-vis spectrophotometer. The DOX was
released from the unimolecular micelles in 0.1 N HCl solution
to cleave the hydrazone bonds. To determine the drug content
in the solution, the absorbance of DOX at 485 nm was measured
using a previously established calibration. The DLC measure-
ments were performed in triplicate for each of the samples.

500 Bioconjugate Chem., Vol. 21, No. 3, 2010
Yang et al.
solution and incubated again for another 3 h with RPMI
containing 250 µg/mL of MTT. After discarding the culture
medium, 200 µL of DMSO was added to dissolve the precipi-
tates, and the resulting solution was measured for absorbance
at 570 nm with a reference wavelength of 690 nm using a
microplate reader (Molecular Devices).
RESULTS AND DISCUSSION
Synthesis and Characterization of Benzyl Malolactonate
Monomer and H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-
FA. Benzyl malolactonate is a ꢀ-substituted ꢀ-lactone monomer
and was prepared by a reported simple and reproducible method
according to Scheme 2 (20). This monomer was purified by
reverse-phase silica (octadecylsilyl silica (ODS)) and then
vacuum-distilled twice (bp ) 90 °C, 10-3 mm Hg). Figure 1
1
shows the H NMR spectrum of the monomer, confirming the
monomer was successfully synthesized (a, 4.0 (d); b, 5.12 (t);
c, 5.34 (s); d, 7.35 (s) in CDCl3).
The synthesis of the H40-(PMA-Hyd-DOX-co-PCL)-MPEG/
PEG-FA copolymer was carried out according to Scheme 3.
First, H40-(PMABz-co-PCL)-OH was prepared by the random
copolymerization of benzyl malolactonate and ε-caprolactone
using H40 as a macroinitiator in the presence of Sn(Oct)2
catalyst. This copolymer was purified by twice precipitating into
diethyl ether from chloroform solution followed by filtering and
was then dried under vacuum. The structure of the H40-
1
(PMABz-co-PCL)-OH copolymer was confirmed by H NMR
(Figure 2a). The molar ratio of the PMABz and PCL repeat
unit was approximately 1:15, which was calculated from the
integral ratio of the characteristic peaks at 5.1 (MABz) and 3.9
(CL) ppm). On the basis of the absolute Mw determined by GPC
(Table 1), it was estimated that each PMABz-co-PCL-OH arm
on the hyperbranched H40-(PMABz-co-PCL)-OH copolymer
had 2 MABz repeat units and 29 CL repeat units. To prepare
the nontargeted unimolecular micelles, which can serve as a
control for the study of cellular uptake and cytotoxicity of FA-
targeted unimolecular micelles, the H40-(PMABz-co-PCL)-
MPEG amphiphilic copolymer was synthesized by reacting H40-
(PMABz-co-PCL)-OH with MPEG-COOH in the presence of
DCC and DMAP. The number of arms in H40-(PMABz-co-
PCL)-MPEG was calculated using the following equation:
Number of arms ) [(Mw of H40-(PMABz-co-PCL)-MPEG -
Mw of H40-(PMABz-co-PCL)-OH)]/Mw of MPEG ) [164 540-
125 460/2000] ≈ 20. This result is consistent with our previous
finding (15).
To achieve active tumor-targeting property, the H40-(PMA-
Hyd-DOX-co-PCL)-MPEG/PEG-FA copolymer was prepared
by reacting H40-(PMABz-co-PCL)-OH with a mixture of
MPEG-COOH and FA conjugated PEG, i.e., FA-PEG-COOH,
in the presence of DCC and DMAP. It is known that folate
moieties with pterin heterocycles can self-assemble in an
aqueous solution by hydrogen bonds and stacking interactions
(21). As a result, introducing too many FA moieties to the
amphiphilic copolymer decreases the stability of the micelles
formed by the amphiphilic copolymer. In addition, the possible
formation of dimers, trimers, or self-assembled tubular quarters
(21) at a higher FA concentration is highly unfavorable for tumor
cell targeting because the folate receptor can bind only one FA
molecule; thus, the self-assembled FA multimers are incapable
of binding to the folate receptors.
Figure 2. 1H NMR spectra of (a) H40-[(poly(benzyl ꢀ-malate)-co-
poly(ε-caprolactone)] (H40-(PMABz-co-PCL)-OH; (b) H40-[(poly-
(benzyl ꢀ-malate)-co-poly(ε-caprolactone)]-methoxy-poly(ethylene glycol)-
poly(ethylene glycol)-folate (H40-(PMABz-co-PCL)-MPEG/PEG-FA);
and (c) H40-[(poly(ꢀ-malic acid-hydrazide)-co-poly(ε-caprolactone)]-
methoxy-poly(ethylene glycol)-poly(ethylene glycol)-folate (H40-
(PMA-Hyd-co-PCL)-MPEG/PEG-FA); (d) H40-[(poly(ꢀ-malic acid-
hydrazone-DOX)-co-poly(ε-caprolactone)]-methoxy-poly(ethylene glycol)-
poly(ethylene glycol)-folate (H40-(PMA-Hyd-DOX-co-PCL)-MPEG/
PEG-FA).
During this study, the molar ratio of the FA targeting ligand
was controlled through the molar ratio between MPEG-COOH
and FA-PEG-COOH, which was kept at 19:1. Thus, the
estimated FA molar ratio in H40-(PMABz-co-PCL)-MPEG/
1
PEG-FA was 1/20, i.e., 5%. Figure 2b shows the H NMR
spectrum of the H40-(PMABz-co-PCL)-MPEG/PEG-FA co-
polymer. The typical NMR peaks from the FA moiety appeared

Unimolecular Micelles as Drug Nanocarriers
Bioconjugate Chem., Vol. 21, No. 3, 2010 501
Table 1. GPC Results of H40, H40-(PMABz-co-PCL)-OH, and
H40-(PMABz-co-PCL)-MPEG
samples
Mn
Mw
Mw/Mn
H40
2 830
76 500
115 506
5 100
125 460
164 540
1.80
1.64
1.42
H40-(PMABz-co-PCL-OH)
H40-(PMABz-co-PCL)-MPEG
at 8.68, 8.17, 7.68, 6.98, and 6.67 ppm. The appearance of these
peaks confirms the successful conjugation of FA to the hyper-
branched copolymer.
The benzyl groups of the H40-(PMABz-co-PCL)-MPEG/
PEG-FA copolymer were substituted with hydrazide groups for
DOX binding through the ester-amide exchange (EAE) ami-
nolysis reaction (22). The reaction was allowed to proceed at
1
40 °C for 24 h. The structure was confirmed by H NMR
spectrum as shown in Figure 2c. The NMR peaks originated
from the benzyl ester groups (5.1 and 7.4 ppm) presented at
the PMABz segments disappeared; meanwhile, the characteristic
NMR peak corresponding to the hydrazine groups (9.0 ppm
broadening peak) was observed. After the H40-(PMA-Hyd-co-
PCL)-MPEG/PEG-FA copolymer was purified, it was reacted
with an excess amount of DOX to form the H40-(PMA-Hyd-
DOX-co-PCL)-MPEG/PEG-FA copolymer. After the reaction
was complete, the polymer solution was dialyzed and further
purified using Sephadex LH-20 to completely remove any
unbound DOX. The structure of the H40-(PMA-Hyd-DOX-co-
PCL)-MPEG/PEG-FA copolymer was confirmed by 1H NMR.
As shown in Figure 2d, the corresponding peaks of DOX
appeared at 0.8, 1.1, and 7.0 ppm, indicating the DOX molecules
were successfully conjugated onto the PMA segments by
hydrazone bonds. The drug loading content determined by the
UV measurement was 14.2 ( 0.3%. Theoretically, if all the
hydrazide groups attached on the PMA segments are conjugated
with the DOX molecules during the DOX conjugation reaction
step, the DOX loading content is close to 15%. Thus, based on
the DOX loading content measured by UV (14. 2%) and the
theoretical maximum DOX loading content (∼15%), it can be
deduced that both PMA repeat units in each arm of the 20-arm
hyperbranched copolymer were conjugated with DOX mol-
ecules. Namely, there were approximately 40 DOX molecules
per H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA molecule.
Micelle Properties of the H40-(PMA-Hyd-DOX-co-PCL)-
MPEG/PEG-FA Copolymer. Figure 3A shows the morphology
of the H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA unimo-
lecular micelles observed by TEM. It can be seen the unimo-
lecular micelles were spherical and most of them had a relatively
uniform size, which was about 20 nm. The size and size
distribution of the unimolecular micelles were further determined
by DLS. The hydrodynamic size of the H40-(PMA-Hyd-DOX-
co-PCL)-MPEG/PEG-FA unimolecular micelles was about 25
( 2 nm, which was somewhat higher than the particle size
Figure 4. Release profile of DOX from the H40-(PMA-Hyd-DOX-co-
PCL)-MPEG/PEG-FA micelles at different pH values.
determined by TEM (i.e., ∼20 nm), as expected. The size
distribution, expressed by polydispersity index, was 0.145 (
0.002. It should be noted that the resulting H40-(PMA-Hyd-
DOX-co-PCL)-MPEG/PEG-FA copolymer micelles were stable
over several weeks and displayed no changes in size or size
distribution within experimental accuracy during this period.
Effect of pH on the in Vitro DOX Release Rate. The pH-
sensitive DOX release behavior of the unimolecular micelles
was evaluated at a series of pH values. As shown in Figure 4,
the DOX release rate from the unimolecular micelles increased
consistently with lower pH values within the pH range of 4.0
to 7.4. A negligible amount of drug release was observed at
pH 7.4, the physiological condition of the bloodstream; thus,
almost no DOX will be released from the unimolecular micelles
during blood circulation, which is a desirable drug delivery
characteristic for drug nanocarriers. The pH values of the
endocytic compartments in which the FA-conjugated unimo-
lecular micelles are located once internalized by the tumor cells
Via FA-receptor mediated endocytosis generally ranges from
4.5 to 6.5. As shown in Figure 4, the DOX released rate
increased significantly within this pH range. In acidic conditions,
the acid-sensitive hydrazone bonds are cleaved releasing
unmodified DOX from the unimolecular micelles, which are
subsequently diffused into the cytoplasm and eventually to the
nucleus. At pH 5.0, about 48% DOX was released after 72 h.
Thus, once the unimolecular micelles are internalized into the
tumor cells, the conjugated DOX can be released effectively to
improve the therapeutic outcome of the chemotherapy.
Cellular Uptake of the Unimolecular Micelles. The behav-
iors of cellular uptake of the FA-conjugated H40-(PMA-Hyd-
Figure 3. (a) TEM micrograph and (b) DLS histogram of unimolecular micelles based on H40-(PMA-Hyd-DOX-co-PCL)-MPEG/PEG-FA
polymer.

502 Bioconjugate Chem., Vol. 21, No. 3, 2010
Yang et al.
Figure 5. Flow cytometry results of 4T1 cells treated with free DOX, H40-(PMA-Hyd-DOX-co-PCL)-MPEG micelles and H40-(PMA-Hyd-DOX-
co-PCL)-MPEG/PEG-FA micelles at 37 °C for 15 and 120 min (DOX concentration 20 µg/mL).
in cellular uptake, which was approximately 1.7 and 1.9 times
(Student’s t test; p < 0.005 and p < 0.0001, respectively) of the
cellular uptake of nontargeted micelles, respectively, while the
cellular uptake of free DOX was approximately 1.3 and 1.7
times (p < 0.05 and p < 0.00005, respectively) the cellular uptake
of FA-conjugated micelles, respectively. The higher cellular
uptake of free DOX compared with DOX conjugated onto
unimolecular micelles can be attributed to the different cellular
uptake mechanism. Free DOX was transported into cells Via
diffusion (4), while unimolecular micelles were taken up Via
an endocytosis process (23). However, the higher cellular uptake
by the 4T1 cells incubated with FA-conjugated micelles
compared with those incubated with nontargeted micelles clearly
demonstrated that FA was an effective tumor-targeting ligand
for tumor cells with positive FR. Moreover, the FA competition
experiment also showed that the cellular uptake level of FA-
conjugated micelles in the presence of free FA was dramatically
reduced and became essentially equivalent to that of nontargeted
micelles indicating the cell uptake of FA-conjugated micelles
was FR mediated endocytosis process (Figure 6). These findings
were further confirmed by the CLSM images as shown in Figure
7. 4T1 cells incubated with free DOX (Figure 7a) showed the
highest DOX fluorescence intensity in both cytoplasm and
nuclear after 2 h incubation. This is because free DOX can
quickly diffuse into the cytoplasm through the cell membrane
and transport into the nucleus, where it can interrupt the DNA
replication process (24). Consistent with the flow cytometry
finding, 4T1 cells incubated with FA-conjugated micelles
showed a much higher DOX fluorescence intensity than those
incubated with nontargeted micelles due to the FA-receptor
mediated endocytosis process.
Figure 6. DOX mean fluorescence intensity (MFI) located in the 4T1
cells incubated with free DOX, nontargeted, and FA-conjugated
unimolecular micelles for 15 and 120 min.
co-PCL)-MPEG/PEG-FA unimolecular micelles and nontargeted
H40-(PMA-Hyd-co-PCL)-MPEG unimolecular micelles against
folate receptor (FR) positive 4T1 cells were studied using both
flow cytometry and CLSM. Figure 5 shows the flow cytometry
histograms of the 4T1 cells incubated with free DOX, FA-
conjugated micelles, or nontargeted micelles for 15 min and
2 h. As the figure shows, 4T1 cells without any DOX treatment
were used as a negative control and showed only the autofluo-
rescence of the cells. The DOX fluorescence intensity (Figure
6) clearly indicated that the cellular uptake of DOX was the
highest in 4T1 cells incubated with free DOX, followed by FA-
conjugated micelles and then nontargeted micelles. After 15 and
120 min incubation, FA-conjugated micelles showed an increase
Cytotoxicity of the Unimolecular Micelles. The cytotoxic
effects of free DOX, H40-(PMA-Hyd-DOX-co-PCL)-MPEG,
Figure 7. CLSM images of 4T1 cells treated with (a) free DOX; (b) H40-(PMA-Hyd-DOX-co-PCL)-MPEG micelles; (c) H40-(PMA-Hyd-DOX-
co-PCL)-MPEG/PEG-FA micelles at 37 °C for 2 h (DOX concentration 20 µg/mL).

Unimolecular Micelles as Drug Nanocarriers
Bioconjugate Chem., Vol. 21, No. 3, 2010 503
targeting, pH-responsive, and potentially in ViVo stable unimo-
lecular micelles may provide a very promising approach for
targeted cancer therapy and greatly improve the quality of cancer
patient care.
ACKNOWLEDGMENT
The authors acknowledge the financial support from the
National Science Foundation (DMR 0906641) for this project.
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